CHEMISTRY AND BIOLOGY

OF PTERIDINES
AND FOLATES
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IRUN R. COHEN, The Weizmann Institute of Science
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RODOLFO PAOLETTI, University of Milan

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CHEMISTRY AND BIOLOGY
OF PTERIDINES
AND FOLATES
Edited by

June E. Ayling
M. Gopal Nair
Charles M. Baugh
University of South Alabama
Mobile, Alabama

SPRINGER SCIENCE+BUSINESS MEDIA, LLC

 Library of Congress Cataloging in Publication Data
Chemistry and biology of pteridines and folates I edited by June E. Ayling, M. Gopal
Nair, Charles M. Baugh.
p. cm.-{Advances in experimental medicine and biology; v. 338)
"Proceedings of the Tenth International Symposium on Chemistry and Biology of
Pteridines, held March 21-26, 1993, in Orange Beach, Alabama"-T.p. verso.
Includes bibliographical references and indexes.
ISBN 978-1-4613-6287-6 ISBN 978-1-4615-2960-6 (eBook)
DOI 10.1007/978-1-4615-2960-6
1. Pteridines-Congresses. 2. Folic acid-Congresses. 3. Prosthetic groups (En-
zymes)-Congresses. I. Ayling, June E. II. Nair, M. Gopal. III. Baugh, Charles M. IV.
International Symposium on Chemistry and Biology of Pteridines (lOth: 1993: Orange
Beach, Ala.) V. Series.
QP801.P69C47 1993 93-29401
599'.019'25---dc20 CIP

Proceedings of the Tenth International Symposium on Chemistry and Biology of Pteridines, held March
21-26, 1993, in Orange Beach, Alabama

ISBN 978-1-4613-6287-6

©1993 Springer Science+Business Media New York

Originally published by Plenum Press, New York in 1993

All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by
any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written
permission from the Publisher
PREFACE

The pteridines in their multitude of forms fulfill many roles in nature ranging from
pigments to cofactors for numerous redox and one-carbon transfer reactions. This
extraordinary diversity of function is unified by the unique chemistry of the pteridine
heterocycle. The International Symposium on the Chemistry and Biology of Pteridines and
Folates is a forum for presenting recent and exciting advances in this expanding field. In
bringing together various disciplines, a synergy of ideas results that has often stimulated
fresh approaches to major problems. The Tenth International Symposium held at Orange
Beach, Alabama, March 21-23, 1993, proved no exception by providing new insights into
folate enzymology, tetrahydrobiopterin and molybdopterin biosynthesis and function,
enzyme synthesis and regulation, along with novel synthetic strategies for producing
compounds that will expedite further study. The many outstanding scientific contributions
found in the following chapters, which represent the work presented at the Symposium, are
a reflection of the significant advances made since the Ninth International Symposium held
in Zurich in 1989.
Since the 7th International Symposium in St. Andrews, Scotland, a tradition has
evolved of honoring scientists who have made outstanding contributions to pteridine
research with a Gowland Hopkins medal and lectureship. Sir Frederick Gowland Hopkins
initiated the first investigation of what later proved to be pteridines in his studies of the
yellow and white colors of butterflies. The selection of Edward Taylor and Wolfgang
Pfleiderer for the 1993 Hopkins award seems especially appropriate in view of the
impending 1OOth anniversary of the first synthesis of a pteridine (lumazine 6,7-dicarboxylic
acid) by Ktihling in 1894. Almost every chemist in the field, as well as many others, have
greatly benefited from the careful and insightful work of these two scientists. Their many
contributions are summarized on the following pages by Peter Beardsley, a former Ph.D.
student of Edward Taylor, and Max Viscontini, still a very active pteridine chemist himself.
We would like to acknowledge members of the Advisory Committee who were of
great assistance in various aspects of planning the Symposium. We would also like to
thank the sponsors, listed on the Acknowledgments page, who made the meeting possible.
The breadth of the support we have received from both governmental and corporate
sponsors is an indication of the continuing recognition of the major role that pteridines play
in biology and medicine. The efforts of members of the finance committee in helping to
obtain this support are also greatly appreciated. Special thanks to Dusti Steelman and Judi
Naylor for so cheetfully helping with the mailings, the preparation of the program and
abstract book and with the registration before and during the meeting. Finally, we would
like to acknowledge the invaluable involvement of Steven Bailey.

v
 We were pleased to host this meeting in Alabama where significant and continuous
contributions have been made to this field over the past 30 years. We look forward to the
11th International Symposium which will be organized by Wolfgang Pfleiderer and Hartmut
Rokos and held in Germany.

June Ayling
Gopal Nair
Charles Baugh

College of Medicine
University of South Alabama

Mobile, April 1993

Vl
 GOWLAND HOPKINS LECTURESHIP
EDWARD C. TAYLOR

The choice of Ted Taylor as one of the two Gowland Hopkins Lecturers for this
International Symposium could not be more appropriate. His contributions to the field of
pteridine chemistry and biochemistry over a career spanning more than 45 years are
equalled only by those of Wolfgang Pfleiderer, the co-recipient of this honor. No one else
even comes close!
While his work in general ts so broad-based to perhaps defy specific
characterization, yet through it all run the traits of exceptional cleverness, imagination and
"elegant simplicity". He has a knack not only for designing and implementing novel
synthetic approaches, but also for noticing the unexpected result and exploiting it in many
variations to synthetic advantage. Taylor is famous for making the complex seem simple.
His work is replete with examples of the synthesis of complex heterocyclic and other
compounds in a short series of steps, many of which involve clever sequences of
condensation, ring cleavage and rearrangement reactions, often occurring "in one pot".
Taylor's contributions to pteridine chemistry include a series of more than 125
papers on pteridines, aza- and deaza-pteridines, numerous patents, and the classic volume
Ptendine Chemzstry, co-edited with Pfleiderer. These describe work which ranges from
the basic chemistry of these ring systems, through development of synthetic methodology,
to applications in the synthesis of natural products and the development of novel folate
antimetabolites. The classtc "Taylor Synthesis" involves elaboration of a pyrazine
intermediate and allows unequivocal synthesis of the biologically important 6-substituted
pteridines. He has applied this approach to the synthesis of natural pteridines including
xanthopterin, folic acid, L-erythro-biopterin, neopterin, urothione, and Form A of the
molybdenum cofactor- only a partial list.

Vll
 Probably the most striking achievement recently has been the synthesis of a wide
variety of deaza analogs of tetrahydrofolate. This work began with the synthesis nearly ten
years ago of 5,10-dideazatetrahydrofolate (DDATHF, Lometrexol). This compound was
shortly found to be a potent inhibitor of de novo purine biosynthesis. Broad spectrum
antitumor activity was then found in pre-clinical models, and Lometrexol is now
undergoing clinical trials with encouraging early results. The initial discovery of DDATHF
has had a major impact on subsequent antifolate drug development. Not only was a new
class of structures opened to further investigation, but added impetus was provided to the
search for other novel inhibitors of previously unexplored enzymatic targets for antifolate
attack. The result has been a major rekindling of interest in antifolate drug development,
an area in which Taylor himself has been a major participant. This work has been full of
new synthetic chemistry and biochemical surprises, and forms the basis for this Gowland
Hopkins Lecture.
Many whose relationship to Taylor is through a common interest in pteridines may
not realize that, despite the magnitude of his achievements in our field, this represents only
a portion of the overall scope of his work. He has also made major contributions to basic
chemistry and synthetic methodology in purines, pyrimidines, triazines and diazetidinones.
In fact, he has contributed very significantly to our knowledge of virtually all heterocyclic
systems. His contributions extend much further than heterocyclic chemistry and include
elegant total syntheses of Chinchona alkaloids. His well known one step synthesis of
tetrahydrocannabinol is a particularly illustrative example of his clever and imaginative
approach to synthetic design. Finally, Taylor has made numerous major contributions to
the whole field of synthetic organic chemistry. While there are many examples,
particularly outstanding has been his pioneering and extensive work in the use of thallium
reagents in organic syntheses. A long and highly productive collaboration between Taylor
and Prof. Alexander McKillop has led to the development of extraordinarily versatile
thallium reagents and reactions which have wide application in virtually every area of
synthetic organic chemistry.
Taylor's role as a teacher and mentor may be almost as important as his own work.
Those who hear his lectures and seminars are treated not only to exciting chemistry, but
to presentations of extraordinary organization and clarity. To students and others who work
with him, he transmits not only some of his profound understanding of how molecules
work and how to assemble them but, perhaps more importantly, a considerable measure of
his boundless enthusiasm and perpetual optimism. For Ted, it's never that a good idea
"doesn't work", it's just that it "hasn't worked yet". Those who come to know him soon
realize that these same qualities of vigor, enthusiasm and optimism apply to his whole
approach to life. Those who have known him over the years realize that these are not
diminishing, rather they are on a continuing upswing. We, therefore, look forward to a
great deal more in the way of exciting new developments and contributions to pteridines
and to chemistry in general.

G. Peter Beardsley
Yale University School of Medicine

viii
 GOWLAND HOPKINS LECTURESHIP
WOLFGANG PFLEIDERER

The year 1955 was a milestone in pteridine chemistry. After four years of intense,
exciting work, we had just published in Helvetica Chimica Acta three articles together with
P. Karrer and E. Hadom, in which we described the isolation of new compounds from the
small fly Drosophila melanogaster, two of which had a deep sky blue (in German "Himmel
J!lau", H ID fluorescence and were therefore baptized HB 1 and HB2 in our laboratory slang.
We found out very quickly that HB 1 corresponded to pterin, while the structure of HB2
gave us a lot of trouble. Experienced chemists like those of Stokstad's team would have
immediately identified HB 2 as L-biopterin. My lack of experience in pterin chemistry
plunged me into an abyss of doubt, because I trusted blindly the elemental analyses, which,
I must admit, in case of pterins, were far from being as accurate as they are today. To end
a long story, our work remained more or less unnoticed - what were we looking for in
these small flies, everybody wondered - especially because some weeks later Stokstad
published the isolation of a growth factor, starting from a thousand liters of human urine,
which he called L-biopterin, and determined its structure. Our work remained more or less
overlooked, except in the eyes of a young chemist who recognized rapidly the importance
of the research which was going on in our laboratory. In the beginning of 1956, a
handsome looking man of good appearance, tall, with a determined face, missing the left
arm, entered my office. A war-wounded man, I thought as I looked at him. I had in front
of me Wolfgang Pfleiderer, not the Professor Pfleiderer we know today, but a young man.
At that time he was 29 years old and well resolved to make his way in life.
"Professor Viscontini, I am a chemist at the University of Stuttgart, where in 1952
I gained my Ph.D. under the supervision of Professor Bredereck. My dissertation was
devoted to the chemical and physicochemical properties of some purine derivatives. Now

IX
I am writing my Habilitation about lumazines. Unfortunately, the topic does not really fill
my supervisor with enthusiasm. As a consequence I am obliged to work by myself without
people with whom I can discuss results, problems or difficulties. Recently I read your
papers on pteridines isolated from Drosophila melanogaster and I was greatly impressed
by the results you published on the research carried out on this fly. I have, therefore,
ventured to come to Zurich in order to make contact with a person who shares my
scientific interest. I would like to know, if I am correct or mistaken in involving myself
so strongly in the field of pteridine chemistry". Here was finally somebody with whom I
was in perfect communication! I felt that my work attracted him. I did my best to
convince him how right he was, taking the way of hard research where results do not
manifest themselves in kilograms of publications, but brings out the author's originality.
I emphasized, that in my opinion, pteridines might play a great role in nature, that he had
to go on in the direction he chose and that he would find later the benefit of it. In a short
time we became close friends, with a very similar scientific philosophy. We separated,
promising to meet again and to discuss as often as possible the results of our research.
Some months later, in 1957 Pfleiderer gained his Habilitation and his appointment as
Dozent at the University of Stuttgart. In 1958 he spent one year as Research Associate
at the University of Princeton with Professor Edward Taylor.
In the meantime we went on with the isolation of three orange pigments,
drosopterin, isodrosopterin, and neodrosopterin from Drosophila. In 1959 we published the
first hypothetical structures that we proposed for these pigments. After one year of absence
Pfleiderer did not hesitate to come to Zurich. "Sir, your structures are wrong. The UV and
the VIS spectra do not match the proposed formulae". I understood perfectly his point of
view, and suddenly it came to me: I felt isolated with my research, my results were not
especially well appreciated, I was short of money and coworkers and now I had in front
of me a promising young chemist, who, as I sensed, had a mind to get involved with the
field of natural pterins. My decision was immediate: "Listen, Herr Pfleiderer, in my
opinion, from the pteridines we just isolated there are two classes of compounds that seem
important, the drosopterins and the L-biopterin. Both are difficult to study, but both will
bring recognition to the chemist who elucidates their properties. I have confidence in you,
devote yourself to the chemistry of drosopterins and I, for my part, will work on the
chemistry of the L-biopterin".
From that day on I stopped working with drosopterin. In 1962 Professor Pfleiderer
formulated his first ideas on their structure. I could have as easily proposed to Pfleiderer
the study of L-biopterin and chosen the drosopterin for myself. Why did I choose L-
biopterin? Don't ask me, I have no idea myself. Anyway, we never regretted our oral
agreement, neither he nor me. We have remained close friends and loyal to our agreement.
As it turned out, destiny and my choice made Professor Pfleiderer the oracle for
drosopterins while I became the expert for tetrahydrobiopterins. Nevertheless, it is quite
certain, that without my encouragement Professor Pfleiderer would have become famous,
because of his personality. Some hundreds of communications and seven books published
between 1953 and 1992 about pteridines and their derivatives bear witness to the wideness
of the research he carried out, and the importance of the results he obtained. Let us cite
succinctly: tautomeric structures of pteridines, photochemistry, electrochemistry, and most
especially the chemistry of natural pteridines: structure of eugleniapterin, partial synthesis
of tetrahydromethanopterin and, to my opinion the most important and the most admirable
of all his works the structure and the synthesis of the drosopterin pigments.
Good luck for the future, dear Wolfgang!

Max Viscontini
University of ZUrich

X
ACKNOWLEDGMENTS

Contributions from the following sponsors are gratefully acknowledged:

Major Corporate Sponsor
Milupa AG., Germany
Other Corporate Sponsors
Acme Business Products, Mobile
American Cyanamid Company
Beckman Instruments, Inc.
Bristol-Myers Squibb
Burroughs Wellcome Co.
Ciba-Geigy Ltd., Switzerland
Coca-Cola Company
Dr. B. Schircks Laboratories, Switzerland
Eli Lilly and Company
F. Hoffmann-La Roche AG., Switzerland
Henning Berlin GMBH, Germany
ICI Pharmaceuticals, UK
Marion Merrell Dow, Inc.
Moravek Biochemicals
Sankyo Corp., Japan
SAPEC S.A., Switzerland
Sigma Chemical Co.
SmithKline Beecham
Swiss Air
The Upjohn Co.
Warner-Lambert Co.
Yamanouchi Pharmaceutical Co., Ltd., Japan
Organizations and Government Agencies
International Union of Biochemistry and Molecular Biology
National Institutes of Health
National Cancer Institute
National Institute of Allergy and Infectious Diseases
National Science Foundation
Ninth International Pteridine Symposium, Switzerland
The Council for Tobacco Research
Federation of American Societies of Experimental Biology
American Chemical Society, Medicinal Chemistry Division

and South Alabama Medical Science Foundation and the

College of Medicine of the University of South Alabama

xi
CONTENTS

CHEMISTRY

GOWLAND HOPKINS LECTURE:

Natural Pteridines - A Chemical Hobby . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
W. Pfleiderer

Properties and Reactions of Pteridines Carrying Functionalized

Side Chains at the 3-Position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
P. Boyle and R. Camier

N2-(N,N-Dimethylaminomethylene)-04-(2-p-Nitrophenylethy1)-Biopterin:
A Versatile Intermediate for a Glycosidation Reaction ................. 21
H. Yamamoto, T. Hanaya, K. Torigoe, and W. Pfleiderer

The Synthesis of Fluorine-Containing Pterins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

C. Dunn, C.L. Gibson, C.J. Suckling, and W. Xing

Formation of Fern or Ferr Complexes with Acetylacetonate and 5,6,7,8-

Tetrahydropterin as Ligands and Their Activation of Oxygen ............ 29
A. Schafer, B. Fischer, R. Bosshard, M. Hesse, and M. Viscontini

Stereoelectronic Effects in the Autooxidative Destruction of

Reduced Folate Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
A.L. Fitzhugh

MIND0/3 Molecular-Orbital Calculations on the 6R-BH4 Molecule

and Its Metabolic Precursors: Conformation and Activity ............... 39
S. Katoh, T. Sueoka, and T. Kurihara

Identification, Stereoconfiguration, Chromatographic and

Fluorescence Properties of Natural Pterins . . . . . . . . . . . . . . . . . . . . . . . . . 43
R. Klein

TETRAHYDROBIOPTERIN DEPENDENT HYDROXYLASES

The Mechanism of Cofactor Regeneration During Phenylalanine Hydroxylation .... 47

S.W. Bailey, S.R. Boerth, S.B. Dillard, and J.E. Ayling

xiii
Structure-Function Studies of the Phenylalanine Hydroxylase Active Site
and a Summary of Structural Features ............................ 55
R.G.H. Cotton, D.W. Howells, J.A. Saleeba, I. Dianzani, P.M. Smooker,
and I.G. Jennings

Expression of Wild Type and Mutant Forms of Human Phenylalanine

Hydroxylase in E. coli ....................................... 59
P.M. Knappskog, H.G. Eiken, A. Martinez, S. Olafsdottir, J. Haavik,
T. Flatmark, and J. Apold

A Re-examination of the Metal Requirement of Chromobacterium violaceum

Phenylalanine Hydroxylase .................................... 63
R.T. Carr and S.J. Benkovic

Histidines 138 and 143 are Copper Binding Ligands in Chromobacterium

violaceum Phenylalanine Hydroxylase ............................ 67
S. Balasubramanian, R.T. Carr, C.J. Bender, J. Peisach, and S.J. Benkovic

Characterization of the Iron Environment in Recombinant Human Tyrosine

Hydroxylase, Using Mossbauer and EPR-Spectroscopy ................ 71
J. Haavik, E. Bill, M. Lengen, A. Martinez, T. Flatmark, and A.X. Trautwein

Interaction of Substrate and Pterin Cofactor with the Metal of Human

Tyrosine Hydroxylase as Determined by 1H-NMR .................... 77
A. Martinez, C. Abeygunawardana, J. Haavik, T. Flatmark, and A.S. Mildvan

Mechanistic Studies of Tyrosine Hydroxylase ............................ 81

P.F. Fitzpatrick.

Alleviation of Catecholamine Inhibition of Tyrosine Hydroxylase by

Phosphorylation at Serine 40 ................................... 87
S.C. Daubner and P.F. Fitzpatrick

Glyceryl Ether Monooxygenase [EC 1.14.16.5]: Stoichiometry and Inhibition ...... 93

B. Kosar-Hashemi, H. Taguchi, and W.L.F. Armarego

TETRAHYDROBIOPTERIN REGENERATING ENZYMES

The Isolation and Characterization of Clones of 4a-Hydroxytetrahydrobiopterin

Dehydratase .............................................. 97
S. Kaufman, B.A. Citron, M. Davis, and S. Milstien

Molecular Cloning and Recombinant Expression of the Human Liver

Phenylalanine Hydroxylase Stimulating Factor Revealed Structural and
Functional Identity to the Dimerization Cofactor for the Nuclear
Transcription Factor HNF-1cx ................................. 103
B. Thony, F. Neuheiser, C.R. Hauer, and C.W. Heizmann

Progress in the Study of Biosynthesis and Role of ?-Substituted Pterins:

Function of Pterin 4a-Carbinolamine Dehydratase ................... 107
H.-Ch. Curtius, S. Ghisla, H. Hasegawa, N. Blau, and I. Rebrin

xiv
Spectroscopic Characterization of Human Liver Pterin 4a-Carbinolamine
Dehydratase ............................................. Ill
I. Rebrin, H.Ch. Curtius, S. Ghisla, and F.H. Herrmann

Is Dihydropteridine Reductase an Anomolous Dihydrofolate Reductase,

a Flavin-Like Enzyme or a Short-Chain Dehydrogenase? . . . . . . . . . . . . . . 115
J.M. Whiteley, N.H. Xuong, and K.I. Varughese

Two Crystal Structures of Rat Liver Dihydropteridine Reductase .............. 123

K.I. Varughese, Y. Su, M.M. Skinner, N.H. Xuong, D.A. Matthews,
and J.M. Whiteley

New Inhibitors of Dihydropteridine Reductase (Human Brain) . . . . . . . . . . . . . . . 127

D. Randles, H. Taguchi, and W.L.F. Armarego

CYS~SER Mutations inch-Human Dihydropteridine Reductase .............. 131

C.M. Hardy, H. Averdunk, B. Paal, R.G.H. Cotton, and W.L.F. Armarego

The Spectrum of Mutations in Dihydropteridine Reductase Deficiency .......... 135

P.M. Smooker, D.W. Howells, I. Dianzani, and R.G.H. Cotton

TETRAHYDROBIOPTERIN BIOSYNTHESIS

Cloning and Characterization of Genes Encoding Tetrahydrobiopterin

Biosynthetic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
R.A. Levine, J.C. States, P.Z. Anastasiadis, and D.M. Kuhn

Drosophila GTP Cyclohydrolase: Multiple Isoform Products of a

Single Gene Derive From Alternate Transcripts that are
Developmentally Regulated and Functionally Specific ................ 147
J.M. O'Donnell, G. Ranganayakula, X. Chen, S. Krishnakumar, and
W.S. Neckameyer

Studies on GTP Cyclohydrolase I of Escherichia coli . . . . . . . . . . . . . . . . . . . . . 157

C. Schmid, W. Meining, S. Weinkauf, L. Bachmann, H. Ritz, S. Eberhardt,
W. Gimbel, T. Werner, H-W. Lahm, H. Nar, and A. Bacher

Partial Purification and Characterization of GTP Cyclohydrolase I

from Spinach Leaves ....................................... 163
Y. Sohta, T. Ohta, and M. Masada

Detection and Quantification of GTP Cyclohydrolase I mRNA 167

M. Giitlich, K. Schott, T. Werner, A. Bacher, and I. Ziegler

Localization of GTP Cyclohydrolase I mRNA in the Rat Brain by

In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
S.I. Lentz, K. Hirayama, and G. Kapatos

Expression of GTP Cyclohydrolase I mRNA in the Rat:

Tissue Distribution and Effect of Reserpine ....................... 175
K. Hirayama, S.I. Lentz, and G. Kapatos

XV
 Regulation of Tetrahydrobiopterin Biosynthesis in Cultured
Hypothalamic and Mesencephalic Neurons by Cyclic
AMP-Dependent GTP Cyclohydrolase I Gene Expression . . . . . . . . . . . . . . 179
K. Hirayama, M. Zhu, and G. Kapatos.

Mycophenolic Acid Simultaneously Reduces Intracellular GTP and

Tetrahydrobiopterin Levels in Neuro-2A Cells ...................... 183
T. Harada, K. Hatakeyama, and H. Kagamiyama

Human Liver 6-Pyruvoyl-Tetrahydropterin Synthase: Expression of the eDNA,

Purification and Preliminary Characterization of the Recombinant Protein .. 187
B. Thony, W. Leimbacher, N. Blau, C.W. Heizmann, and D. Biirgisser

Enzymatic Properties of 6-Pyruvoyl-Tetrahydropterin Synthase

Purified from Fat Bodies of Silkworm Larvae ...................... 191
M. Masada

Northern Blot Analysis of Sepiapterin Reductase mRNA In

Mammalian Cell Lines and Tissues ............................. 195
J. Maier, K. Schott, T. Werner, A. Bacher, and I. Ziegler

Purification and Properties of Human Sepiapterin Reductase from Placenta ...... 199
J. Maier and I. Ziegler

TETRAHYDROBIOPTERIN REGULATION

Stimulation of Tetrahydrobiopterin Synthesis by Cytokines in Human

and In Murine Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
E.R. Werner, G. Wemer-Felmayer, G. Weiss, and H. Wachter

Interferon-y and Kit-Ligand are Primary Regulators of GTP Cyclohydrolase

Activity: Mechanisms and Implications .......................... 211
I. Ziegler, K. Schott, and L. Hiiltner

Differential Metabolism of Tetrahydrobiopterin in Monoamine Neurons:

A Hypothesis Based Upon Clinical and Basic Research ............... 217
G. Kapatos, K. Hirayama, S.l. Lentz, M. Zhu, and S.L. Stegenga

Tissue Distribution of Tetrahydrobiopterin Generating Enzymes . . . . . . . . . . . . . . 223

M. Hoshiga, K. Hatakeyama, and H. Kagamiyama

Co-Induction of Tetrahydrobiopterin Levels and Tyrosine Hydroxylase

Activity in Cultured PC12 Cells ............................... 227
P.Z. Anastasiadis, J.C. States, D.M. Kuhn, and R.A. Levine

Long-Term Treatment of PC12 Pheochromocytoma with Dibutyryl

Cyclic AMP Increases Biopterin Content in the Cells but
Decreases that in the Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
N. Nakanishi, S. Onozawa, A. Isono, M. Hara, H. Hasegawa, and S. Yamada

xvi
Nicotinic Cholinergic Regulation of Tetrahydrobiopterin Levels in
Bovine Adrenal Chromaffin Cells .............................. 235
J.C. Waymire, J.E. Ayling, and G.L. Craviso

Inter-Relationships Between Pterins and Cytokines Produced During

Allogeneic Immune Reactions and Possible Use as Early Markers
of Immune Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
J.J. Rippin and D.C. Henderson

An Example of the Detection of an Esophageal Carcinoma in Its

Very Early Stage by Urinary Xanthopterin Determination .............. 243
T. Iino, H. Watanabe, W.L. Gyure, T. Mazda, H. Mieno, and M. Tsusue

Neopterin in Subacute Sclerosing Panencephalitis ........................ 247

H. Shintaku, R. Murata, H. Hattori, 0. Matsuoka, T. Nakajima,
T. Imamura, and Y. Sawada

The 7-Deazaguanine Derivative, Queuine, Regulates Mammalian Cell

Proliferation Depending on the Metabolic State . . . . . . . . . . . . . . . . . . . . . 251
W. Langgut, M. Haupt, and T. Reisser

TETRAHYDROBIOPTERIN DEFICIENCY

Tetrahydrobiopterin Deficiency and an International Database of Patients . . . . . . . . 255

N. Blau and J.-L. Dhondt

Tetrahydrobiopterin Deficiency in Portugal: Results of the Screening

for Hyperphenylalaninemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
LT. de Almeida, P.P. Leandro, R. Portela, A. Cabral, F. Eusebio,
T. Tasso, A. Matasovic, and N. Blau

A Microtitre Plate Method for Measuring Biopterin with Cryopreserved

Crithidia fasciculata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
R.J. Leeming, S.K. Hall, H. Friday, P. Hurley, and A. Green

Oral Administration of Liposomally Entrapped Tetrahydrobiopterin . . . . . . . . . . . . 271

Y. Sawada, H. Shintaku, T. Nakajima, T. Imamura, Y. Tsubakio,
C. Iwamura, G. Isshiki, and T. Oura

Experimental Research on a Fetal Treatment for Tetrahydrobiopterin Deficiency . . . 273

H. Shintaku, T. Nakajima, T. Imamura, Y. Sawada, G. Isshiki, and T. Oura

Experimental Research on a New Treatment for Maternal Phenylketonuria . . . . . . . 277

T. Imamura, H. Shintaku, T. Nakajima, Y. Sawada, G. Isshiki, and T. Oura

NITRIC OXIDE SYNTHASE

Nitric Oxide Synthase: Function and Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . 281

M.A. Marietta

xvii
Macrophage Nitric Oxide Synthase: Tetrahydrobiopterin Decreases
the NADPH Stoichiometry ................................... 285
J.M. Hevel and M.A. Marietta

Role of Tetrahydrobiopterin in Cytokine-Stimulated Metabolism of Tryptophan

and Hydroxylation of Arginine ................................ 289
S. Milstien, N. Sakai S. Kaufman, K. Saito, and M.P. Heyes

Tetrahydrobiopterin Synthesis is Induced by LPS in Vascular Smooth

Muscle and is Rate-Limiting for Nitric Oxide Production . . . . . . . . . . . . . . 295
S.S. Gross, R. Levi, A Madera, K.H. Park, J. Vane, and Y. Hattori

6R-[ 3H] Tetrahydrobiopterin Binding Activities in Rat Brain ................. 301

Yu. Watanabe, H. Morii, Y. Nemoto, B. Mayer, E.R. Werner,
S. Miwa, and Ya. Watanabe

Inducible Nitric Oxide Synthase Activity in Hepatocytes is Dependent

on the Coinduction of Tetrahydrobiopterin Synthesis . . . . . . . . . . . . . . . . . 305
M. Di Silvio, D.A. Geller, S.S. Gross, A Nussler, P. Freeswick,
R.L. Simmons, and T.R. Billiar

Modulation of Nitric Oxide Synthase Activity in Intact Cells by

Intracellular Tetrahydrobiopterin Levels .......................... 309
G. Werner-Felmayer, E.R. Werner, G. Weiss, and H. Wachter

OTHER FUNCTIONS OF TETRAHYDROPTERINS

6R-L-Erythro-5,6,7,8-Tetrahydrobiopterin: A Regulator of
Neurotransmitter Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
K. Koshimura, T. Ohue, Ya. Watanabe, and S. Miwa

Positron Emission Tomography Studies on Some Neurotransmitter

Receptor Systems with 6R-Tetrahydrobiopterin Pretreatment ............ 321
Ya. Watanabe, Y. Tani, T. Kanai, P. Hartvig, 0. Inoue,
J. Andersson, A Lilija, and B. Ungstrom

Positron Emission Tomography (PET) Study: The Effects of 6R-L-Erythro-

5,6,7,8-Tetrahydrobiopterin (R-THBP, SUN 0588) on the Central
Dopamine D 1, D2, and D 3 Receptors in Rhesus Monkey ............... 327
Y. Tani, T. Ishihara, T. Kanai, T. Ohno, H. Onoe, Ya. Watanabe,
J. Andersson, A Lilija, G. Westerberg, P. Hartvig, and B. Ungstrom

6R-L-Erythro-5,6,7,8-Tetrahydrobiopterin (R-THBP, SUN0588) Acts on the

Brain Muscarinic and Nicotinic Cholinergic Receptors as Evaluated
by Positron Emission Tomography (PET) Studies in Rhesus Monkey . . . . . . 331
Y. Tani, T. Ishihara, T. Kanai, T. Ohno, Ya. Watanabe, J. Andersson,
A Lilija, G. Westerberg, P. Hartvig, and B. Ungstrom

Effect of 6R-Tetrahydrobiopterin on the Central Muscarinic Cholinergic

Receptor as Evaluated by Positron Emission Tomography Studies
Using Rhesus Monkey ...................................... 335
Ya. Watanabe, H. Onoe, M. Tanaka, K. Kobayashi, K. Suziki, Y. Tani,
S. Miwa, and 0. Inoue

xviii
Increase of Tetrahydropterins in Cell-Free Retinal Extracts in Response
to Light Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
G. Cremer-Bartels, H. Gerding, and K. Krause

Effect of Triamterene on the Electroretinogram of Long Evans Rats .....•.••.• 343

G. Cremer-Bartels, H. Gerding, L. Hanneken, and K. Krause

Immunoenzymatic Labeling of Biopterin and Neopterin in the

Pigment Epithelium of Bovine Retina ........................... 347
H. Gerding, E. Vollmer, G. Cremer-Bartels, H. Rokos,
K. Krause, and H. Busse

Reduced Pterins as Scavengers for Reactive Oxygen Species . . . . . . . . . . . . . . . . 351

R. Shen and Y. Zhang

MOLYBDOPTERIN COFACTORS

Chemistry and Biology of the Molybdenum Cofactors ..................... 355

K.V. Rajagopalan, J.L. Johnson, M.M. Wuebbens, D.M. Pitterle,
J.C. Hilton, T.R. Zurick, and R.M. Garrett

Studies on the Molybdenum Cofactor. Synthesis of (±)-Form B (Dephospho) .... 363

E.C. Taylor and I.S. Darwish

Molybdenum-Pterin Complexes: A Functional and Structural Model for the

Binding Site in the Enzyme Dimethyl Sulfoxide Reductase . . . . . . . . . . . . 369
B. Fischer, H. Schmalle, E. Dubler, and M. Viscontini

Human Molybdenum Cofactor Deficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

J.L. Johnson, K.V. Rajagopalan, and S.K. Wadman

Molybdopterin Biosynthesis in Man. Properties of the Converting Factor

in Liver Tissue from a Molybdenum Cofactor Deficient Patient . . . . . . . . . 379
J.L. Johnson and K.V. Rajagopalan

Cloning of a Eukaryotic Molybdenum Cofactor Gene . . . . . . . . . . . . . . . . . . . . . 383

P. Kamdar, M.E. Shelton, and V. Finnerty

SYNTHESIS AND BIOLOGICAL EVALUATION OF ANTI-FOLATES

GOWLAND HOPKINS LECTURE:

Design and Synthesis of Inhibitors of Folate-Dependent Enzymes
as Antitumor Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
E.C. Taylor

Synthesis and Antitumor Activity of LY288601, the 5,6-Dihydro

Analog of LY231514 ....................................... 409
C.J. Barnett, T.M. Wilson, and G.B. Grindey

Synthesis and Preliminary Biological Evaluation of Analogues of

5,8-Dideazaisofolic Acid and its 2-Desamino-2-Methyl
Derivative Containing Fluorine at Position 5 ....................... 413
J.B. Hynes, O.S. Fetzer, B. Shane, and J.H. Freisheim

xix
Synthesis and Biological Evaluation of Analogues of 5,8-Dideazaisofolic
Acid (IAHQ) Modified at Positions 2, 4 and 9 . . . . . . . . . . . . . . . . . . . . . 417
J.B. Hynes, R.L. Hagen, B. Shane, and J. Freisheim

New Thiophene Substituted 10-Deazaaminopterins: Synthesis and

Biological Evaluation ....................................... 421
A. Abraham, J.J. McGuire, J. Galivan, B.R. Vishnuvajjala, and M.G. Nair

Evaluation of the Anti-Arthritic Activity and an Alternate Synthesis

of a Thiophene-Substituted 10-Deazaaminopterin .................... 425
A. Desai and M.G. Nair

Lipophilic Antifolates as Candidates Against Opportunistic Infections . . . . . . . . . . 429

J.R. Piper, C.A. Johnson, C.A. Hosmer, R.L. Carter,
E.R. Pfefferkorn, S.E. Borotz, and S.F. Queener

Analogues of Classical Antifolates Bearing Naphthoyl in Place of Benzoyl ...... 435

J.R. Piper, C.A. Johnson, J.A. Maddry, J.J. McGuire,
G.M. Otter, and F.M. Sirotnak

Synthesis and Biological Activity of Tricyclic, Conformationally Restricted

Analogs of Lipophilic Pyrido[2,3-d]Pyrimidine Antifolates ............. 441
A. Gangjee, F. Mavandadi, and S.F. Queener

Novel 2,4-Diamino-5-Substituted Furo[2,3-d]Pyrimidines as Potential Antifolates .. 445

A. Gangjee, R. Devraj, S.F. Queener, and R.L. Kisliuk

Bicyclic Conformationally Restricted Analogs of Nonclassical Pyrido[2,3-d]

Pyrimidines as Potential Inhibitors of Dihydrofolate Reductases . . . . . . . . . 449
A. Gangjee, A. Vasudevan, and S.F. Queener

Synthesis, Structural and Biochemical Characterization of Cytostatic

Methotrexate-y-Glutamyl-Glutathione Conjugates . . . . . . . . . . . . . . . . . . . 453
M. Kussmann, D. Wiehr, T. Knepper, and M. Przybylski

Activation by Peptidases and Cytotoxicity of 2-(L-a-Aminoacyl)

Prodrugs of Methotrexate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
H.T.A. Cheung, Z. Dong, L. Escoffier, M.A. Smal, and M.H.N. Tattersall

Effect of a Novel Antifolate, N"-(4-Amino-4-Deoxypteroyl)-~-Hemiphthaloyl­

L-Omithine (PT523) on Growth of H35 Rat Hepatoma and
HEPG2 Human Hepatoma Cells ............................... 461
M.S. Rhee, J. Galivan, E.M. Tyobeka, M.L. Sherman, and A. Rosowsky

Tubulin Binding Properties of Two Chiral Isomers with

1-Deaza-7 ,8-Dihydropteridine Structure .......................... 465
D. Leynadier, V. Peyrot, M. Sarrazin, J.M. Andreu, C. Temple,
G.A. Rener, and C. Briand

Effects of Folic Acid on Pyrimethamine Teratogenesis in Rats . . . . . . . . . . . . . . . 469

G. Kudo, K. Tsunematsu, M. Shimoda, and E. Kokue

XX
 DIHYDROFOLATE REDUCTASE

Mutations of Human Dihydrofolate Reductase Causing Decreased

Inhibition by Methotrexate ................................... 473
R.L. Blakley, J.R. Appleman, S.K. Chunduru, T. Nakano,
W.S. Lewis, and S.E. Harris

Conformational Analysis of Human Dihydrofolate Reductase Inhibitor

Complexes: Crystal Structure Determination of Wild Type and F31
Mutant Binary and Ternary Inhibitor Complexes .................... 481
V. Cody, A. Wojtczak, T.l. Kalman, J.H. Freisheim, and R.L. Blakley

Computer-Aided Design of Mechanism-Based Pterin Analogues and MD/FEP

Simulations of Their Binding to Dihydrofolate Reductase . . . . . . . . . . . . . . 487
J.E. Gready, P.L. Cummins, and P. Wormell

Does R67 Dihydrofolate Reductase Possess a Proton Donor? . . . . . . . . . . . . . . . . 493

J.C. Holland, C.E. Linn, E. DiGiammarino, R.J. Nichols, and E.E. Howell

Laser-Sensitized Tautomers in Dihydrofolate Reductase . . . . . . . . . . . . . . . . . . . . 499

J.W. Ledbetter, W. Pfleiderer, and J.H. Freisheim

Methotrexate-Insensitive Mutants of Human Dihydrofolate Reductase (hDHFR)

Constructed by Site-Directed Mutagenesis at Phenylalanine 34 .......... 503
T. Nakano, J.R. Appleman, and R.L. Blakley

Kinetic Investigation of Methotrexate Resistant Human Dihydrofolate

Reductase (hDHFR) Mutants at Phenylalanine 31 . . . . . . . . . . . . . . . . . . . 507
S.K. Chunduru, J.R. Appleman, and R.L. Blakley

The Effect of Codon 31 on the Relative Affinities for the Binding of

Designed 8-Alkyl-Pterins to Dihydrofolate Reductase: A Statistical
Perturbation Theory and Molecular Dynamics Simulation Study ......... 511
P.L. Cummins and J.E. Gready

Effect of Codon 22 Mutations on Substrate and Inhibitor Binding

for Human Dihydrofolate Reductase ............................ 515
E. Ercikan, M. Waltham, A. Dicker, B.I. Schweitzer, and J.R. Bertino

Thermodynamic Study of Folate Analogue Binding to Dihydrofolate

Reductase from Different Species .............................. 521
S. Sasso, R. Gilli, C. Lopez, J.C. Sari, and C. Briand

Comparison of Binding and Activity of 8-Aikyl-Pterins and 8-Aikyl-N5-

Deaza-Pterins with Dihydrofolate Reductase ....................... 525
M.T.G. Ivery and J.E. Gready

Development of a Spectrofluorimetric Method for Determining the pK,.

of Pterin-Analogue Ligands Bound to Dihydrofolate Reductase .......... 529
S-S. Jeong and J.E. Gready

xxi
Selective Inhibition of Dihydrofolate Reductase from Problem Human Pathogens . . 533
R.L. Then, P.G. Hartman, I. Kompis, and D. Santi

Translational Regulation of the Synthesis of Dihydrofolate Reductase .......... 537

E. Ercikan, D. Banerjee, M. Waltham, B. Schnieders, K.W. Scotto,
and J.R. Bertino

Expression of the Trimethoprim Resistant Dihydrofolate Reductase

Encoded by Transposon TN4003 in a Soluble Form and its Subsequent
Purification to Homogeneity .................................. 541
G.E. Dale, D. Stuber, C. Broger, and H. Langen

Effect of Genomic Position on Amplification of the DFR1

Gene in Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
B.J. Barclay, N.K. Ondrusek, Y.D. Wildenhain, T. Huang, R.L. Carlone,
J-M. Clement, and G.M. Wahl

Dihydrofolate Reductase is not the Target of Trimethoprim in

Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
B.J. Barclay, M.G. Nagel, and T. Huang

Point Mutations in the Dihydropteroate Synthase Gene Causing

Sulfonamide Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555
G. Swedberg, C. Fermer, and 0. Skold

Frequent Amplification of a Short Chain Dehydrogenase Gene

in Methotrexate Resistant Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
B. Papadopoulou and M. Ouellette

THYMIDYLATE SYNTHASE

Enzyme Interactions Involving T4 Phage-Coded Thymidylate

Synthase and Deoxycytidylate Hydroxymethylase . . . . . . . . . . . . . . . . . . . 563
C.K. Mathews, L.J. Wheeler, C. Ungermann, J.P. Young, and N.B. Ray

Isolation of cDNAs Encoding Thymidylate Synthase from Soybean

Seedlings and Expression of the Protein in E. coli . . . . . . . . . . . . . . . . . . . 571
M. Wang, S. Ratnam, and J.H. Freisheim

Use of 10-Propargyl-5,8-Dideazafolate and Directed Mutagenesis to

Probe the Catalytic Mechanism of Thymidylate Synthase .............. 575
H.T. Spencer, J.E. Villafranca, and J.R. Appleman

y-Linked Dipeptide Analogues of 2-Desamino-2-Methyl-N10-Propargyl-5,8-

Dideazafolate as Antitumour Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
A.L. Jackman, G.M.F. Bisset, D.I. Jodrell, W. Gibson, R. Kimbell,
V. Bavetsias, A.H. Calvert, K.R. Harrap, T.C. Stephens,
M.N. Smith, and F.T. Boyle

Substituted-2-Desamino-2-Methyl-Quinazolinones: A Series of Novel

Antitumour Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
F.T. Boyle, Z.S. Matusiak, L.R. Hughes, A.M. Slater, T.C. Stephens,
M.N. Smith, M. Brown, R. Kimbell, and A.L. Jackman

xxii
Use of Murine L5178Y Lymphoma Thymidine Kinase Mutants for
in Vitro and in Vivo Antitumour Efficacy Evaluation of Novel
Thymidylate Synthase Inhibitors ............................... 589
T.C. Stephens, M.N. Smith, S.E. Waterman, M.L. McCloskey, A.L. Jackman,
and F.T. Boyle

Quinazoline Antifolates Inhibiting Thymidylate Synthase: Synthesis of

y-Linked Peptide and Amide Analogues of 2-Desamino-2-Methyl-N10-
Propargyl-5,8-Dideazafolic Acid (ICI 198583) ..................... 593
V. Bavetsias, A.L. Jackman, T.J. Thornton, K.P. Pawelczak,
F.T. Boyle, and G.M.F. Bisset

The Duration of the Inhibition of Thymidylate Synthase in Intact L1210 Cells

Exposed to Two Different Classes of Quinazoline Analogues . . . . . . . . . . . 597
R. Kimbell, A.L. Jackman, F.T. Boyle, A. Hardcastle, and G.W. Aherne

The Toxicity of ICI D1694 in Man and Mouse .......................... 601

S.J. Clarke, A.L. Jackman, and I.R. Judson

Evaluation of Immunohistochemical Staining and Activity of

Thymidylate Synthase in Cell Lines ............................. 605
C.L. van der Wilt, K. Smid, G.W. Aherne, H.M. Pinedo, and G.J. Peters

The Interval Between Methotrexate and Leucovorin Determines the

Efficacy of 5-Fluorouracil Modulation in Vitro and in Vivo ............. 609
C.L. van der Wilt, M. de Jong, B.J.M. Braakhuis, H.M. Pinedo, and
G.J. Peters

Potentiation of 5-Fluorouracil Induced Inhibition of Thymidylate Synthase

in Human Colon Tumors by Leucovorin is Dose Dependent ............ 613
G.J. Peters, K. Hoekman, C.J. van Groeningen, C.L. van der Wilt,
K. Smid, S. Meijer, and H.M. Pinedo

Interaction with 2(4)-Thio-5-Fluoro-dUMP of Thymidylate Synthases

with Differing Sensitivities to 5-Fluoro-dUMP ..................... 617
J.M. Dzik, Z. Zielillski, J. Ciesla, W. Rode, M. Bretner,
T. Kulikowski, and D. Shugar
Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-
(N4-Hydroxy-5-Fluoro)-dCMP in View of the Structure and
Conformation of N4-Hydroxy-(N4Hydroxy-5-Fluoro )-Cytosine
Calculated by the Ab Initio Quantum Mechanical Methods ............. 621
A. Les, L. Adamowicz, and W. Rode

Sulphonamide Antifolates Inhibiting Thymidylate Synthase. Synthesis,

Enzyme Inhibition and Cytotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
K. Pawelczak, M. Makowski, M. Kempny, J.M. Dzik, M. Balmska,
and W. Rode

FOLYLPOLYGLUTAMATE SYNTHETASE AND HYDROLASE

Folylpoly-y-Glutamate Synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629

B. Shane, T. Garrow, A. Brenner, L. Chen, Y-J. Choi,
J-C. Hsu, and P. Stover

xxiii
Increased Activity of y-Glutamyl Hydrolase in Human Sarcoma Cell Lines:
A Novel Mechanism of Intrinsic Resistance to Methotrexate . . . . . . . . . . . . 635
W.W. Li, M. Waltham, W. Tong, B.l. Schweitzer, and J.R, Bertino

Mechanism-Based Approaches to Inhibition of the Synthesis and Degradation

of Folate and Antifolate Polyglutamates . . . . . . . . . . . . . . . . . . . . . . . . . . 639
T.l. Kalman

Polyglutamate Product Formation by Lactobacillus casei Folylpolyglutamate

Synthetase in Vztro and in Vzvo in Recombinant Escherichia coli . . . . . . . . . 645
J. Toy and A.L. Bognar

Development of a Simple Folylpolyglutamate Synthetase Assay in

Tissues and Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651
G.J. Peters, C.L. van der Wilt, J. Cloos, and H.M. Pinedo

Two Novel HPLC Methods which Rapidly Detect the Substrates and
Cleavage Products of y-Glutamyl Hydrolase . . . . . . . . . . . . . . . . . . . . . . . 655
Y. Wang, R.F. Rotundo, Z. Nimec, T.J. Ryan, and J. Galivan

Purification and General Properties of Human Folylpolyglutamate Synthetase . . . . . 659

T.A. Garrow and B. Shane

Antitumor Efficacy of Classical Non-Polyglutamylatable Antifolates

that Inhibit Dihydrofolate Reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 663
A. Abraham, M.G. Nair, J.J. McGuire, J. Galivan, R.L. Kisliuk,
and B.R. Vishnuvajjala

Studies on the Cross-Resistance of Folylpolyglutamate Synthetase-Deficient,

Methotrexate-Resistant CCRF-CEM Human Leukemia Sublines ......... 667
J.J. McGuire, K.J. Heitzman, W.H. Haile, C.A. Russell,
D.E. McCloskey, and J.R. Piper

Variable Pharmacodynamics of Antifolates in Squamous Cell

Carcinoma of the Head and Neck . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671
B.J.M. Braakhuis, G. Jansen, and G.J. Peters

Metabolism of Folate Glutamates in Aspergillus nidulans ................... 675

I. Lewandowska, E. Sikora, I. Szablewska, M. Balmska and A. Paszewski

ONE-CARBON METABOLISM

Evidence that 5-Formyltetrahydropteroylglutamate has a Metabolic

Role in One-Carbon Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679
P. Stover, H. Kruschwitz, and V. Schirch

Cobalamin-Dependent and Cobalamin-Independent Methionine Synthases in

Escherichia coli: Two Solutions to the Same Chemical Problem ......... 687
J.T. Drummond and R.G. Matthews

Folate Metabolites as Modulators of Antitumor Drug Activity . . . . . . . . . . . . . . . . 693

D.G. Priest, J.C. Schmitz, and M.A. Bunni

xxiv
MTX Does Not Affect Enhanced Biosynthesis and Metabolism of S-Adenosyl-
Methionine in Testosterone-Induced Hypertrophic Mouse Kidney ........ 699
W. Chmurzynska, M. Manteuffel-Cymborowska, M. Szlazak, and
B. Grzelakowska-Sztabert

Thermolability of Residual Methylenetetrahydrofolate Reductase (MR)

Activity, Methionine Synthase Activity and Methyl-Cobalamin Levels
in Cultured Fibroblasts from Patients with MR De(iciency ............. 703
D.S. Rosenblatt, H. Lue-Shing, N. Matiaszuk, L. Low-Nang,
A. Arzoumanian, and B.A. Cooper

Enzymes For Synthesis of 10-Formyltetrahydrofolate in Plants: Characterization

of a Monofunctional 10-Formyltetrahydrofolate Synthetase
and Co-Purification of 5,10-Methylenetetrahydrofolate Dehydrogenase
and 5,10-Methenyltetrahydrofolate Cyclohydrolase Activities ........... 707
E.A. Cossins, C.D. Kirk, H.C. Imeson, and L. Zheng

Cloning of the Genes Encoding the Serine Hydroxymethyltransferases

from Saccharomyces cerevisiae ................................ 711
B.V. Taylor, J.B. McNeil, E.M. Mcintosh, F. Zhang and A.L. Bognar

Serine Hydroxymethyltransferase: Role of the Active Site Lysine

in the Mechanism of the Enzyme .............................. 715
D. Schirch, S. Delle Pratte, S. Iurescia, S. Angelaccio,
R. Contestabile, F. Bossa, and V. Schirch

Purification of Neurospora crassa Cytosolic Serine Hydroxymethyltransferase .... 719

H. Kruschwitz, D. McDonald, E. Cossins, and V. Schirch

Purification and Properties of Rabbit Liver 5,10-Methenyltetra-

hydrofolate Synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
P. Stover, T. Huang, V. Schirch, B. Maras, S. Valiante, and D. Barra

FOLATE NUTRITION AND TRANSPORT

Folate Metabolism in Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 727

J.M. Scott, J. McPartlin, A. Molloy, H. McNulty, A. Halligan,
M. Darling, and D.G. Weir

Influence of Gestation and Lactation on the Levels of Plasma Folates in Sows . . . . 733
M. Natsuhori, E. Kokue, and M. Shimoda

Identification of Endogenous Tetrahydrofolate and 10-Formyltetrahydrofolate

as Major Folates in Rat Bile .................................. 737
H-C. Shin, M. Shimoda, E. Kokue, and Y. Takahashi

Development of a Sensitive Assay for Detection of Uracil in DNA ............ 741

B.C. Blount and B.N. Ames

5-Methyltetrahydrofolate Urinary Excretion: Modeling by Cultured

Human Kidney Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745
K.E. McMartin, K.M. Morshed, D.J. Hazen-Martin, and D.A. Sens

XXV
Contribution of Plasma Protein Binding to the Stability of
Tetrahydrofolate in Pig Plasma ................................ 749
K. Sasaki, M. Shimoda, Y. Aoki, M. Natsuhori, and E. Kokue

Ligand Induced Conformation Change in Folate Binding Protein .............. 753

N.C. Kaarsholm, A-M. Kolstrup, S.E. Danielsen, J. Holm, and S.I. Hansen

The High-Mfinity Folate Binding Protein in Normal and

Malignant Mammary Gland Tissue ............................. 757
J. Holm, S.I. Hansen, K. Sjijndergaard, and M. Hl<1ier-Madsen

Detergent-Insolubility During the Biosynthesis of Membrane Folate Receptor-2 ... 761

S. Rijnbout, G. Strous, E. Bijleveld, G. Posthuma, M. Ratnam,
J.H. Schomagel, and G. Jansen

The Reduced Folate/Methotrexate Carrier and a Membrane-Associated

Folate Binding Protein as Transport Routes for Novel Antifolates:
Structure-Activity Relationships ............................... 767
G. Jansen, G.R. Westerhof, J.H. Schomagel, A.L. Jackman, and F.T. Boyle

Identification of a Reduced Folate/Methotrexate Carrier in Human KB-Cells

Expressing High Levels of Membrane-Associated Folate Binding Protein ... 771
G.R. Westerhof, J.H. Schomagel, S. Rijnboutt, H.M. Pinedo, and G. Jansen

Altered Transport of Folic Acid and Antifolates Through the Carrier Mediated
Reduced Folate Transport System in a Human Leukemia Cell Line
Resistant to (6R)5,10-Dideazatetrahydrofolic Acid (DDATHF) .......... 775
M. Pavlovic, J.J. Leffert, 0. Russello, M.A. Bunni, G.P. Beardsley,
D.G. Priest, and G. Pizzomo

Transformation of an L-Cell Line with the DNA Coding for the

Reduced-Folate/Methotrexate Transporter Protein from a CCRF-CEM
Human Leukemia Cell Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 779
F.E. Williams, M. Ratnam, T.P. McAlinden, G. Jansen, J.H. Schomagel,
and J .H. Freisheim

Determinants of the Disparate Antitumor Effects of (6R)5,10-Dideaza-5,6,7,8-

Tetrahydrofolate and Methotrexate Toward Methotrexate Resistant
CCRF-CEM Cells, Characterized by Severely Impaired Antifolate
Membrane Transport ....................................... 783
L.H. Matherly and S.M. Angeles

Up-Regulated Transport of Methotrexate and 5-Methyltetrahydrofolate

in a Human Breast Cancer Cell Line ............................ 787
F. Mandelbaum-Shavit

Participants ................................................... 791

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809

Subject Index ................................................. 815

xxvi
NATURAL PTERIDINES - A CHEMICAL HOBBY

Wolfgang Pfleiderer

Fakulti=it fUr Chemie

UniversWit Konstanz
D-7750 Konstanz I Germany

INTRODUCTION

The natural pteridines can be regarded as the origin of pteridine chemistry

in general and have initiated and stimulated research in this field to a very
large extent ever since Frederick Gowland Hopkins has focussed for the first
time more than 100 years ago 1 the attention to butterfly pigments in trying to
isolate the yellow pigments from the English brimstone butterfly and the white
pigment from the cabbage butterfly six years later2. The unusual physical and
chemical properties of these substances, however, did not allow to get pure
materials suitable for structure elucidations. From 1924 - 1926 Clemens
Schopf3A took up Hopkins' observations in Heinrich Wieland's laboratory and
named the pigments according to their colors and appearance in nature
xanthopterin (2) and leucopterin (4). In 1933 a third component,
isoxanthopterin (3)5, was isolated, but his constitution remained also unknown
until Robert Purrmann was able to elucidate the structures of all three
pigments 6-8, which turned out to be derivatives of the pyrazine [2,3-
d]pyrimidine (1) ringsystem termed by Wieland9 pteridine.

NflY~~
~L,lsJJ
N N
1

2 3 4

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993
 The period of 15 years of investigations in two laboratories on the
structures of the butterfly pigments indicates already some principal difficulties
encountered with pteridines which are attributed to their incomplete
combustibility in elemental analysis, their high melting and decomposition
points, their poor solubilities in water and most organic solvents and hence
problems in purification.
Nevertheless the natural pteridines reveal so many exceptional physical
and extraordinary chemical properties that we cannot only learn basic
principles of heterocyclic chemistry, in general, but also get detailed knowledge
and under-standing why special structures are a prerequisite for specific
biochemical and enzymatic reactions. The fascination to discover the
unexpected and anomaly is one of the reasons why I have devoted already 40
years of research efforts to a large extent to pteridine chemistry.
I, therefore, see in the adjudication of the Frederick Gowland Hopkins
Award a great honor and I am very pleased that my contributions to the field of
pteridines have been recognized by the scientific community in such a noble
fashion. I thank all the many students, research associates and colleagues who
have encountered over all these years the spectacular and common results
published in joint papers.

BUTTERFLY PIGMENTS

Detailed investigations with tropical butterflies revealed two more deeply

coloured pigments of which erythropterin has been described earlier10 . The
proposed structure11, which seemed to have been proven by various
syntheses12, had to be revised on the basis of studies with model substances
indicating a different constitution to explain the long wavelength absorption at
450 nml3. The orange coloured South American butterfly Catopsilia argante
contains relatively large amounts of erythropterin in the wings, which is a good
source for isolation of the pigment. Since the basic structure of erythropterin as
7-pyruvoyl-xanthopterin (5) does not coincide with the observed spectroscopic
data we learned that simple tautomerism of the side-chain to the
thermodynamically more stable vinylogous amide (6) 14 makes all the
difference.

:;..J::x:a,
0 H

H
2
to
I
5 COOH 6

From the dark red coloured butterfly Appias nero native in Java a violet
pigment was isolated and identified as pterorhodin15. This finding was very
interesting regarding the fact that the chemistry of pterorhodins is related to the
beginning of pteridine chemistry, in general, when Frederick Gowland
Hopkins2 noticed already in 1895 during his attempts to isolate butterfly
pigments the formation of a dark-red to violet coloured component which he
called "lepidoporphyrin". This artifact, which is formed under acid catalysis

2
from the simple naturally occurring butterfly pigments, has also troubled
SchOpf's and Wieland's group during their investigations, but only in 1942
Hopkins16 described more details about his studies with coloured material
which he renamed now "rhodopterin". Purrmann et al.17 were able to elucidate
the structure of this artificial component by oxidative degradation and called it
"pterorhodin" in 1944. The structure of this highly insoluble substance was also
proven by chemical synthesis from xanthopterin and 7-methylxanthopterin by
oxidative coupling in acidic medium18. Two structures, the linear (7) and the
bent, hydrogen-bound form (8) are still under discussion accounting for the
deep violet colour of this substance.

We are dealing with a dipterinylmethane dyestuff of which also two more

isomers exist connecting either xanthopterin or isoxanthopterin with 6-methyl-
isoxanthopterin via a 6,7- or 6,6-methine bridge, respectively, to form iso-ptero-
rhodin and allo-pterorhodin.

/SO-PTERORHODIN ALLO-PTERORHODIN

It is interesting to note that pterorhodin has recently been found also as a

natural pigment in various insects, worms and amphibia. The red eye-pigment
of Ephestia and Ptychopoda l9 as well as of Platynereis dumerilli20 and and the
leaf-frog melanophore pigment21 turned out to be identical with pterorhodin,
which seems to be widely distributed in the animal kingdom.
We can deduce from the reported physical data that the proposed
structures of pterorhodin and its isomers are principally correct, but no
statements have been made regarding the fine-structural features of these
molecules. It is, for example, of interest to know which tautomer (7, 8, 9 or 10)
will be the most stable one and which equilibria will exist in various solvents
and under different pH conditions, respectively.

3
 There is also the theoretical possibility that the formation of pterorhodin
by oxidative coupling progresses further giving rise to condensation products
of type 11 or 12 .

11

Unfortunately, the low solubility of pterodrhodin and its analogs in

common solvents do not allow any studies of the neutral species, but spectra in
formic acid trifluoroacetic acid and sulfuric acid are available without knowing
which mole- cule form is present under these conditions.
In order to get more soluble derivatives of pterorhodin we synthesized
several N-methyl derivatives such as 3,3'-dimethyl-(13), 3,3',5-trimethyl-(14)
and 3,3',5,5'-tetramethyl-pterorhodin (15) from the appropriate starting
materials.

13 Me H H Me
14 Me Me H Me
15 Me Me Me Me

5.0

4.5

4.0 \

lgE

3.5
0 1'1 " 0
"''·•A..,-"l.o oy"v'-•·""•
H,NJ...N..lN C,I...IIIIJ...H...t..NH 1 - - """"''
H H
3.0

• --.-. ICt'II.H,SQ.

2.5

······••••··• ICt'll. H.S0+

2.0+......--.-...........~~..-...-.......~~T"""'"~...,......~.......-,........,..........~.........,....,.....~..........,.........~
200 250 300 350 400 450 500 550 600 650 700

~ [nm]

Figure 1 UV I VIS spectra of pterorhodm (8) and 1ts methyl denvanves 13,14,15 m 80% sulfuriC aCid

4
 The UV I VIS spectra of pterorhodin (8) and its N-methyl derivatives 13,
14, and 15 in cone. formic acid and 80 % sulfuric acid, respectively, are almost
super-imposed with each other indicating that the same molecular species,
presumably the dication of the hydrogen chelated form 9 is present. The shape
of the curves is of a cyanine-type structure accounting for the strong absorption
in the visible region.
Analogous studies with iso- and allo-pterorhodin, respectively, and their
N-methyl derivatives look even more complex and do not yet allow to draw
obligatory conclusions regarding the fine-structural assignments.
From solubility reasons we then continued other investigations in the
lumazine series where several N-methyl derivatives have been available to
form under acid catalyzed oxidative coupling reactions the corresponding
lumarhodin, isolumarhodin, and allo-lumarhodin analogs. We synthesized
first the 1,1',3,3'-tetramethyl derivatives 20, 21, and 22 from 1,3-dimethyl-(16)
and 1,3,7-trimethyl-6-oxo-5,6-dihydrolumazine (17) as well as from 1,3-
dimethyl(18) and 1,3,6-trimethyl-7-hydroxy-lumazine (19), respectively.

----
16 17

16 19

It was found that 20 and 21 form again dark-red to violet microcrystalline

powders showing that 20 and 21 are dilumazinylmethine dyestuffs, whereas 22
turned out to be colourless after reprecipitation from formic acid and water. Its
UV spectrum reveals an absorption band only at 335 nm in formic acid
indicating the presence of a 6,6'-methylene tautomer (Fig. 2). We furthermore
have to assume that the lactim structure22 of the starting materials 18 and 19
has tautomerized to the normal lactam configuration due to the fact that the
condensation products of 1,3,8-trimethyl-7-oxo-7,8-dihydrolumazine (25) with
19 and of 1,3,6,8-tetramethyl-7-oxo-7,8-dihydrolumazine with 25, respectively,
afforded the corresponding 8-monomethyl-(23) and 8,8' -dimethyl (24) dimer
analog of very similar UV absorption.

5
 +

y 19

R R1
22 H H
23 CH3 H
24 CH 3 CH 3

5.0 , - - - - - - - - - - -- - - - -- - - - - - - - - - . . ,

log~

3.0

2.5

2.0+--~--.....--~~~~~~---.--~~~-r-~~ ...........---.--.....--.........- i
200 250 300 350 400 450 500
'). (nm]

Figure 2. UV spectra of 22, 23, and 24 in HCOOH.

In order to get an even more soluble pterorhodin analog we reacted 1,3,5-

trimethyl- (27) and 1,3,5,7-tetramethyl-6-oxo-5,6-dihydrolumazine (28) in 4 N
HCI under oxygen atmosphere and obtained 29 as a dark violet crystalline
powder. If the same reaction was performed analogously in 1 N HCl a mixture
of 29 and its tautomer 30 was formed. Both components could be separated into

6
the pure forms which showed the expected physical properties consistent with
the pro-posed structures (Fig. 3).

27 •• I ..
~
+

29 30

5.0 . , . . - - -- - - - - -- - -- -- - -- - - - - - - -,

4.5

I I

!\.'\ I '\

\
1~ '' ~
-.-'
I I
I
I

~ 1"--

\
- I
I
3.5
I
I
I

' '

\
3.0

2.5+--~........~~"'T"""'~"""'""T"""'""T""T""'""T"""""T""'.........,....;..................~........,,...........,....,,_,.,,.......;;....,....,,.......~-;
200 250 300 350 400 450 500 550 600 650 700
nm

Figure 3. UV I VIS spectra of 29 and 30 in HCOOH,

Since from the so far available physical data an exact structural assignment
of 29 could not be made regarding the four most probable conformers 29 and 31
and the cis-trans isomers 32 and 33 respectively, more studies had to be 1

performed.

7
 33

Emission spectra of 29 in ethanol at 80°K clearly point to a highly

symmetrical structure as does also the 1H-NMR spectrum in formic acid by
showing the methine proton at low field and three N-methyl signals in the
normal range. To prove finally structure 32 unambiguously we crystallized the
compound from formic acid and got nice crystals for X-ray analysis. These
studies are in absolute agreement with structure 32 indicating further that the

Figure 4. X-ray analysis of 1,3,5-trimethyl-6-oxo-5,6-dihydrolumazinyl-7-methylidene-1,3,5-trimethyl-

6-oxo-5,6,7,8-tetrahydro-lumazine (32).

high symmetry is reflected by finding actual half of the electron density tor the
chelate hydrogen at both N-8 and N-8' positions (Fig. 4).
This structure reveals very nicely that the two 1,1'-methyl groups do not
interact sterically, which should give rise to some other isomeric form missing
the intramolecular stabilization by hydrogen bonding. In order to find out,

8
which of the proposed 5 structures 29 - 33 will be the second stable isomer, we
synthesized from 3,5-dimethyl-1-sec.butyl-6-oxo-5,6-dihydrolumazine (34) and
the corresponding 3,5,7-trimethyl derivative 35 in 4 N HCl in the usual manner
the pterorhodin analog 36, which turned out to consist, as expected, of slightly
yellow but not red to violet crystals. It is obvious that the bulky sec.butyl groups
in 1- and 1'-position do not allow a stable chelated structure (36) but
tautomerizes to bis-3,5-dimethyl-1-sec.butyl-6-oxo-5,6-dihydrolumazin-7-yl-
methane (37).

CHa 0

OtNJLN'CH 3
+
N N~O
I
CH2
I
H3C-CH-CH 3

34 35

I 02

36 37

During the synthesis of 37 the reaction solution turned red, from which
yel-lowish crystals separate on cooling. In solution an equilibrium between 36
and 37, preferring very strongly the latter tautomeric form, must exist according
to the red colour, but it is not possible to isolate the hydrogen chelate. The UV I
VIS spectrum in MeOH also proved the presence of a small amount of 36 or its
anion spectrophotometrically, whereas in HCOOH a colourless solution was
observed.
In conclusion it was proven by these extended studies with various model
substances that the structure of the natural butterfly pigment pterorhodin (8) is
most likely also represented by a hydrogen-bonded hydrogen chelate.

DROSOPHILA PIGMENTS

Another interesting group of natural pteridine pigments is present in the

eyes of Drosophila melanogaster. Lederer23 supposed already in 1940 that we
are dealing here with pterin derivatives and called them "drosopterins".
Viscontini et ai.24 developed in 1957 chromatographical methods to separate the
complex mixture into yellow and orange to red fluorescing components.
Chromatography on cellulose gave two orange-coloured compounds which
have been called droso--pterin and isodrosopterin whereas the red pigment

9
was named neodrosopterin. After the determination of the chiroptical
properties of droso- and isodrosopterin 25 it was obvious that these pigments are
not composed of isomers but of enantiomers showing mirror image ORD and
CD spectra 26. two-dimensional paper chromatography of the drosopterin
fraction finally led to the discovery of two more orange coloured pigments
designated as aurodrosopterin 27 and present again in form of an enantiomeric
mixture28.
Several attempts were made to elucidate the structure of droso- and
isodrosopterin29,30 but only in 1978 Theobald and Pfleiderer31 were able to solve
this difficult problem by proposing the following constitution from NMR and
mass-spectral data.

38 Drosopterin 39

A simple synthesis for this pentacyclic ringsystem was found from 7,8-
dihydropterin (40) and a-hydroxy-13-ketobutyric acid under special pH
conditions 31,32 to become independent from the tedious isolation from
Drosophila heads. The proposed mechanism 32 of this intersting reaction has to
be revised since 6-acetyl-homopterin (41) has been found as a natural
intermediate 34,34 in the biosynthesis of drosopterins.

COOH
I
CHOH -C~

bo
I
CH 3
40

H3C H3C'c
'CHOH HOH

- --
I
0 H~:O o ,.....C-OH

- HN~N~H ~Ni H
H N~.J,
-- H
2
N~N I NJc:::
H
~ H
2
N~N N
H ~

10
 41

:z;~NH,
0H,:TtN NHN

HN~ H

H
2
N~N).__NH H

Drosopterin

This mechanism is supported by the fact that 6-acetyl-homopterin (41) and

7,8-dihydropterin (40) react very cleanly at pH 3 to form in a nonenzymatic
synthesis the two enantiomers drosopterin and isodrosopterin 35 . We concluded
recently from these findings that it may be possible to elucidate the structure of
aurodrosopterin by condensation of 41 with other 7,8-dihydropteridines. High
resolution 1H-NMR spectra of natural drosopterin and aurodrosopterin,
respectively, which look very much alike regarding the aliphatic hydrogens
indicated that the pentacyclic ringsystem is the same whereas the substituents at
the periphery may alter to some extent. Comparisons of the UV-spectra finally
told us that the small hypsochromic shift of the visible absorption band of
aurodrospterin was similar to the change of pterins into lumazines.
Under these aspects it was not too surprising that equimolar amounts of 41
and 7,8-dihydrolumazine afforded at pH 3 after 24 h in quantitative yield
aurodrosopterin (42) 36 (Fig. 5). The natural and the synthetic materials were
identical in every respect by checking the UV and NMR spectra and the
chromatographical behaviour.

42 43

11
 1 5
16

~
·"f :/
~NH2

HN:.XN H H H H

O~N N H 1.2
" "
E

'h
o.a 08

300 SOD 6CG

Figure 5. Condensation reaction of 41 and 7,8-dihydrolumazine at pH 3.

Furthermore we condensed 6-acetyl-homopterin (41) with 2,4-diamino-7.8-

dihydropteridine in acidic medium and observed in a much slower reaction the
formation of a red, drosopterin-like product which we call aminodrosopterin
(43) but it is not identical with the naturally occurring neodrosopterin.
Finally there is still one unsolved problem in this series regarding the
stereochemistry of the chiral centers C-6a and C-6b. Since all attempt the
crystallize either of these compounds failed it was not yet possible to achieve an
X-ray analysis for absolute proof of the structure. We tried to elucidate the
stereochemistry by NMR spectral means to figure out whether the two
hydrogens at C-6 and C-6a position are in a cis- or trans-configuration. It was
possible to simulate the NMR spectrum of drosopterin very nicely but the
obtained coupling constants of the two hydrogens in question are too similar to
differentiate between a cis- or trans-arrangement. Therefore, the cis-(39) and
trans-drosopterin (38) have been designed by the Alchemy II program and after
minimizing the structures to 44 and 45 the dihedral angles of the various
adjacent hydrogen atoms were determined and the corresponding coupling
constants calculate according to the Karplus equation.

44 45

12
 These results (Table 1) again revealed no significant differences which
could be used for a structural assignment. The problems encountered with
these studies are due to the distorted rings in the drosopterins which do not
allow to correlate the dihedral angles with the experimentally determined
coupling constants. From the structures 44 and 45 as well as from Dreiding
models it can be seen that the cis-drospterin exhibits a much more planar shape
of the rings then trans-drosopterin with its bent and distorted conformation.
Presumably the cis-form is thermodynamically more stable.

Table 1. Experimental and calculated NMR data of cis- and trans-drosopterin.

DROSOPTERIN
cis trans 1H-NMR
------------------------- - -------- ---------------------------- - ---------
Proton Dihedral Coupling Dihedral Coupling Coupling
Position Angle Constant Angle Constant Constant
H-C 44 calc. 45 calc. experim.
------------------------------------------------------
6a6b 108.5 11-14 108.5 11-14 12.5
6a 6a 169 9.0 10 9.0 12.0
6b 6a 50 3.6 130 3.6 4.7
6a 6b 8 9.1 172 9.1 5.0
6b 7a 155 7.5 22 8.9 8.2
6b7b 85 0 92 0 2.4
7a 7b 109.5 11-14 108.5 11-14 13.0

The 250 MHz NMR spectrum of natural drosopterin was taken in D20 I
DCl and revealed informative resolution and spitting of the signals of the
aliphatic region (Figure 6). Neodrosopterin is the most difficult Drosophila eye
pigment to work with since its purification is associated with partial
decomposition. Very little material has so far been available which did not
allow to get a high resolution NMR spectrum. Further studies have to be made
to determine its molecular composition and the chemical structure.
·--- - - - - - -- -- - -- - - - - - - - - - ,

..
....
~

....
~ ...
....,,.
.._~

4..$ u •3 u •·• •.o 3.t 3.t 3.7 ~e 3.5 :u 3..3 u 3., l .O 2.e 2.a u 2s
PPm

Figure 6. 250 MHz NMR spectrum of drosopterin measured in 0 20 I DC! .

13
Acknowledgments

I would like to thank Prof.J.Yim, Seoul I Korea for the isolation of the
natural eye pigments from Drosophila melanogaster, Dr.G.Kollmannsberger-
v.Nell forthe simulation and calculations of the NMR spectra and Dr.B.Fischer,
University of Zurich I Switzerland for the X-ray analysis of the pterorhodin
analog. The experimental help of Mr.L. Kyriases during the synthetic efforts and
of Mr.E. Krienitz in measuring various NMR spectra is also appreciated.

REFERENCES

1. F.G.Hopkins, Note on a yellow pigment in butterflies, Proc.Chem.Soc. 5: 117,

(1889). F.G.Hopkins, Dber den gelben Farbstoff bei Schmetterlingen,
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in Butterflies (Note), Nature, (London) 40: 335 (1889).
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3. H. Wieland and Cl. Schopf, Dber den gelben Fliigelfarbstoff des
Citronenfalters (Gonepteryx rhamni), Ber.Deut.Chem.Ges. 58: 2178 (1925).
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5. H. Wieland, H. Metzger, Cl. Schopf and M. BUlow, Leukopterin, das
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(1951).
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mentes "Erythropterin", Chem.Ber. 95: 2195 (1962).
14. W. von Philipsborn, H. Stierlin, and W. Traber, Zur Struktur von 9-
Acetonylxanthopterin und Erythropterin, Helv.Chim. Acta 46: 2592 (1963).
15. W. Pfleiderer, Pterorhodin, ein neues natiirliches Schmetterlingspigment,
Z. Naturforsch. 14b: 654 (1963).
16. F.G. Hopkins, A contribution to the chemistry of pterins, Proc.Roy.Soc.
London (B), 130: 359 (1942).

14
17. R. Purrmann and M. Maas, Zur Kenntnis des Pterorhodins, Liebigs
Ann.Chem. 556: 186 (1944).
18. P.B. Russell, R. Purrmann, W. Schmitt and G.H. Hitchings, The Synthesis of
Pterorhodin (Rhodopterin), J.Am.Chem.Soc. 71: 3412 (1949).
19. A. Kuhn and A. Egelhaf, Der rote Augenfarbstoff von Ephestia und Ptycho-
poda- ein Pterinpigment, Z. Naturforsch. 14b: 654 (1959).
20. M. Viscontini, W. Hummel and A.Fischer, Isolierung von Pterindimeren
aus den Augen von Platynereis dummerilli 1833, Helv.Chim. Acta 53: 1207
(1970).
21. G. Misuraca, G. Prota, J.T. Bagnara and S.K. Frost, Identification of the leaf-
frog melanophore pigment, Rhodomelanochrome, as pterorhodin, Comp.
Biochem.Physiol. 57B: 41 (1977).
22. W. Pfleiderer, Ober 7-Hydroxy- und 7-Hydroxy-6-methyl-2,4-dioxo-
tetrahydropteridine, Chem.Ber. 90: 2588 (1957).
23. M. Lederer, Les Pigments des Invertebres (a !'Exception des Pigments Respi-
ratoires), Biol.Rev. Cambridge Phil.Soc. 15: 273 (1940).
24. M. Viscontini, E. Hadron and P. Karrer, Fluoreszierende Stoffe aus
Drosophila melanogaster: die roten Augenfarbstoffe, Helv.Chim. Acta 40:
579 (1957).
25. M. Viscontini and P. Karrer, Fluoreszierende Stoffe aus Drosophila melano-
gaster, Helv.Chim. Acta 40: 986 (1957).
26. H. Schlabach and W. Pfleiderer, Die physikalischen Eigenschaften der roten
Augenpigmente aus Drosophila melanogaster, einer neuen Klasse atropiso-
merer Naturstoffe, Helv.Chim. Acta 55: 2525 (1972).
27. I. Schwinck and M. Mancini, The drosopterin pattern in various eye color
mutants of the fruit fly Drosophila melanogaster, Arch.Genetik 46: 41 (1973).
28. K. Rokos and W. Pfleiderer, Isolierung, physikalische Eigenschaften und al-
kalischer Abbau der Augenpigmente Neodrosopterin und
Aurodrosopteriaus Drosophila melanogaster, Chem.Ber. 108: 2728 (1975).
29. M. Viscontini, Beitrag zur KonstitutionsaufkHirung der Drosopterine,
Helv.Chim. Acta 41: 1299 (1958).
30. H. Schlabach and W. Pfleiderer, Isolierung,,Molekulargewichtsbestimmung
und alkalischer Abbau von Drosopterin und Isodrosopterin-
Augenpigmente der Fruchtfliege Drosophila melanogaster, Helv.Chim.Acta
55: 2518 (1972).
31. N. Theobald and W. Pfleiderer, Ein neuer Strukturvorschlag fur die roten
Augenpigmente Drosopterin und Isodrosopterin aus Drosophila melano-
gaster, Chem.Ber. 111: 3385 (1978).
32. H.Schlobach and W. Pfleiderer, Synthese und Reaktionen von Droso- und
Isodrosopterin, Helv.Chim. Acta 55: 2533 (1972).
33. G.J. Wiederrecht, D.R. Paton and G.M. Brown, The Isolation and Identifica-
tion of an Intermediate involved in the Biosynthesis of Drosopterin in Dro-
sophila melanogaster, J.Biol.Chem. 256: 10399 (1981).
34. K.B. Jacobson, D. Dorsett, W. Pfleiderer, J.A. McCloskey, S.S. Sethi, M.V.
Buchanan and I.R. Rubin; A Naturally Occurring Pyrimidodiazepine in
Drosophila: Chemical and Spectral Properties and Relationship to
Drosopterin, Biochemistry 21: 5700 (1982).

15
35. D.R. Paton and G.M. Brown, The nonenzymatic synthesis of' drosopterin'
from dihydropterin and 2-amino-4-oxo-6-acetyl-3H,9H-7,8-
dihydropyrimido-4,5-b][1,4]diazepine, in: "Chemistry and Biology of
Pteridines 1986", B.A. Cooper and V.M. Whitehead, ed., W. de Gruyter,
Berlin, New York (1986), p. 295.
36. W. Pfleiderer, S.J. Kim and J. Yim, The nonenzymatic Synthesis of Droso-
pterins, in: "Pteridines and Related Biogenic Amines and Folates", ed.,
N.Blau, H.Ch. Curtius, R. Levine and J. Yim, Hanrim Publ. Co. Seoul,
Korea, 1992), p. 54.

16
PROPERTIES AND REACTIONS OF PTERIDINES CARRYING
FUNCTIONALISED SIDE CHAINS AT THE 3-POSITION.

Peter Boyle and Russell Camier

University Chemical Laboratory, Trinity College, Dublin 2, Ireland

It is already known that some pteridines can act as photosensitisers, 1 and it has been
observed recently 2 that ultraviolet irradiation of DNA in presence of a pteridine can cause
widespread but random cleavage of the DNA chain. The chemistry described here arose out
of a requirement to attach a pteridine to a synthetic oligonucleotide for purposes of inducing
a photochemical lesion on a DNA chain at a specific targeted position. This paper reports
on some new compounds, reactions, and ring systems, which were investigated during the
preparation of a pteridine with a functionalised side chain at position 3. This pteridine was
successfully attached to the 5' -OH of a synthetic oligonucleotide.
The starting point for the study was 5-amino-7-(3-chloropropoxy)furazano[3,4-d]-
pyrimidine (1), obtained by treating 5-amino-7-methylthiofurazano[3,4-d]pyrimidine with
3-chloropropanol in presence of bromine.3 On hydrogenolysis4 in tetrahydrofuran solution
followed by treatment with benzil, (1) was readily converted via 2,4,5-triamino-6-(3-chloro-
propoxy)pyrimidine (2) into 2-amino-4-(3-chloropropoxy)-6,7-diphenylpteridine (3).

Tetrahydro·
luran

A
0

HO~ N~NXPh
I ""' ......1 -HO"
--
~ , Benzil
H2N N N Ph

(6) (4) (5)

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 17
In contrast to this, however, if water was added to the reaction medium, pyrimidine (2) was
found to be unstable, and cyclised spontaneously to give the highly resonance stabilised
multident cation (4). Because of electron delocalisation, the two amino groups in this are of
widely different nucleophilicities, and (4) can condense with a dicarbonyl compound with a
high degree of regiospecificity cf the classical problem of the Isay pteridine synthesis.
Thus, reaction of (4) with pyruvaldehyde led to a single isomer, presumed to be
2-amino-4-(3-hydroxypropoxy)-7-methylpteridine (5), of at least 95% regiochemical purity,
as shown by n.m.r. When cation (4) was treated with hydroxide ion and benzil, the only
product obtained was 3-(3-hydroxypropyl)-6,7-diphenylpterin (6). This was probably
formed by attack at position A (as shown in the diagram) rather that at position B, since when
(4) was treated with methoxide ion and benzil, only products (6) and (7) were obtained. No
N3-(3-methoxypropyl) product (10) was isolated.
When pteridine (6) was refluxed with triphenylphosphine in a mixture of dry carbon
tetrachloride and tetrahydrofuran its hydroxypropyl side chain was chlorinated, giving (8) in
66% yield. With triphenylphosphine in pure dry carbon tetrachloride solution, however,
the yield of (8) dropped to 39%, and significant amounts (46%) of a new product (9) were
found to be formed as well. Proton n.m.r. spectroscopy showed signals for five phenyl
groups in the new compound, suggesting the presence of a triphenylphosphorus moiety,
and this was confirmed by 31 P n.m.r. spectroscopy, which revealed one peak at o 20.4
(relative to phosphoric acid). This and other spectroscopic evidence, together with mass
spectroscopic data and elemental analysis, allowed the identification of (9) as 3-(3-chloro-
propyl)-6,7-diphenyl-2-triphenylphosphazino-4(3H)-pteridinone. To our knowledge this is
the first phosphazene to have been reported in the pteridine series. It is noteworthy for its
ready solubility in organic solvents such as dichloromethane and ethyl acetate. It is stable
in refluxing aqueous ethanol, but suffers cleavage of the phosphazene group in refluxing
ethanolic hydrochloric acid, regenerating (8). It decomposes in aqueous ethanolic sodium
hydroxide.

MeO

Benzt

(7)

(10)

A marked red shift in the u.v. spectra of both (6) and (8) was observed on going from
neutral to basic conditions in ethanol solution. With the chloropropyl compound (8),
however, the spectrum changed further with time, and revealed the formation of a new
compound (14), containing a tricyclic ring system, only a few examples of which have been
reported previously in the literature. 5 The structure of (14) follows from its spectroscopic
properties, and from several chemical interconversions as shown in the diagram. For
example, when (6) was refluxed in methanolic potassium hydroxide it underwent a Dimroth
rearrangement to give the isomeric compound (17), with the 3-hydroxypropylamino side
chain attached at position 2 of the pteridine. Treatment of (17) with triphenylphosphine in
carbon tetrachloride gave tricyclic compound (14), identical with the product obtained from

18
(8), and presumably formed via chloropropyl compound (15). Acetylation of (8) afforded
its N-acetyl derivative (11). Under different reaction conditions, the unusual
2-diacetylamino derivative (13) was obtained. Both (11) and (13) cyclised under basic
conditions to the same tricyclic compound (12), and this was identical with the product
obtained by acetylation of (14).
In alkaline solution the tricyclic compound (14) underwent further rearrangement, via
lumazine (16), to give another tricyclic compound (18). This was unstable in solution, so
that an analytically pure sample of it could not be obtained. Its structure could be assigned,
however, from its high resolution mass spectrum which included the required molecular
ion, from its n.m.r. spectra, and from its u.v. spectrum which corresponded with that
reported 6 for compound (20), having a similar chromophore. To our knowledge the ring
system in (18) has not been reported before. Pteridine (16) was also unstable, since it
showed a marked propensity to cyclise to (18). It was characterised as its N-acetate (19).

CNt.J!.;XPh
~N ~~N
;..t.J!..XPh
Ph

+--
Ph

Ao t (12) Ao (13)

(14) t (15)

/~H Jro )H JrON Ph

1 I :X -+
~ N Ph LHN ~
HN''.(N I N~Ph
H2N N N Ph
(6) (17)

(18)

Finally, pteridine (6) was successfully attached to a synthetic oligonucleotide. Its

2-amino group was first protected by treatment of it with N,N-dimethylformamide
dimethylacetal, to give the formamidine derivative (21). The two N-methyl groups in this
compound have slightly different chemical shifts in the proton as well as in the carbon
n.m.r. spectra. Reaction of the side chain hydroxyl group in (21) with chloro-
(2-cyanoethoxy)(di-iso-propylamino)phosphine afforded the activated derivative (22),
which was then attached to a synthetic oligonucleotide using an Applied Biosystems 380 B
DNA synthesiser? The initial condensation product (23) was oxidised to phosphate with
iodine, cleaved from the solid support by base, and deprotected by aqueous ammonia
treatment, to give the final product (24). Interestingly, when the 2-amino group of (6) was
protected after the side chain hydroxyl group had been allowed to react with a

19
chlorophosphine, rather than before, the phosphorus atom was found to have undergone
oxidation to the (V) oxidation state. For example, when (6) was treated with
chloro(di-iso-propylamino)(methoxy)phosphine the phosphine oxide (25) was obtained,
which could then be converted into its formamidine derivative (26). Both (25) and (26)
were stable, and were obtained in analytically pure form. Their elemental analysis and
spectroscopic properties are in accordance with the assigned structures. Of particular
importance are the 31 P n.m.r. spectra, showing signals at() 14.5 and() 22.4 for (25) and (26)
respectively (relative to phosphoric acid).
0

.J)~-):x:
~NMe2 (21)

(24)

A )-£ :J[O N
X Ph A )-~ :J[O N
X Ph

o=P1'0
N
HN~N
N
I, I """
N Ph
o=P1'0
N
N~N
N
I, I """
N Ph
0 2
0 ~
CHa (25) CH3 NMe 2 (26)

ACKNOWLEDGEMENTS

We are indebted to Dr. R. J. H. Davies and Mr C. Stevenson of Queen's University,

Belfast, who carried out the DNA synthesiser experiment, and to Professor J.M. Kelly and
Mr. M.M. Feeney of Trinity College Dublin, for invaluable discussions on the DNA aspects
of this work.

REFERENCES

1. C. Chahidi, M. Giraud, M. Aubailly, A. Valla and R. Santus, Photochem. Photobiol.,

1986, 44, 231.
2. J.M. Kelly and S. Meschwitz, personal communication.
3. P.H. Boyle and R.J. Lockhart, J. Org. Chem., 1985,50,5127.
4. E.C. Taylor and A. Cocuzza, J. Org. Chem., 1979, 44, 302.
E.C. Taylor, S. Martin, Y. Maki and G.P. Beardsley J. Org. Chem., 1973,38, 2238.
5. R.B. Angier and W.V. Curran, J. Am. Chem. Soc., 1959,81,5650.
6. H. Lutz and W. Pfleiderer, Croat. Chem. Acta, 1986,59, 199.
7. The DNA synthesiser experiment was carried out by Dr. R.J.H. Davies and Mr. C.
Stevenson, in the biochemical laboratories of Queen's University, Belfast.

20
N2-(N ,N-DIMETHYLAMINOMETHYLENE)-04·(2-p-NITROPHENYL-
ETHYL)-BIOPTERIN: A VERSATILE INTERMEDIATE FOR A
GLYCOSIDATION REACTION

Hiroshi Yamamoto, I Tadashi Hanaya,l Kiyoshi Torigoe,l and

Wolfgang Pfleiderer2

!Department of Chemistry, Okayama University, Okayama 700, Japan

2Universitat Konstanz, Fakultat ftir Chemie, 7750 Konstanz, Germany

INTRODUCTION
A certain number of pteridines having a glycosidated side-chain occur in nature; e.g.,
biopterin glucoside 1 isolated from a blue-green alga, Anacystis nidulansl and riboside 2
from a blue alga, Spirulina platensis.2 For the structural proof, some efforts to prepare these
compounds were made by direct glycosidation. However, the overall yields remained unsat-
isfactory; e.g., 1 and 2 were produced in less than a 1% yield by the condensation of bio-
pterin 3 respectively with D-glucose and D-ribose in DMF in the presence of TsOH at 60 °C
for 2 days.3,4 A slightly improved preparation of the D-Ribofuranoside 2 was achieved
through the sequences shown below (3 --? 4 --? 6 --? 2).4
0 OH
HN~N~
H2N~N'!l.N~ "
HO~
f
HOOH
2 (mp 200 "C decomp.)

1 NaOMe I MeOH
r.t, 24 h, 81%

0 OH

"1
Biopterin
(Me3 SihNH HN~N~
3
125 "C, 3d
H2N,k.NJlN~
4 BzO~
BzO OBz
6 (11% yield from 3)

Because various types of glycosides of biopterin and related pteridines were considered
to be of interest in view of potent biological activities, we have -attempted to explore more
efficient and versatile glycosidation methods of biopterin and found that an N2,04-protected
biopterin can conveniently be subjected to various reactions including glycosidation.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 21
RESULTS AND DISCUSSION

The observation4 that the tetrakis-trimethylsilyl compound 4 appeared to be too labile

under the glycosidation conditions employed, to regenerate a considerable amount of the
insoluble starting material3 led us to devise the preparation of slightly more acid-withstand-
ing 2,4-protected biopterin which, preferably, is readily soluble in non-polar aprotic sol-
vents. As a simple approach, we sought to prepare N2-acetyl-04-NPE-biopterin [NPE = 2-
(p-nitrophenyl)ethyl],S by starting with the triacetyl derivative 7.6 Thus, 7 was converted
into 04-NPE derivative 8. However, upon treatment with methanol even at 20 °C, the N2-
acetyl group of 8 was preferentially cleaved to give 9.7 Conversion of 9 into other N2-
protected derivatives 10 (e.g., R = Boc, benzyl, etc.) has somehow remained unsuccessful.

~OH
NPEO OAc NPEO OAc
o2Nv
PPh,. EtO,CN-NCO,Et NJ......N~ MeOH, r.t,15h NJ......N~
/1 ,4-Dioxane, r.t., 30 h AcHNkN!lNJ OAc 82% H2NkN!lNJ OAc
72%
S(mp 140 °C) 9 (mp 163-4 °C)
I
I
NPE = -CH~HAH4NQip) I

NPEO f OAc
NJ......N~
RHNkN!lNJ OAc

10 R = Boc, Bn, eta.

The reaction of 3 with N ,N-dimethylformamide diethylacetal in DMF at 20 °C for 11 hr

gave N2-(N,N-dimethylaminomethylene)-biopterin 11, which was then converted into the
1',2'-di-0-acetyl derivative 12. This was led to the 04-NPE compound 13, which, in turn,
upon methanolysis at 50 °C, successfully furnished versatile N2,04-protected biopterin 14.8
This compound is soluble in various nonpolar aprotic solvents and can be subjected to a
variety of the dial reactions of the side-chain.

Me2NCH(OEtk I DMF
r.t., 11 h
80-85%

~NCs~C~CHp-1
Ac20 I Py, r.t., 15 h PPh 3, E102CN=NC02Et
11 ,4-Dioxane, r.t., 24 h
90%
12 ( mp 194-5 °C) 85%

·:~·~
'~~NAN N_. OAc
MeOH, 50 °C, 15 h

94%
14 (mp 122 °C)
13 (mp 205 °C)

Glycosidation9 of 14 (via di-0-trimethylsilylated 15) was studied by employing D-ribo-

furanose 5 as a model substrate, thus affording a mixture of biopterin 2 1 -0-~-D-ribofuran-

22
oside 16 and 1',2'-di-0-~-D-ribofuranoside 17,10 the yields of which depended upon the
molar ratio of 5 to 14.

I CH2CI2 , 40 "C, 20 h

14

Bzol0~Ac
\-( + SoCI4
BzOOBz
5
15 +
I CH,c12, r.t., 18-24 h

17 (mp 98-9 °C}

Yield from 14

5 ( equiv.) 16(%) 17(%)

R= BzO~
12 25 11
1.7 52 15 BzO OBz
2.0 27 31
3.0 8 38

Various efforts were made to selectively obtain a mono-glycoside of type 16, but so far
without appreciable success. For example, an application of Hanessian's methodll to the
1',2'-0-(N,N-dimethylaminomethylene) derivative 1812 (prepared from 14) resulted in only
a low yield of the expected mono-furanoside 20, besides a much larger proportion of the
non-glycosidated product 19.

M9:2NCH(OEtl2

BzOl-o.?Ac
\-( + SoC4 NPNE:):O Nn:;OCHO
BzOOBz I • +
'N~ AN No& OH
I N
19 (42%) BzO O

BzOl--0~
+ 14 (4%) + )--( Ac BzO OBz

BzO OBz 20 (5%)

(a:P=1:3)

Selective deprotection of 16 and 17 was sought under various conditions. The 04-
NPE group of 16 was cleaved by the action of DBU in DMF5 to give 21 (R = H), whereas
the treatment with DBU in pyridine produced N2-deprotected 22 (R =H) and N2,04-depro-
tected 23. On the other hand, debenzoylation was effected by treatment of 16 with sodium
methoxide at 25 oc to give initially 24 (R" = H), then slowly several products including 2'-

23
P-D-riboside 2. Similar results were obtained for deprotection of the his-riboside 17. Opti-
mization of these deprotection steps, as well as glycosidation of 14 with other sugars, are in
progress.

0.5M DBU I DMF

r.t, 1 d

16 R·H
17 R·R'

R·~
H~:t~~ 22

"6:·rY
BzO OBz
1. MeONa I MeOH
1,4-Dioxane, r.t, 1 d

:~::· ~
2. Amberlite IRC-50

~0-ribofuranosyl HO OH

We are grateful to Kanegafuchi Chemical Co. Ltd. (Osaka) for the supply of biopterin to us. The
present work was partially supported by a Grant-in-Aid for Scientific Research No. 03045035 from the
Japanese Ministry of Education, Science, and Culture.

REFERENCES
1. H.S. Forrest, C. van Baalen, and J. Myers, Science, 125, 699 (1957); C. van Baalen, H.S. Forrest, and
J. Myers, Proc. Nat/. Acad. Sci. USA., 43, 701 (1957); F.I. MacLean, H.S. Forrest, and J. Myers,
Arch. Biochem. Biophys., 114, 95 (1966).
2. H.S. Forrest, C. van Baalen, and J. Myers, Arch. Biochem. Biophys., 78, 95 (1958).
3. W. Pfleiderer and M. Beeck, unpublished results; M. Beeck, Dissertation, Univ. Konstanz (1981).
4. W. Pfleiderer and M. Kappel, unpublished results; M. Kappel, Dissertation, Univ. Konstanz (1981).
5 F. Himmelsbach, B.S. Schulz, T. Trichtinger, R. Charubara, and W. Pfleiderer, Tetrahedron, 40, 59
(1984) and references cited therein.
6 H. J. Furrer, J.H. Bieri, and M. Viscontini, Helv. Chim. Acta, 62, 2577 (1979); 7 was reported as an
oil which solidifies on standing. We obtained 7 as pale yellow crystals. A partial hydrolysis of 1' ,2'-0-
acetyl groups of 7, desirably to provide N2-acetyl-biopterin, was attempted under various conditions:
e.g., 0.5 N LiOH (1-3 equiv.) in 80% aqueous t-BuOH, or 2-2.5 equiv. K2C03 in aqueous t-BuOH
at 20 oc for 1-2 h. In all cases, the hydrolysis resulted in the formation of biopterin 3.
7 It should be noted that 7 was found to give 1',2'-di-0-acetyl-biopterin, only upon refluxing with MeOH
(after 36 hr, 76% yield).
8 Prolonged heating of 13 in methanol slowly caused cleavage of the N2-aminomethylene group as well.
9 The glycosidation procedures essentially followed those for related pteridines: cf., .Ref. 4; P.R. Boyle
and W. Pfleiderer, Chern. Ber.,l13, 1514 (1980). We found that Lewis acid SnCLJ gave a more satis-
factory resulL
10 Separation of 16 and 17 was achieved by silica gel column chromatographl with EtOAc-hexane -+
MeOH-CHCl3. The structures of 16 and 17 were established by 500-MHz H-NMR spectroscopy in
CDCl3, as well as by the spectral analysis of the 1'-0-acety1 derivative of 16.
11 S. Hanessian and J. Banoub, Tetrahedron Lett. 1976, 661.
12 Compound 18 was obtained as a ca. 1:1 mixture of two diastereomers with respect to the MezN-1 group
on the dioxolane ring (by NMR). Purification of 18 by silica-gel column chromatography with MeOH-
CHCl3 as an eluent resulted in a facile decomposition. Thus the crude product 18 had to be immedi-
ately subjected to the subsequent glycosidation.

24
THE SYNTHESIS OF FLUORINE-CONTAINING PTERINS

Caroline Dunn, Colin L. Gibson, Colin J. Suckling, and Weifan Xing

Department of Pure and Applied Chemistry, University of Strathclyde

Glasgow, Gl lXL, Scotland

IINTRODUCTION

The introduction of fluorine into molecules has been well demonstrated to modifY
the biological properties significantly!. In heterocyclic compounds this effect is clearly
demonstrated through the action of 5-fluorouracil and its nucleosides as inhibitors of
thymidylate synthetase. In folic acid derivatives, fluorine has been introduced into the
glutamate side chain2 and into the aminobenzoic acid ring to promote nmr observations of
interactions with dihydrofolate reductase3. Some side-chain fluorinated pteridinones have
been prepared previously'! but analogues of natural products appear to be novel. It would be
of interest to investigate the properties of pterins with fluorine as a substituent of the
pyrazine ring and also at C-9 of the methylene group. To approach this study we have
examined the reactions of 2,4,5-triaminopteridin-6(1H)-one (1) with a number of readily
available di- and trifluoromethyl carbonyl compounds. We have also examined the
electrostatic potentials of the molecules associated with the introduction of fluorine into the
pyrazine ring and its substituents.

SYNTHESIS OF PTERINS

The triaminopteridine 1 was reacted with bromotrifluoroacetone, ethyl

chlorodifluoroacetate, chlorodifluoroacetaldehyde, and chlorodifluorotrifluoroacetone. With
bromotrifluoroacetone in dimethylformamide in the presence of triethylamine at 80°C, a
chloroform-soluble product was obtained together with a precipitate of triethylamine
hydrobromide. This solubility of the product suggested that the strongly hydrogen bonding
amino-amide functions of the pyrimidine ring had been modified. Assuming that the first
step in the reaction is the condensation of the 5-amino group of 1 with the highly
electrophilic carbonyl group of bromotrifluoroacetone to afford the imine (2), subsequent
alkylation of oxygen by the bromomethyl group would lead to an organic soluble product
(3, scheme 1).

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 25
 !

Scheme 1 Condensation ofbromotrifluoroacetone with 1

Structure 3, a 7H-pyrimido[4,5-b][l,4]oxazine5, was consistent with the

spectroscopic properties of the product (be 56.22 (OCH2) in DMSO-d6; Vmax 3 peaks
centred on 1370 cm-1 (CF 3 )). The alternative mode of cyclisation via alkylation on nitrogen
cannot be conclusively ruled out on the basis of the available data. However on standing in
dimethylsulphoxide solution (an nmr sample), 3 was transformed into a further product
provisionally characterised as 4 on the basis of the movement of the resonance of 56.22 ppm
(OCH2) to 151-155 ppm indicative of oxidation to a conjugated unsaturated structure.
Clearly the reaction of bromotrifluoroacetone with 1 is not a suitable route to a
representative of the pterins of interest.
The remaining examples of fluorine-containing precursors contained the less reactive
chloride as leaving group in place of bromide and also one carbon atom less so that
cyclisation to a six-membered ring would be impossible. Treatment of the
triaminopyridimidine 1 with each of these compounds in methanolic solution in the presence
of triethylamine afforded highly insoluble products typical of amino-amide pteridines. The
products were purified by dissolution in aquous ammonia and slow evaporation. All three
products had similar electronic spectra (Amax 245, 405, 450(s) nm in aqueous ammonia), ir
absorptions at 1300 - 1350 cm-1 assignable to C-F stretching vibrations, and acceptable
microanalyses. The products of these reactions (5-7) are unsuitable for further elaboration
into analogues of more complex natural products because of their low solubility but show

26
 Scheme 2 Reactions of 1 with chlorodifluorocarbonyl compounds

that standard methods for the synthesis of pterins can be adapted to afford compounds with
fluorine in the pyrazine ring or at the C-9 substituent.

THEORETICAL EVALUATION OF FLUOROPTERINS

If the fluorpterins of interest are to be useful in the study of folate transport and
metabolism, it is important that the introduction of fluorine should not perturb the normal
hydrogen bonding properties of the amino-amide pyrimidine system. To provide some
reassurance that this would not be the case, we have carried out semi-empirical molecular
orbital calculations (AM14) of electrostatic potentials of representative fluoropterins in
comparison with 6-methyl-7,8-dihydropterin. The results shown in the table below make it
clear that the influence of fluorine, even in polysubstitution, is limited to two atoms from the
fluorine-bearing atom; there is essentially no change in the potentials observed in N-1 to 0-4
of the pyrimidine ring. The results obtained from these calculations indicate that the
insertion of fluorine will have a highly localised effect, a property that is important in the
planned investigation ofbiological mechanisms and action.

27
 Table AMI electrostatic potentials of 6-methyl-7,8-dihydropterin ( 8), 6- trifluoro
methyl-7 ,8-dihydropterin (9), and 7, 7-difluoro-7,8-dihydropterin (10)

Compound 8 9 10

Atom

Nl -0.384 -0.388 -0.370

C2 0.323 0.338 0.315
N(H2) -0.398 -0.389 -0.381
N3 -0.345 -0.347 -0.318
C4 0.371 0.375 0.374
0= -0.332 -0.312 -0.328
C4a -0.290 -0.304 -0.324
N5 -0.055 0.010 0.001
C6 -0.116 -0.225 -0.216
C(H3)(F3) -0.152 0.406
C7 0.014 0.028 0.409
N8 -0.317 -0.306 -0.344
N8a 0.243 0.267 0.249

REFERENCES

1. J.T. Welch and S. Eswarakrishnan. "Fluorine in Bioorganic Chemistry", Wiley-

Interscience, New York, (1991).
2. J.K. Coward, J.J. McGuire, and J.Galivan. "Chemistry and Biology of Pteridines",
H.C. Curtius, ed., de Gruyter, Berlin, (1990), p. 1190.
3. G. Fendrich and R.H. Abeles, Biochemistry, 21:6685 (1982).
4. M. Tsuchiya, Nippon Kagaku Zasshi, 92:1177 (1971).
5. G.B. Elion and G.H. Hitchings, J.Am.Chem.Soc., 74:3887 (1952).
6. Calculations carried out using Hyperchem package using AMI routines. J.J.P.
Stewart, J.Computer-AidedMol.Design, 4:1 (1990).

28
FORMATION OF Fem OR Fen COMPLEXES WITH ACETYLACETO-
NATE AND 5,6,7,8-TETRAHYDROPTERIN AS LIGANDS AND THEIR
ACTIVATION OF OXYGEN

Andrea Schafer 1 , Berthold Fischer2 , Rene Bosshard3 , Manfred Hesse 1 ,

Max Viscontini 1

1 0rganisch-chemisches Institut der Universitat, Winterthurerstrasse 190,

CH-8057 Zurich, Switzerland
2 Anorganisch-chemisches Institut der Universitat, Winterthurerstrasse

190, CH-8057 Zurich, Switzerland

3 Kinderspital der Universitat, Abteilung Klinische Chemie, Stein-

wiesstrasse 75, CH-8032 Zurich, Switzerland

INTRODUCTION
The formation of a Felli ~ Fen complex 3a ~ 3b with acetylacetonate [acac] and
N(5)-methyl-5,6,7,8-tetrahydropterin [N(5)MTHP] (1) as ligands was demonstrated by
electrospray ionization mass spectrometry (ESI-MS) 1 •2 •

H
o" o
+
3a ~

1 2 4
mw: 181 mw: 353 mw: 100

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 29
 The [N(5)MTHP] (1) coordinates in this complex (3) the iron-center with the
atoms 0(4) and N(5). The methyl group at the N(5)-position stabilizes 3 and with this
ligand at pH 2-3 no activation of oxygen is observed. It was of interest to investigate
the behaviour of 5,6,7,8-tetrahydropterin [THP] (5) a compound without a stabilizing
group at N(5). The question is of importance because numerous tetrahydropterins are
part of oxidoreductase coenzymes.

RESULTS

5 6 7
mw: 167 mw:254 mw:254

THP (5) displays in the ESI-MS a more complicated behaviour than the [N(5)MTHP].
Immediately after adding THP · 2 HCl (5 · 2 HCl) to a equimolar solution of Feiii(acach
(2) (10- 3 M, acetonitrile, argon) the ESI-MS given in Figure 1 can be observed.

254 E+ 06
100
6 1.69
255
7

80

60 8

~
421

]
40

10
286 9
20 I
4
101
I

100 150 200 250 300 350 400 450 m/z

Figure 1. ESI-MS of 5 · 2 HCl and 2 (lo- 3 M, acetonitrile, argon).

We propose the following structures for the compounds (or clusters) explaining the
most prominent peaks in this spectrum.

Peak 101: corresponds to [Hacac+H]+, (cf. 4).

Peak 168: corresponds to [THP+H]+, (cf. 5).

30
Peak 254: corresponds to [Feiii(acach]+, (cf. 6).
Peak 255: corresponds to [Ferr(acach 0 +H]+, (cf. 7).
Peak 320: corresponds to [Ferr(acac)-quinonoid-(6H)-7,8-dihydropterin]+, (cf. 9).
Peak 354: corresponds to [Fem(acach 0 +H]+, (cf. 2).
Peak 419: corresponds to [Feiii(acac)Tquinonoid-(6H)-7,8-dihydropterin]+, (cf. 10).
Peak 421: corresponds to [Feiii(acach(THP)]+, (cf. 8).

9
mw: 320

8
mw: 421
10
mw: 419

The ESI-MS in Figure 1 is not stable and changes within minutes. In particular
the relative intensity of the signal at m/z 421 drops rapidly. This indicates dynamical
processes in the reaction mixture. The THP in 8 looses two electrons and two protons to
form the two Fe-quinonoid-dihydropterin complexes 9 and 10. We assume the electrons
and protons to react with traces of oxygen dissolved in the solution. To verify this hy-
pothesis, we measured the oxygen uptake versus time, to oxidize THP to dihydropterin
and pterin with and without Feiii(acac)J. The results of these experiments are shown
in Figure 2.
The oxidation of THP in the presence of air is accomplished at a slower rate without
Feiii(acach (curve B) than with Fem(acac)3, and needs approximately one molecule of
oxygen. Oxidation with Feiii(acach is, in particular at the beginning, much faster and
uses almost three atoms of oxygen. There is a break in the oxidation curve after the
consumption of one atom of oxygen. This fact can be related to the formation of the
Fe-quinonoid-dihydropterin complexes 9 and 10. Further oxidation of these complexes
involve mechanisms, that are unknown so far. The formation of Fe-oxo species and/or
higher oxidized pterins could explain the additional consumption of oxygen. This in-
terpretation is strengthened by thin layer chromatography (TLC). Without Feiii(acach
TLC shows only pure pterin in the reaction mixture; with Fem(acach, TLC reveals a
series of other, not yet identified products beside small amounts of pterin.

31
 1.5 '-j-----+-----+~-----t---- I
1
!
-----r--- Curve A
~I
0"'
1 ~ i j

10 20 30 40 50 60 time (hours)
Figure 2. Oxidation curves of 10-3 M THP (5) in dioxan/water (9:1) solution, pH range
1.5-7.5, 22-24°C, 725 Torr, air. Curve A: with Felii(acac)a. Curve B: without Feiii(acac)a.

We summarize that the reaction of N(5)MTHP with Fe1u(acac) 3 leads to stable, but
not yet isolable complexes, whereas that of THP with Felli( acac )a results in very unstable
complexes and the ESI-MS shows dynamical processes. The measurements of the oxygen
uptake of THP solutions with and without Feiii(acac)a confirm this assumption.

REFERENCES
1. B. Fischer, A. Schii.fer, R. Bosshard, M. Hesse, and M. Viscontini, "6th International Confer-
ence on Pteridines and Related Biogenic Amines and Folates", N. Blau, H. Ch. Curti us,
R. Levine, J. Yim, eds., Hanrim Publishing Comp., 56-1 Soopydong, Chung-ku, Seoul
100-230, South Korea (1992), 35.
2. A. Schii.fer, B. Fischer, H. Paul, R. Bosshard, M. Hesse, and M. Viscontini, Helv. Chim.
Acta 75:1955, (1992).

32
STEREOELECTRONIC EFFECTS IN THE AUTOXIDATIVE
DESTRUCTION OF REDUCED FOLATE DERIVATIVES

Anthony L. Fitzhugh

PRI/DynCorp, National Cancer Institute-FCRDC

Medicinal Chemistry Section, CSAL
P.O. Box B
Frederick, Maryland 21702-1201

INTRODUCTION

One of the most well studied and remarkable properties of reduced folate derivatives is
their varying sensitivity to autoxidative destruction. I Depending on the substituent(s)
attached to the N5 and/or NlO positions, they are either quite stable to or rapidly destroyed
by 02. Noteworthy examples of derivatives displaying such contrasting sensitivities are
tetrahydrofolic acid, 5-methyl tetrahydrofolic acid, and 5-formyl tetrahydrofolic acid. The
chemical basis for this divergence in reactivity is unknown. Stereoelectronic theory ,2
however, provides a qualitative means of reconciling the experimentally observed pattern of
02-reactivity.

DISCUSSION
Tetrahydrofolate
Stereoelectronic theory is a more recent molecular orbital concept in organic chemistry.
It argues that chemical intermediates breakdown under the constraints imposed on them by
the precise stereo orientation of their substituents and nonbonded electron pairs. For
tetrahydrofolate the relevant chemical intermediate for such analysis is the 4a-hydroperoxy
dihydrofolate derivative. This intermediate is formed following exposure of tetrahydrofolate
to 02 (Fig. 1, 1a and 1b) The specific nature of 02 addition to the 4a-position is due to the
extensive resonance delocalization provided by the three electron pairs at positions N2', N4,
and N8. These atoms redistribute their electron density in such a manner that the 4a-position
becomes more electron dense. Theoretically, four 4a-hydroperoxy dihydrofolate
intermediates are possible (Fig. 1, 2a-d). Stereoelectronic theory argues, however, that
only two of the four intermediates (2a and 2c) can displace the hydroperoxy group from the
4a-position. The reason for this is simple. Intermediates in which the N5 nonbonded
electron pair is oriented antiperiplanar to the departing hydroperoxy group (2a and 2c)
proceed along a much lower energy pathway than synperiplanar derivatives (2b and 2d).
The breakdown of 2b and 2d therefore is not competitive with 2a and 2c. In reality,
however, the negligible barrier to inversion between these derivatives means that 2b and 2d
are in equilibrium with 2a and 2c. Accordingly, stereoelectronic theory predicts that the
autoxidation of tetrahydrofolate should be a facile process. Appreciable experimental
evidence supports this postulate. I
5-Methyl Tetrahydrofolate
From lH NMR studies, the N5 and C6 substituents of 5-methyl tetrahydrofolate occur
in a trans configuration relative to one another.3 This factor and the unfavorable steric

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 33
 r4f1;H
H
N N

J HN'O
!
02
re H I
la

----
---

----
---
2c

Figure 1. Formation and Breakdown of 4a-Hydroperoxy Dihydrofolate

interactions between the 4a-hydroperoxy, NS-methyl, and C6 substituents indicate that only
one intermediate is likely to be formed following exposure of this derivative to 02 (Fig. 2).
Stereoelectronic analysis indicates that this intermediate cannot breakdown to a quinonoid 5-
methyl dihydrofolate derivative given the synperiplanar orientation of its NS nonbonded
electron pair and 4a-hydroperoxy group. Cleavage of the C3-4a bond, however, is
stereoelectronically allowed. Remarkably, there is ample experimental evidence indicating

34
that the autoxidation of 5-methyl tetrahydrofolate proceeds through cleavage of this bond to
give a pyrazino[1,2-a]-s-triazine derivative.4

~~~~
6 OH

N~N~
H,N)lN~N'-cu,
0
Figure 2. Breakdown of 4a-Hydroperoxy 5-Methyl Dihydrofolate

5-Formyl Tetrahydrofolate
lH NMR studies of 5-formyl tetrahydrofolate5 indicate that the formyl group of this
derivative occurs as two slowly interconverting conformers (Fig. 3, Ia and lb).

Figure 3. Two Conformers of 5-Formyl Tetrahydrofolate

Sterle interactions between the carbonyl oxygen of the tetrahydropteridine ring and the formyl
group prevent Ia from adopting a planar conformation. As a result, the attack of 02 on the si
face of the 4a-position is blocked by overwhelming steric interactions (Fig. 4). In addition,
like 5-methyl tetrahydrofolate, the re face attack of 02 on Ia yields a 4a-hydroperoxy

35
 si

~~~H
H'(s H

..
O\ NH
OH

Figure 4. Attack of 0 2 on the Si andRe Faces of Conformer Ia

derivative that is incapable of undergoing autoxidative cleavage (Fig. 4).

Since Ib can adopt a planar conformation, it hypothetically is a good candadite for 4a-
hydroperoxy bond cleavage. However, the significant ylide-like character of its 4a-N5 bond
makes the attachment by 02 to either face of the 4a site an unfavorable process (Fig. 5).

Figure 5. Ylide-like character of 4a-N5 bond in Conformer Ib

Therefore, on stereoelectronic and electronic grounds, neither conformer Ia nor Ib of 5-

formyl tetrahydrofolate is likely to undergo autoxidation. A postulate that is supported by
considerable experimental evidence.!

CONCLUSION
Previously, the chemical basis for the relative susceptibility of reduced folate derivatives
to 02-mediated autoxidation was not clear. The stereoelectronic and electronic principles
outlined above provides a simple, qualitative means of understanding of this phenomenon.

ACKNOWLEDGMENT
The content of this publication does not necessarily reflect the views or policies of the
Department of Health and Human Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S. Government.

36
REFERENCES
1. R L. Blakely."Frontiers of Biology, V.13: The Biochemistry of Folic Acid and Related
Pteridines," American Elsevier, New York, 58 (1969); C. Temple, Jr. and J.A.
Montgomery." Folates and Pterins," R.L. Blakey and S.J. Benkovic, ed., J.
Wiley & Sons, New York, 61(1984).
2. P. Deslongchamps."Stereoelectronic Effects in Organic Chemistry,"Pergamon, New
York, 1983.
3. M. Poe, O.D. Hensens, and K. Hoogsteen, J. Biol. Chern., 254:10881(1979).
4. J.A. Jongejan, H.I.X. Mager, and W. Berends."Chem. Biol. Pteridines," R.L. Kisliuk
and G.M. Brown, ed., Elsevier, New York, 241(1979); J.M. Whiteley and A.
Russell, Biochem. Biophys. Res. Commun., 101:1259(1981).
5. M. Poe and S.J. Benkovic, Biochemistry, 19:4576(1980); J. Feeney et al., J. Chern.
Soc., Perkin Trans II, 176(1980).

37
MIND0/3 MOLECULAR-ORBITAL CALCULATIONS ON THE 6R-BH4 MOLECULE AND ITS

METABOLIC PRECURSORS : CONFORMATION AND ACTIVITY

Setsuko Katoh 1 , Terumi Sueoka 1 , and Teruo Kurihara 2

1 Department of Biochemistry, Meikai University School of

Dentistry, Sakado, Saitama 350-02, Japan
2 Department of Organic Chemistry, Faculty of Science, Josai

University, Sakado, Saitama 350-02, Japan

INTRODUCTION

Tetrahydrobiopterin [6-(1',2'-dihydroxypropyl)-5,6,7,8-tetrahydro-
pterin ; BH4] acts as a cofactor of aromatic amino acid hydroxylases and
nitric oxide synthase. Of the total of eight configurational isomers of
the BH4 molecule (L-erythro,L-threo, D-erythro and D- threo forms for
each of the 6R- and 6S-enantiomers), naturally-occurring BH4 cofactor in
mammals is the 6(R)-L-erythro isomer (6R-BH4) (Fig. 1). The stereo-

H
1 H H H He
4•
~):'
s c-c
1,• 12• :s;../
-~..-Hb
-C-C-CH3
II II [1]
1 I I '
0 0
OH OH Ha
N
• 7
Hb H
1 Ha -C-C-CH3
H II I [2]
0 OH
H
-<r-R-cH3 [31
OH 0
Fig. 1. Natural tetrahydrobiopterin (6R-BH4). Structure {2-Amino-(6R
l'R,2'S)-6-(1',2'-dihydroxypropyl)-5,6,7,8-tetrahydropteridln-4(3H)-onej
and its metabolic precursors: [1]6-Pyruvoyl PH4, [2] 6-Lactoyl PH4, [3]
6-Hydroxyacetonyl PH4. PH4;tetrahydropterin.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 39
structure of the tetrahydropterin ring and the alkyl side chain of the
6R-BH4 molecule and the reactivity of atoms constituting it supply
specific reguratory process in exhibiting the cofactor activity. 6R-BH4
molecule, however, is unstable at physiological pH under oxidative
conditions. Previous studies on the stereochemistry of this molecule
were generally performed at acidic pH'' 2 or with the dihydrochloride
crystal of it'. In these studies, the atoms of N5 or N1 and N5 of the
pterin ring were protonated.
We have determined the absolute conformation and quantum chemical
properties of neutral form of 6R-BH4 molecule(as a physiological confor-
mation) by a method of semiempirical Molecular-Orbital calculations.
To know the enzymatic procedure of the formation of the conforma-
tion of the 6R-BH4 molecule, we also attempted about the molecules of
tetrahydropterin (PH4) precursors of 6R-BH4. In the final step of BH4
biosynthesis, 6R-BH4 is formed stepwise from 6-pyruvoyl PH4 [1] by the
function of sepiapterin reductase and NADPH through hydroxyketo interme-
diates, 6-lactoyl PH4 [2] and 6-hydroxyacetonyl PH4 [3] 3 (Fig. 1).
Results of calculations about 6R-BH44- and 6-pyruvoyl PH4 5 have been
described in detail.

RESULTS AND DISCUSSION

The structures of 6R-BH4 [2-Amino-(6R, 1'R,2'S)-6-(1' ,2'-dihydroxy-

propyl)-5,6, 7,8-tetrahydropteridin-4(3H)-one] and its precursors, [1],
[2] and [3] were set up as the structure in solution that are accord
with experimental findings and general chemical considerations 1 ' 2 ' s, 7 :
Tetrahydropyrazine ring was in a half chair conformation,and an alkyl
side chain attached at position 6 in the pseudo-equatorial posit1on. All
of the calculations were performed with the MOPAC on an FACOM M-360
computer with the MIND0/3 framework. The first optimisation of 6R-BH4
was performed for the conformation in which the bonds of 01'-c1' and C6-
H6 were in the trans position. Then the reaction pass was done on the
dihedral [01'-c1'-C6-H6] at intervals of 30· (Table 1). Finally two
different conformations were optimized as the lowest energy conformer
of the 6R-BH4 molecule. Formation energies (~H) of Form A([01'-C1'-c6-
H6)= -sa· ) and Form B ([01'-C1'-C6-H6]= 30· ) were -174.21 and -174.46

Table 1. Reaction pass for conformations of the alkyl side chain

of 6(R)-L-erythro-BH4.
Angle# ~H (kcal/mole) Angle ~H(kcal/mole)
178. 12· (start) -172.76 o· -172.79
-15o· -173.61 30° -174.45**
-120° -173.44 60° -170.71
-90° -174.14* 90° -167.89
-60° -173.54 120° -167.47
-30° -172.04 15o· -170.86
#Reaction coordinate angle:[01'-cl'-C6-H6].
*Optimized to Form A (-88. ), **Optimized to Form B (30° ).

40
 (a)

(c)
(d)

(e)

Fig. 2. The lowest energy conformations of natural tetrahydrobiopterin

and 1ts metabolic precursors. (a)6R-BH4 [Form A] 4 , (b) 6R-BH4 [Form B] 4 ,
(c) 6-Lactoyl PH4, (d) 6-Hydroxyacetonyl PH4, (e) 6-Pyruvoyl PH4 5 •

kcal/mole, respectively. The ~H values of the lowest energy conforma-

tions of the optimized forms of [1], [2], and [3] were -152.18, -165.23,
and -162.22 kcal/mol, respectively.
Computer generating perspective structures of 6R-BH4 and its
precursors were illustrated in Fig. 2. The molecular width (H2a-H3'b) of
them was about 11 A. Structures of the precursors imply that Form A is a
more favorable conformation of a natural 6R-BH4 than Form B. Theoretical
conformation of 6R-BH4 was recently predicted by other workers 8 • 9 by
different tools than we did. In addition to the determination of the
conformation, we calculated atomic net charges of atoms constituting the
optimized molecules of 6R-BH4 (Fig. 3) and its precursors. Results
showed that atoms of N(l), C(2), C(4), 0(4), C(4a), and C(8a) may be
important for the approaching of the substrate to the specific site of
the enzyme for pteridine binding. Net charge of C(4a) of 6R-BH4, at
which position an oxygen molecule may bind first on the hydroxylation of
aromatic amino acids, was - 0.32 (Fig. 3). Calculations of net charges
of atoms of 6R-BH4 (Fig. 3) and 6-pyruvoyl PH4 5 , and of LUMO
coefficients of 6-pyruvoyl PH4 5 suggest that, in these molecules, C2' is
more reactive (=) thar. C1' toward NADP+ or NADPH in the reversible
reaction of enzymes such as sepiapterin reductase and aldose reductase:

[1] [2] [3] [6R-BH4]

PH4-CD-CO-CH 3 ~ -CO-CHOH-CH 3 -CHOH-CO-GH 3 ~ -CHOH-GHOH-CH 3
~ (NADPH) (NADP+) ~

41
 +0.24

0-0.58 '!Lo.49
+0.04 \ -0 04 -0.01
+o.o8
\
+0.66 \ +0.33 / ~o.o1 I
'-.. /f.""-.. /o.No~ /C~ -c\o.o1
c c/
-o. 25/t/.

+0.44
-o. 32

+0.34
I - 0 · 06
l ,-0.07/\+.
+o. 10 c, 0
-0.12
36 -o. o1

+o.1o ,(: :c 'c+o.18 0 -o.4s

'-N /
-0. 191
"-R/
-0.40
'-.IN -0.13
/ " -0.06
' +0.25
+0. 11 +0. 07

Fig. 3. Net charges of atoms constituting the natural tetrahydrobio-

pterln (6R-BH4 [Form A] 4 ) . Expression of H atoms was omitted.

REFERENCES

1. S.Matsuura, T.Sugimoto, S.Murata, Y.Sugawara, and H.Iwasaki,

J. Biochem. 98:1341-1348 (1985).
2. W.L.F.Armerego, P.Waring, and B.Paal, Aust.J.Chem. 35:785-793 (1982).
3. S.Katoh,and T.Sueoka.in:Chemistry and Biology of Pteridines 1989,
pp.324-327, Walter de Gruyter, Berlin, New York, (1990).
4. S.Katoh, T.Sueoka, and T.Kurihara, Pteridines 4(1):27-31 (1993).
5. S.Katoh, T.Sueoka, and T.Kurihara, Biochem. Biophys. Res. Commun.
176:52-58 (1991).
6. J.E.Gready, J.Mol. Struct.,Theochem.,109:231-244 (1984).
7. M.Viscontini, in:Biochemical and Clinical Aspects of Pteridines,
2:21-34, Walter de Gruyter, Berlin, New York (1983).
8. J.E.Ayling, S.B.Dillard, S.W.Bailey, in:Pteridines and Biogenic
Amines in Neurology, Pediatrics and Immunology, pp.269-282,
Lake Shore Publishing Co., Grosse Pointe (1991).
9. I.Ziegler,M.Borchert, F.Heaney, A.P.Davies, P.H.Boyle, Biochim.
Biophys. Acta, 1135:330-334 (1992).

42
IDENTIFICATiON, STEREOCONFIGURATION, CHROMATOGRAPHIC
AND FLUORESCENCE PROPERTIES OF NATURAL PTERINS

Roger Klein

Institut Curie, Section de Physique et Chimie,

Laboratoire de Physique et Chimie Biomoleculaires (UA 198 CNRS)
11 rue P. et M. Curie, 75231 Paris Cedex 05, France

INTRODUCTION

Several years ago, we reported the identification of dictyopterin as 6-[D-threo]-1 ', 2'-
dihydroxypropyl-pterin1. Such a result had been obtained after numerous and tedious
purification steps. In order to facilitate the determination of the stereoconfiguration of natural
pterins, which are found in very small amounts in living organisms, we aimed to find a
sensitive method. This was achieved in using chiral HPLC. The separation of D- and
L-enantiomers of 6-(polyhydroxypropyl)-pterins was obtained by ligand exchange
chromatography in using a reversed-phase column with a mobile phase containing
D-phenylalanine as the chiral modifier and Cu(II) as the metal ion. This enantiomeric
separation allowed the stereoconfiguration of some natural pterins to be determined in the
picomole range, by comparison of their chromatographic and fluorescence properties with
those of reference enantiomeric pterins2. The present paper briefly reports an aspect of the
method dealing with the fluorescence quantum yields of pterins and describes one application
which answers the following questions : what is the stereoconfiguration of urinary
monapterin ? Are there any differences in human urine from cancer and non-cancer patients?

RESULTS AND DISCUSSION

Fluorescence Quantum Yields of Natural Pterins

The fluorescence quantum yield of unconjugated pterins with an unsubstituted amino

group in position 2 (neopterin, monapterin, biopterin, dictyopterin) is 0.16 at pH 3, whereas
the fluorescence quantum yield of euglenapterin (N2,N2-dimethylamino-4-hydroxy-6-[D- or
L-threo]-1',2',3'-trihydroxypropyl-pteridine) is only 0.003 (R. Klein et al., unpublished).
Auorescence quantum yields play a key role in the sensitivity of our new chiral HPLC
method. Figure 1 shows a chromatogram obtained from one nanomole of each enantiomer of
euglenapterin and only 25 picomoles of each enantiomer of monapterin. Monapterin
enantiomers were previously separated at low temperature (l2°C)2. In the present work, the
enantiomers were separated at room temperature (24°C}. A double-detection was used based
on the absorption and fluorescence properties of the pterins of interest (as summarized in
Table 1). The absorbance of D- and L-monapterins could barely be detected (Figure la),
whereas the fluorescence signals of monapterins and euglenapterins were of similar intensity
(Figure lb). The ratio of the fluorescence signal over the absorbance signal increases as does
the fluorescence quantum yield. The latter ratio might be useful for the identification of
fluorescent pterins, in addition to fluorescence excitation and emission maxima.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 43
 Table 1. Absorption and fluorescence properties of monapterin and euglenapterin.
monapterin euglenapterin
Molar absorption coefficient (e) at360 nm, pH 3 (M-1 cm-1) S.Ox103 5.6x1o3
fluorescence excitation maxima, wavelength (nm) 276 &362 286 &375
fluorescence emission maximum, wavelength (nm) 444 482
fluorescence quantum yield, pH 3, excitation wavelength : 360 nm 0.16 0.003

01.02
D-E a
w L-E
0
z
<(
CD
a:
0
(/)
CD
Figure 1. Separation of enantiomers of monapterin
<( and euglenapterin by HPLC.
Conditions: reversed-phase column LC18DB
A
(Supelco), SIJffi, 4.6x250 mm, 24°C.
Mobile phase: 8 mM D-phenylalanine + 4 mM
D-M b
~

:::) CuS04, pH 3.1, 1.2 mllmin.

Double-detection: a) absorbance at 360 nm; b)
~ fluorescence, excitation wavelength: 360 nm, emission
w wavelength: 460 nm. Injection of about 1 nanomole
0 L-M D-E
z of each euglenapterin enantiomer and 25 picomoles of
w L-E each monapterin enantiomer. D-E: D-euglenapterin, L-
0
(/)
w E: L-euglenapterin, D-M: D-monapterin, L-M: L-
a: N monapterin.
0
:::)
..J
u. \J L
5 10 15 20 25 30
TIME (min)

Urinary Pterins

Using the chiral HPLC method, the stereoconfiguration of optically active pterins
could be obtained from only 25 microliters of human urine. D-neopterin, D-monapterin and
L-biopterin were found in urines from healthy individuals2. The determination of D-
neopterin and L-biopterin agreed with determinations obtained earlier from as much as 1000
liters of human urine3.4, whereas the determination of D-monapterin in urines from healthy
individuals was in disagreement with the finding of L-monapterin by Fukushima and
Shiota5. This result was in press2 when Ogiwara et al.6 reported that D-monapterin was
present in the urine of cancer patients. The authors6 suggested that the urinary D-monapterin
might be considered as an eventual biochemical marker of cancer, since the enantiomeric
L-monapterin had been previously found in the urine of healthy individualsS. The suggestion
of using urinary D-monapterin as a biochemical marker of cancer prompted us to investigate
the origin of the disagreement about the stereoconfiguration of urinary monapterin. The use
of a bioassay method based on the growth of Crithidia fasciculata by Fukushima and Shiota5
could be questionable. Another hypothesis might explain the above contradiction : an urinary
infection might be at the origin of L-monapterin, since this compound is excreted by bacteria,
for example, Escherichia coli 7 and Serratia indica 8.
The main goal of the present work was to compare, by using the same chiral HPLC
method, the stereoconfiguration of optically active pterins in urine samples collected from 25

44
cancer patients with various carcinomas, from 2S patients with bacterial, fungal or parasitic
urinary infections and from 4 healthy individuals. Urine samples from cancer patients under
therapeutic treatment were collected at the lnstitut Curie, Section Medicale et Hospitaliere,
Paris; urine samples from patients with infectious deseases, before or under therapeutic
treatment, were collected at the Centre Medico-Chirurgical de laPorte de Choisy, Paris.
Urine was deproteinized with perchloric acid and oxidized with acidic iodine as
described elsewhere2. Neutralized samples were chromatographed as shown in Figure 2.
Four fluorescent peaks, labeled Ul to U4 were found in the urine of a healthy individual
(Figure 2a). In the urine of a cancer patient a fifth peak, (US), was found in addition to peaks
Ul to U4 (Figure 2b).
The peak US, was not specific for cancer patients. It was present in ten urine samples
from cancer patients, in two urine samples from non cancer infected patients and, at a very
low level, in the urine from one healthy individual. This peak, characterized by fluorescence

::J
U1 a us b
U3
:5. U1
w
0
z
w U3
0
UJ
w
a: U4
0 U2
::J
...I
IL

::J U1
U3+L-B c us d
:5. U3+L-B
w U1
0
z L-N
L-D
w L-M
0
UJ
w L-D
a:
0
::J
...I
IL

::J
U1+D-N
e us f
:5. U1 +D-N
w
0 U2+D-M U2
z
w +
0 U3 D-M
UJ
w D-D U3
a:
0
::J
...I
IL

0 s 10 1S 20 2S 30 0 s 10 1S 20 2S 30 3S
TIME (min) TIME (min)

Figure 2. Determination of the stereoconfiguration of natural pterins in the urine

from a healthy individual compared to the urine from a cancer patient.
Conditions as in Figure 1 except temperature 20°C and flow rate: 1 ml/min from 0
to 15 minutes then 1.5 ml/min from 16 to 35 minutes. Fluorescence detection:
excitation wavelength, 360 mn, emission wavelength, 450 mn. a) U1 to U4 :
fluorescent peaks in 8 j..ll of urine from a healthy individual. b) Ul to US:
fluorescent peaks in 8 j..ll of urine from a cancer patient. c) co-injection of the urine
from a healthy individual with the four L-enantiomers. L-N: L-neopterin. L-M:
L-monapterin. L-B : L-biopterin. L-D : L-dictyopterin. d) co-injection of the urine
from a cancer patient with the four L-enantiomers. e) co-injection of the urine from
a healthy individual with the four D-enantiomers. D-N : D-neopterin. D-M : D-
monapterin D-B : D-biopterin. D-D: D-dictyopterin. f) co-injection of the urine
from a cancer patient with the four D-enantiomers.

45
excitation and emission maxima at 320 nm and 420 nm, respectively, remained so far
unidentified. By comparison with the chromatographic and fluorescence properties of model
compounds2, the peaks Ul to U4 were identified as neopterin, monapterin, biopterin and
?-xanthopterin respectively. The fluctuations observed in the retention times showed that the
retention time alone is not an accurate parameter to determine the stereoconfiguration by
comparison with that of reference compounds. The method, systematically adopted, was to
co-inject each sample, first with the four L-enantiomers, and then with the four
D-enantiomers of the stereoisomers of biopterin and neopterin. Figure lc and Figure ld
compared with Figure le and Figure 1f clearly show that Ul coeluted with D-neopterin, U2
with D-monapterin and U3 with L-biopterin in the urines of both a healthy individual and a
cancer patient. L-monapterin has never been observed, even in the urines with high densities
of bacteria. Regardless of the origin of the urine, from cancer or non cancer patients, ill or
healthy individuals, pterins were always found with the following steroconfigurations :
D-neopterin, D-monapterin, and L-biopterin. Clearly, D-monapterin is not specific for cancer
patients. Rather, it is the normal stereoconfiguration of human urinary monapterin. Recently,
Ogiwara et al.9 have also confirmed, by using circular dichroism measurements, that
D-monapterin was present in human urine of both cancer patients and non-cancer controls.
The main advantage of the chiral HPLC method over the previous physico-chemical
ones is its greater sensitivity. Using circular dichroism, 50 liters of urine were required,
pooled from several cancer patients, whereas chiral HPLC could be applied by using only 25
microliters of urine collected from a single individual. Identification of compounds by on-line
fluorescence spectroscopy and comparison with pure enantiomeric pterins makes the chiral
HPLC method more reliable than the method based on the growth of Crithidia fasciculata.
The latter advantage is obvious in the case of urinary monapterin and in the case of ciliapterin
as well. Indeed, the major pterin of Tetrahymena pyriformis had been deduced from the
Crithidia test as L-threo-biopterin and named ciliapterinlO. In contrast to that conclusion,
D-monapterin, clearly identified for the first time as a natural compound by using chiral
HPLC, was proven to be the major pterin in four strains of Tetrahymena!! and in another
ciliate protozoan, Colpidium campylum12.

Acknowledgements

This work was supported by Centre National de la Recherche Scientifique (U. A. 198
and Comite National de la Chimie). I am grateful to Professor Pfleiderer (University of
Konstanz, Germany), to Professor Sugimoto (University of Nagoya, Japan) and to
Professor Viscontini (University of Zurich, Switzerland) for their generous gifts of reference
compounds. I thank Dr P. Pouillart, L. Deneux (lnstitut Curie) and M. Finetti (CMC Choisy)
for supplying with urine samples. I also wish to thank Brad Factor and Professor P. Y.
Turpin for critical reading of the manuscript and G. Tham for skilfull technical assistance.

REFERENCES
1. R. Klein, R. Thiery, and I. Tatischeff, Eur. J. Biochern. 187 : 665 (1990)
2. R. Klein, Anal. Biochern. 203 : 134 (1992)
3. E. L. Patterson, M. H. von Saltza, and E. L. R. Stockstad, J. Arner. Chern. Soc. 78: 5871 (1956)
4. A. Sakurai and M. Goto, J. Biochern. 61 : 142 (1967)
5. T. Fukushima and T. Shiota, J. Bioi. Chern. 247: 4549 (1972)
6. S. Ogiwara, T. Nagatsu, R. Teradaira, K. Fujita, and T. Sugimoto,Tetrahedron letters 33: 1341 (1992)
7. H. Wachter, A. Hausen, E. Reider, and M. Schweiger, Naturwissenschaften 67: 610 (1980)
8. K. Iwai, M. Kobashi, and H. Fujisawa, J. Bacterial. 104: 197 (1970)
9. S. Ogiwara, T. Nagatsu, R. Teradaira, K. Fujita, and T. Sugimoto, Bioi. Chern. Hoppe-Seyler 373 : 1061
(1992)
10. G. W. Kidder and V. C. Dewey, J. Bioi. Chern. 243: 826 (1968)
11. R. Klein, I. Tatischeff, G. Tham, and C. A. Groliere, Biochirnie 73 : 1281 (1991)
12. R. Klein and C. A. Groliere, Chrornatographia 37 : (1993), in press.

46
THE MECHANISM OF COFACTOR REGENERATION DURING
PHENYLALANINE HYDROXYLATION

Steven W. Bailey, Scott R. Boerth, Shirley B. Dillard, and June E. Ayling

Department of Pharmacology
College of Medicine
University of South Alabama
Mobile, AL 36688

INTRODUCTION

Although the ability of the liver to convert phenylalanine to tyrosine has been known
since 1913 1, characterization of the components of the system did not begin until the mid
1950's. Tetrahydrobiopterin was identified as the cofactor for the hydroxylase reaction2,
which uses molecular oxygen as the source of the new hydroxyl group, Figure 1. It was
first thought that only one other enzyme, dihydropteridine reductase (DHPR), was involved
in the overall process. This converts the oxidized form of cofactor, a quinoid
dihydropterin3, back to the starting tetrahydrobiopterin at the expense of NADH allowing
cofactor to be used catalytically. The clinical symptoms of patients with a deficiency of
DHPR have shown that cofactor regeneration is essential4•
It is now well accepted, however, that the initial product of phenylalanine hydroxylase
is the C4a,N5-hydrate of quinoid dihydrobiopterin*, which was first detected by its UV
spectrums. Isotope labeling studies using pyrimidine cofactor analogs6 and 6-methyl-
tetrahydropterin7 then definitively identified this structure. Further, a protein was
discovered which was at first called phenylalanine hydroxylase stimulating factors, but was
later shown to be capable of catalyzing the dehydration of the C4a,N5-hydrate to give a
quinoid dihydropterin8• Thus we would seem to have all of the enzyme reactions needed
for cycling of cofactor. However, the dehydration reaction proceeds quite well non-
enzymatically. In fact, almost all assays that suggest a need for a dehydratase enzyme have
been done at alkaline pH where the quinoid dihydropterin C4a,N5-hydrate is more stable.
As a result one might question whether this dehydratase activity is physiologically relevant.

*This compound has also been referred to as C4a-hydroxy-tetrahydrobiopterin. Although this is a

correct nomenclature based on the number of double bonds in the system, the use of the term quinoid
dihydrobiopterin C4a,N5-hydrate provides a better understanding of its redox state and chemical
properties.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et at., Plenum Press, New York, 1993 47
 PHENYLALANINE TYROSINE

O - y N H2 HO--o-vNH 2

C02H C02 H

~ ~~
s~~Y'("'
H

8~ rr~l(~~NH2
+ o.
~~~NH HYDROXYLASE
H 0 H H 0

\
TETRAHYDROBIOPTERIN QUINOID DIHYDROBIOPTERIN C4a,N5-HYDRATE

DIHYDROPTERIDINE
REDUCTASE
NAD
~ DEHYD;.,.TASE

H20
NADH
H

H N~~H
~ II
2
N~ N

0
QUINOID DIHYDROBIOPTERIN

Figure 1. Cofactor regeneration during phenylalanine hydroxylation.

For many years one of our main goals has been to elucidate the rate limiting step in
overall phenylalanine hydroxylation. Therefore, we felt it necessary to determine if an
enzyme is really necessary to perform the dehydration reaction in a physiological
environment. Further, in any event is this reaction rate limiting? Probably the main reason
this question has not already been clearly answered is that the only way to generate quinoid
dihydropterin C4a,N5-hydrates has been by using the phenylalanine or tyrosine hydroxylase
reaction9•10•11 • Therefore, a general synthesis for this class of compounds was devised.

The Synthesis of Quinoid Dihydropterin C4a,N5-Hydrates

This procedure is similar to that recently described for the generation of the
C6-stereoisomers of tetrahydropterins 12, except that the cyclization conditions are modified
to accumulate the C4a,N5-hydrate. The central intermediate is a triamino-4-pyrimidinone
containing an aminoethyl side-chain (1). This intermediate can be assembled in four or five
relatively simple steps starting from an amino acid enantiomer. The chirality of the
a-carbon is retained through the entire synthesis thus determining the stereochemistry of
the pteridine-C6. The triaminopyrimidine is rapidly oxidized by halogen, and the resulting
iminoquinone (II) then hydrolyzed in mild acid. By carefully taking a solution of the
ketone (III) to about pH 9, a Schiff's base cyclization occurs giving the desired
C4a,N5-hydrate. Figure 2 illustrates this reaction for the production of the (6S)-methyl-
derivative (IV) (which has the same C6-configuration as in (6R)-tetrahydrobiopterin), where
the initial chirality has been incorporated using L-alanine. The sequence starting with I is
performed in one pot over about 45 minutes. In this way gram quantities of
C4a,N5-hydrates can be conveniently generated. However, due to their instability (solutions
can be kept for about 2 days at -80"C) typical reactions are done on a 10 mg scale. The
synthesis of tetrahydropterin stereoisomers differs primarily in that the dehydration is
promoted by a shift in pH and temperature, and the resulting quinoid dihydropterin is then
reduced12 •

48
 6-Methyl- (65)-Methyl-qulnold· (65)-Methyl-qulnold·
7,8-dlhydropterin dlhydropterin dlhydropterln C4a,N5-hydrate

Figure 2. Oxidative cyclization of 6N-((2'S)-aminopropyl)-2,5,6-triamino-4-pyrimidinone to

6(S)-methyl-quinoid-dihydropterin-C4a,N5-hydrate, and subsequent dehydration.

Nonenzymatic Dehydration of C4a,N5-Hydrates

The spectral changes accompanying the dehydration of the C4a,N5-hydrate of

(6S)-methyl quinoid dihydropterin are shown in Figure 3. Previous studies of
C4a-carbinolamine dehydration have generally used the large change in extinction near
246 om to follow this reaction. However, quinoid dihydropterins which are not
disubstituted13 at C6 are subject to tautomeric rearrangement to 7,8-dihydropterins (e.g.
V ~ VI, Figure 2). Due to the mild conditions of the reaction illustrated in Figure 3, only
a small amount of tautomerization has occurred by the last scan. However, the generation
of the 7 ,8-dihydropterin and other products can be significant at higher temperatures and
with increased buffer catalysis. In such cases the complexity of the absorbance changes in
the low UV can hinder accurate quantitation of dehydration rates. Reactions monitored at

Q)
u
c::::
ctS
..0 200 400 600
0en Seconds
..0
<

250 300 350 400

Wavelength (nm)

Figure 3. UV spectra of (6S)-methyl-quinoid dihydropterin C4a,N5-hydrate dehydrating in O.oi M Tris-Cl

pH 8.4 at lO"C, 2 min between scans; inset: change in Abs34() vs time for a similar reaction, superimposed on
the best fit exponential decay curve.

49
the isosbestic point between quinoid and 7,8-dihydropterin, usually near 340 nm, showed
first-order increases in absorbance over three or four half-lives. HPLC analysis of the
dehydration products of 6-alkyl-quinoid-dihydropterin-C4a,N5-hydrates indicates that the
quinoid- and 7,8-dihydropterins are the only significant compounds that absorb in this
wavelength region. The inset to Figure 3 is the progress of the absorbance increase at
340 nm which has been digitized and fitted to an exponential decay to give the rate
constant.
Several C4a,N5-hydrates with varied C6-substituents were synthesized and their
spontaneous dehydration studied under a variety of conditions. Figure 4 illustrates the
effect of pH and buffer concentration (insert). The data using several buffers were fitted
simultaneously to a model equation for hydrogen ion and buffer catalysis. The dashed line
represents the rate at each pH extrapolated to zero buffer concentration.

0.12

0.033
0.08
~

'u 'o
(])
.e ! ~
0.04
~ 0.023

0.003 +---+----+---+- ---4

7.0 8.0 9.0 10.0 11.0
pH
Figure 4. The effect of pH on the rate of dehydration of (6R,S)-methyl-quinoid-dihydropterin C4a,N5-hydrate
at l7°C in the absence of buffer catalysis; Inset: The effect of buffer concentration on dehydration in Tris-Cl
(circles) and trimethylamine-Cl (triangles) with the same compound at l7°C.

As would be anticipated from the literature on carbinolamine dehydration, the reaction

is promoted both by hydrogen ion and general acid catalysis. The pH of maximum stability
depends on the buffer and its concentration. In the absence of buffer, the hydrate is most
stable at about pH 8.2. The more effective catalysis by Tris-Cl than Tris-base shifts the
point of optimal stability to higher pH with increasing buffer concentration (e.g. pH 8.6 in
0.1 M Tris-Cl). One remarkable feature is the faster rate of dehydration under more
alkaline conditions even in the absence of buffer. Such base enhanced dehydration has not
been observed before with carbinolamines formed by the reaction of a basic amine (such
as the ethylamine side-chain in Ill) with a ketone. The mechanism of base catalysis
appears to be correlated with the formation of the anion of the C4a,N5-hydrate 14 • Although
the anionic species of a hydrate of simple aromatic pteridines have not been observed 15 , the
C4a,N5-hydrate of a quinoid dihydropterin is analogous to alloxan hydrate which has an
acidic dissociation.
The temperature dependence of dehydration for the C4a,N5-hydrates of quinoid
dihydrobiopterin and the 6-methyl analog was investigated in 0.025M Tris-Cl, pH 8.4. In
this buffer the two compounds behaved the same within experimental error. The entropy
of activation was found to be negative, suggesting the occurrence of solvent organization
in the transition state. At pH 7.4 the enthalpy of activation decreased, but at the expense

so
of a further decrease in AS*. At 37° in 0.025M Tris-Cl pH 7.4 the rate of nonenzymatic
dehydration of the C4a,N5-hydrate of quinoid dihydrobiopterin was 0.038 sec· 1• Even
considering buffer catalysis by bicarbonate, various forms of phosphate and other buffers
present in the cytoplasm, it is unlikely that spontaneous dehydration in vivo would be much
greater than this.

Comparison of the Rate of Spontaneous C4a-carbinolamine Dehydration with the Rate

of Phenylalanine Hydroxylation

Whether a dehydration rate of about 0.04 sec· 1 would be rate limiting for overall
tyrosine formation in vivo depends upon the rate of the phenylalanine hydroxylase reaction.
In the presence of sufficient DHPR activity the hydroxylase and dehydration reactions
compete with each other to establish a partitioning of the available pool of cofactor (about
6 pM in rat liver) between the reduced (6R)-tetrahydrobiopterin and the C4a,N5-hydrate.
The instantaneous rate of the phenylalanine hydroxylase reaction in an animal subject
to a phenylalanine load is difficult to determine. The time course of phenylalanine
clearance from a rat is shown in Figure 5. A number of studies indicate that phenylalanine
exchanges with several compartments 16• For example, there appears to be a significant pool
of free phenylalanine in the peripheral tissues. Therefore, one cannot reliably interpret the
downward slope in Figure 5. On the other hand, typically by about 3 hours the resting
level of plasma phenylalanine is restored. At this point, presumably, all the pools will have
re-equilibrated. Since comparatively little L-phenylalanine would be excreted in the urine
or used for protein synthesis 16•17 , the only mechanisms for clearance are by hydroxylation
or transamination. However, experiments in which phenylalanine hydroxylase activity in
rats has been mostly abolished by pretreatment with p-chloro-phenylalanine 18 have shown
that tyrosine formation is responsible for the majority of the clearance.

1.4

1.2
:?
§.
Q)

·c:c::
.!!! 0.8
~
c::
Q) 0.6
.s::::.
a..
ctl
E 0.4
Ul
ctl
a: 0.2

40 80 120 160 200 240

Minutes
Figure 5. Clearance of plasmaL-phenylalanine from a 380 g male rat after i.p. injection of 510 )!moles.

A minimum rate of hydroxylation in the liver of 5 pM/sec, which is an average over

the entire curve in Figure 5, can thus be estimated by dividing the total load by 180 minutes
and again by the liver weight (9g). This value is lower than the maximal rate not only
because it is averaged over time, but also over the whole volume of the liver. (Note: many
reports of phenylalanine tolerance, especially in humans, are given in terms of per Kg body
weight, rather than liver weight.) Other clearance curves in the literature for rats
administered similar loads 18•19 give rates between 3 to 8 pM/sec when analyzed in this

51
manner. This agrees well with the rate of phenylalanine removal by isolated rat livers
perfused with 1 mM phenylalanine20• The rate of appearance of 0 20 in the blood of rats
given phenylalanine-Dt, when expressed in pmoles/g liver/sec, indicates a slightly faster
rate within the first 60 minutes of injection that may represent the rate of hydroxylation
during the load peak**.
At these rates of hydroxylation, spontaneous dehydration would cause almost all of
the available cofactor to reside in the form of the C4a,N5-hydrate. In other words, if 99%
of cofactor is to be maintained in the reduced form, a rate of dehydration of between 50
to 130 sec·' would be required. This is greater than 103 faster than that provided by
nonenzymatic dehydration under physiological conditions. A typical dietary load in a
human raises plasma phenylalanine levels only by a factor of 2 or 3, rather than by about
25-fold as in Figure 5. Still, spontaneous dehydration is at least two orders of magnitude
too slow to prevent depletion of the pool of reduced cofactor during a meal. Clearly, there
is a need for a dehydration catalyst.
One must then ask whether the previously described phenylalanine hydroxylase
stimulating protein fulfills this need. This is of particular concern since it has recently been
shown that the protein to which dehydratase activity has been attributed is identical to
DCoH, a regulator of hepatic nuclear homeodomain transcription factor 1a dimerization21 .22 •

Dehydratase Mechanism and Kinetics

The physical chemistry of the nonenzymatic dehydration reaction gives several clues
to the mechanism of enzymatic dehydration. On first consideration, it would be reasonable
to expect that simple enzyme promoted protonation of the C4a-hydroxyl group might be
involved. On the other hand, as shown in Figure 4, base catalysis can also be quite
effective. There is the additional possibility that the mechanism of the dehydratase may
also involve obviation of the negative entropy of activation of the spontaneous reaction.
Sequestration of substrate by the catalytic site in a manner that can avoid solvent
organization in the transition state of hydroxide elimination might also provide some rate
acceleration. Recent measurements of dehydratase kinetics have revealed the relative
importance of these mechanisms 14•
As a result of the new method for the synthesis of substrates, steady-state kinetics of
the dehydratase with several quinoid dihydropterin C4a,N5-hydrates have been measured
including (6R,S)-methyl-, (6S)-methyl-, (6S)-propyl-, and (6R)+erythro-dihydroxypropyl.
The ~ for the C4a,N5-hydrate of quinoid (6R)-dihydrobiopterin is close to the
concentration of tetrahydrobiopterin in the liver. The dehydratase activity in rat liver is
such that even given the rate of hydroxylation calculated for the high phenylalanine load
illustrated in Figure 5, less than 1% of the biopterin pool would be present as the quinoid-
dihydrobiopterin C4a,N5-hydrate 14 • Also, the tissue distribution of dehydratase activity has
been reported to parallel hydroxylase activity in several organs23 • All of these factors
strongly suggest that despite the other role of the protein in regulating transcription, that the
dehydratase activity specifically evolved to participate in the regeneration of
tetrahydrobiopterin. A dehydratase does not typically appear to be significant in vitro, since
reactions are rarely performed at rates as fast as those in the liver during a dietary load, and
the nonenzymatic rate of dehydration, especially at neutral pH and high buffer
concentrations, is adequate to regenerate cofactor.
Thus we can now conclude that the carbinolamine dehydratase is an important

•• From reference 17, Figure 1, the rate of 0 20 liberated into the blood was 4 mM/hr for a female rat
injected i.p. with 0.2 g/kg L-phenylalanine. Assuming nearly complete metabolism of the phenyl ring to
C02 and 2.5 0 20, and equilibration of this 0 20 with the total body volume of the rat (100-150 g), this
equals about 55 nmols/sec/animal, which for a -4 g liver gives about 14 pM/sec.

52
component of phenylalanine metabolism. This adds support to the proposal that a transient
form of hyperphenylalaninemia not yet associated with any of the known defects in
aromatic amino acid hydroxylation24•25 could be due to an absence of this activity. Having
a ready source of the C4a,N5-hydrate substrates has enabled us to develop a sensitive assay
which is linear with dehydratase activity. This will facilitate the evaluation and kinetic
characterization of the defect in patients suspected of being deficient in this enzyme.

ACKNOWLEDGMENTS

This work was supported by NIH grants GM30368 and NS26662. SRB was recipient of
a Juvenile Diabetes Foundation summer research fellowship.

REFERENCES

1. G. Embden and K. Baldes, Ober den Abbau des Phenylalanins in Tierischen Organismus, Biochim.
Z. 55:301-322 (1913).
2. S. Kaufman, The Structure of the Phenylalanine-Hydroxylation Cofactor. Proc. Nat/. Acad. Sci.
USA 50:1085-1093 (1963).
3. S. Kaufman, Studies on the Structure of the Primary Oxidation Product Formed from
Tetrahydropteridines during Phenylalanine Hydroxylation, J. Bioi. Chern. 239:332-338 (1964).
4. R.G.H. Cotton, Inborn errors of pterin metabolism, in "Folates and Pterins", Vol.3 R.L. Blakley and
V.M. Whitehead, eds. Wiley & Sons, New York, (1986).
5. S. Kaufman and D.B. Fisher, Pterin-requiring aromatic amino acid hydroxylases, in "Molecular
Mechanisms of Oxygen Activation, 0. Hayaishi, ed., Academic Press, New York, (1974).
6. S.W. Bailey, S.T. Weintraub, S.M. Hamilton, and J.E. Ayling, Incorporation of Molecular-Oxygen
into Pyrimidine Cofactors by Phenylalanine-Hydroxylase, J. Bioi. Chern. 257:8253-8260 (1982).
7. T.A. Dix, G.E. Bollag, P.L. Domanico, and S.J. Benkovic, Phenylalanine Hydroxylase: Absolute
Configuration and Source of Oxygen of the 4a-Hydroxytetrahydropterin Species, Biochemistry
24:2955-2958 (1985).
8. R.A. Lazarus, S.J. Benkovic, and S. Kaufman, Phenylalanine Hydroxylase Stimulator Protein is a
4a-Carbinolamine Dehydratase, J. Bioi. Chern. 258:10960-10962 (1983).
9. R.A Lazarus, C.W. DeBrosse, and S.J. Benkovic, Phenylalanine Hydroxylase: Structural
Determination of the Tetrahydropterin Intermediates by Carbon-13 NMR Spectroscopy, J. Am.
Chern. Soc., 104:6869-71 (1982).
10. J. Haavik, K.A. Andersson, and T. Flatmark, Native and Phosphorylated Bovine Adrenal Tyrosine
3-Monooxygenase. Interactions with Tetrahydropterins and Substrate and Stability of the Formed
4a-Hydroxy-Tetrahydrobiopterin, Pteridines 1:11-16 (1989).
11. M.D. Davis, and S. Kaufman, Evidence for the Formation of the 4a-Carbinolamine During the
Tyrosine-dependent Oxidation of Tetrahydrobiopterin by Rat Liver Phenylalanine Hydroxylase,
J. Bioi. Chern. 264:8585-8596 (1989).
12. S.W. Bailey, R.Y. Chandrasekaran, and J.E. Ayling, Synthesis of Tetrahydropteridine
C6-Stereoisomers, Including N5-Formyl-(6S)-tetrahydrofolic Acid, J. Org. Chern. 57:4470-4477
(1992).
13. S.W. Bailey and J.E. Ayling, 6,6-Dimethylpterins: Stable Quinoid Dihydropterin Substrate for
Dihydropteridine Reductase and Tetrahydropterin Cofactor for Phenylalanine Hydroxylase,
Biochemistry 22:1790-1799 (1983).
14. S.W. Bailey, S.R. Boerth, and J.E. Ayling, manuscript in preparation (1993).
15. A. Albert, Covalent hydration of pteridines, in "Pteridine Chemistry", W. Pfleiderer and
E.C. Taylor, eds. Macmillan Co., New York (1964).
16. S. Kaufman, Phenylketonuria: biochemical mechanisms, in "Advances in Neurochemistry",
B.W. Agranoff and M.H. Aprison, eds., Plenum Press, New York, (1977).
17. S. Milstien and S. Kaufman, Studies on the Phenylalanine Hydroxylase System in Vivo, J. Bioi.
Chern. 250:4782-4785 (1975).
18. K.N. Antonas, W.F. Coulson, and J.B. Jepson, Simulation of Phenylketonuria in Rats by Extended
p-Chlorophenylalanine Treatment, Biochem. Soc. Trans. 2:105-107 (1974).

53
19. M.A. Lipton, R. Gordon, G. Guroff, and S. Udenfriend, p-Chlorophenylalanine-lnduced Chemical
Manifestations of Phenylketonuria in Rats, Science 156:248-250 (1967).
20. M.B. Youdim, B. Mitchell, H.F. Woods, The Effect of Increasing Phenylalanine Concentration on
Phenylalanine Metabolism in Perfused Rat Liver, Biochem. Soc. Trans. 3:683-684 (1975).
21. B.A. Citron, M.D. Davis, S. Milstien, J. Gutierrez, D.B. Mendel, G.R. Crabtree, and S. Kaufman,
Identity of 4a-Carbinolarnine Dehydratase, a Component of the Phenylalanine Hydroxylase System,
and DCoH, a Transregulator of Homeodomain Proteins, Proc. Natl. Acad. Sci. USA 89:11891-11894
(1992).
22. C.R. Hauer, I. Rebrin, B. Thony, F. Neuheiser, H.C. Curtius, P. Hunziker, N. Blau, S. Gisla, and
C.W. Heizmann, Phenylalanine Hydroxylase-stimulating Protein/Pterin-4a-carbinolarnine
Dehydratase from Rat and Human Liver, J. Bioi Chern. 268:4828-4831 (1993).
23. M.D. Davis, S. Kaufman, and S. Milstien, Distribution of 4a-Hydroxytetrahydropterin Dehydratase
in Rat Tissues - Comparison with the Aromatic Amino Acid Hydroxylases, FEBS Letters 302:73-76
(1992).
24. H.C. Curtius, C. Adler, I. Rebrin, C. Heizmann, and S. Ghisla, 7-Substituted Pterins: Formation
During Phenylalanine Hydroxylation in the Absence of Dehydratase, Biochem. Biophys. Res.
Commun. 172:1060-1066 (1990).
25. M.D. Davis, S. Kaufman, and S. Milstien, Conversion of 6-Substituted Tetrahydropterins to
7-Isomers via Phenylalanine Hydroxylase Generated Intermediates, Proc. Natl. Acad. Sci. USA
88:385-389 (1991).

54
STRUCTURE FUNCTION STUDIES OF THE PHENYLALANINE
HYDROXYLASE ACTIVE SITE AND A SUMMARY OF STRUCTURAL
FEATURES

R.G.H. Cottont, D.W. Howells2, J.A. Saleeba3, I. Dianzani4 P.M

Smooker and I. G. Jennings 1
1Murdoch Institute, Royal Children's Hospital, Parkville 3052 Australia
2Austin Hospital, Heidelberg 3084 Australia
3Dartmouth College, Hanover NH 3755-3576 USA
4Istituto Di Clinica Pediatrica, Torino Italy

There is a paucity of detailed structural information on mammalian

phenylalanine hydroxylase (PAH) mainly due to the fact that this enzyme has been
difficult to crystallize. Equally there is a paucity of knowledge of the amino acids
residues necessary for function. The main structural features described so far are
shown in Table 1.

Table 1 - Known Structural Features

1. Iron Content (1 mole Fe/mole PAH subunit)

2. Chymotrypic fragment (catalytic) and regulatory tail
3. Phosphorylation of regulatory sequence at Ser 16
4. Lys 198 and Cys 236 reactions
5. Putative pterin binding site (PPBS)
6. Homology with other aromatic amino acid hydroxy lases

It has been known for some time that PAH contains iron (1) however there is as
yet no indication where the iron is bound although the concentration of iron ligands
from amino acids 263 to 289 makes this a likely iron binding location. Once all three
aromatic amino acid hydroxylases were cloned, sequence comparisons allowed
localization of a strongly conserved region in theN terminal portion of the C terminal
half of the molecule (2) and see below). Limited chymotryptic digestion produces a
36Kd fragment which retains PAH catalytic activity but is unaffected by
phosphorylation or phenylalanine activation (3). This cleavage removes 11Kd from the
N-terminal of PAH including the phosphorylation site Ser 16 (4) and 5Kd from the
C-terminus of PAR. More recently a photoaffinity pterin analogue was used to label
PAH from which a labelled peptide was isolated with subsequent identification of lys
198 as the labelled amino acid (5). The same paper describes identification of the
reactive cysteine residue associated with activation of PAH enzyme activity (5). Also
use of a pterin mimicking antiidiotype antibody has allowed the identification of a 27
amino acid peptide which binds pterin (6). This peptide is referred to as the putative
BH4 binding site (PPBS) and is in the same region as the concentration of potential iron
binding ligands mentioned above. The sequence of this peptide is as follows:

263 HCTQYIRHGSKPMYTPEPDICHELLGH 289

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 55
 Table 2 - Domains of Phenylalanine Hydroxylase
DOMAIN HOMOLWITH AMINO ACIDS EXONS CHARGE CHANGE FEATURES
TYH WRTTYH

A 20 1-116 (117) 1,2,3 Radical Chymo

Digested

B 58 117-183 (66) 4,5,6 (25%) 14% Charge Density 30%

PHS

c 65 184-240 (56) 6 (75%) 18% Charge Density 20%

Lys 197,
2X Cons CYS

D 87 241-334 (93) 7,8,9,10 (33%) 4% PPBS

3X Cons Cys

E 40 335-452 (117) 10 (67%) 11,12,13 17% Chymo Digested

Consideration of the whole PAH sequence and its relationship to that of tyrosine
hydroxylase (TYH) has suggested a series of 5 domains within the linear sequence of
PAH (Table 2).

Domain A has a radically different sequence to TYH and has only 20%
homology. This domain ends near the N-terminal chymotrypic and V8 protease
cleavage sites (6). Domain B stretches through a region of 30% charged amino acids
(66 amino acids) with an abrupt change to 20% at the start of Domain C. Domain
B&C have 58 and 65% homology with respect to TYH. They also have 14&18%
charge changes with respect to TYH respectively. Domain D is the most conserved
(87%) with respect to TYH and is marked by only a 4% charge change with respect to
TYH over the 93 amino acids. Domain E contains the remaining 117 amino acids and
is 40% homologous to TYH and has 17% charge changes with respect to TYH. About
half of this domain is removed on chymotrypic digest. These features can be seen
mapped together with monoclonal antibody binding sites in Fig 1.

Domain A B c D E

TyH
9 7
I
(PJ

I
v
te {! •• ~
PPBS V
t
homology(%) 1 20 I 58 65 87 40

Residue *I *
100 *
200 *
300 *
400 *
452

Figure 1. Map of features of phenylalanine hydroxylase showing 5 domains based on

comparison with tyrosine hydroxylase. Numbers represent monoclonal antibody
binding sites, V: limits of V8 protease fragment, arrows pointing down: limit of
chymotryptic fragment, K lysine 197, C cysteine 236, (P) phosphorylation site, PPBS
putative BH4 binding site, arrows pointing up: conserved cysteine residues.

The map shows that the putative pterin binding site maps in domain D, the 87%
conserved region. The conserved cysteine residues map in Domains C&D. V8
protease digestion leaves a core of domain B,C and 2/3 of D. Lysine 198 is in domain
C well away from the PPBS and must be brought into its vicinity by folding. The
cysteine labelled on activation of PAH is that at 236 (5).

56
 We have initiated a range of studies to further define the amino acid residues
binding tetrahydrobiopterin. These include (a) alteration and deletion of amino acids of
a synthetic peptide corresponding to PPBS (see above) (b) sequence comparisons with
other known pterin binding enzymes and (c) in vitro mutagenesis.
Cleavage of the PPBS peptide with cyanogen bromide at met 275 renders the
two pieces (mixed or alone) incapable of binding pterin. Removal of 2 N terminal
amino acids also eliminates binding except under certain conditions, but removal of 3
amino acids from the C terminal only reduces binding 40%. However removal of a
further 4 amino acids from the C-terminal end renders the peptide inactive (in
preparation). This suggests (a) that amino acids at each end of the peptide may be
needed for pterin binding and (b) secondary structure of this peptide may be involved.
In the search for a motif which may bind reduced biopterin derivatives in
proteins we used the sequence of PPBS to search the sequence of proteins in the data
base (Howells et al submitted) (Fig 2).

FIRST SEQUENCE
RESIDUE

Figure 2. Comparison of sequences of rat PAH, TYH, DHPR and GTPGH aligned to
show regions demonstrating a common motif. A synthetic peptide corresponding to
PAH peptide 263-289 has been shown to bind reduced pterins (6).

As expected phenylalanine, tyrosine and tryptophan out hydroxylases were

homologous in the region of PPBS. However with dihydropteridine reductase (DHPR)
we found 4 conserved residues (showing conservation with PAH as reference). Four
others were present with either conservative or space changes. This conservation
extended C terminal from PPBS for another 3 conserved amino acids with a further 5
conservative changes and two single space changes showing. We believe this both
supports the contention that PPBS binds tetrahydrobiopterin and indicates this identified
region of DHPR binds the pterin substrate. This latter finding is supported by the fact
that when this region of DHPR equivalent to PPBS is synthesized it is bound by the
pterin mimicking antibody (Howells et al submitted), although this binding is not
inhibited by the addition of pterin.
We also found a motif in rat GTP cyclohydrolase which binds
tetrahydrobiopterin as an end product inhibitor (7). (Fig 2). However the homology is
only to DHPR and is stronger in the C terminal direction from PPBS. PAH and
GTPCH have only 2 identical residues plus 5 with spacing or conservative changes.
Currently we are performing in vitro mutagenesis of those amino acids
conserved between PAH and DHPR followed by mutant protein expression with a view
to defining the pterin binding amino acids.
Concerning iron binding, it is possible that those amino acids such as histidine,
tyrosine and cysteine which bind iron in other systems and which are conserved in the
hydroxylases, but not in DHPR, may be those which bind iron in PAH.

REFERENCES
1. D. B. Fisher, R. Kirkwood and S. Kaufman, 1. Biol. Chern. 247:5161-5167 (1972).
2. H.E. Grennet, F.D. Ledley, L.L. Reed and S.L.C. Woo, PNAS 84:5530-5534 (1987).
3. D.B. Fisher and S. Kaufman, 1. Biol. Chern. 248:4345-4353 (1973).
4. M. Wretbom, E. Humble, U. Ragnarsson and L. Engstrom, Biochern. Biophys. Res. Comrnun.
93:403-408 (1980).
5. B.S. Gibbs and S. Benkovic, Biochem. 30:6795-6802 (1991).
6. I.G. Jennings, B.E. Kemp and R.G.H. Cotton, PNAS 88:5734-5738 (1991).
7. G. Schoedon, V. Redweik, F. Gerhard, R.G.H. Cotton and N. Blau, Eur. 1. Biochern.
210:561-568 (1992).

57
EXPRESSION OF WILD TYPE AND MUTANT FORMS
OF HUMAN PHENYLALANINE HYDROXYLASE IN E. COLI

PerM. Knappskog1•2, Hans G. Eiken 1, Aurora Martinez2, Sigridur

Olafsdottir, Jan Haavik2, Torgeir Flatmark2 and Jaran ApoW

1Department of Medical Genetics, Haukeland Hospital

2Department of Biochemistry and Molecular Biology
University of Bergen, N-5021/N-5009 Bergen, Norway

INTRODUCTION

Phenylketonuria (PKU) is an autosomal recessive disease caused by the absence or

severely reduced enzymatic activity of the hepatic enzyme phenylalanine hydroxylase
(phenylalanine 4-monooxygenase, EC 1.14.16.1, PAH). The loss of enzymatic activity found
in PKU patients is a result of single base substitutions or small deletions in the PAH gene.
Presently, more than 70 different mutations associated with the disease are known. PKU and
non-PKU hyperphenylalaninemia (HPA) patients show extensive clinical heterogeneity. To
understand the molecular mechanism of this heterogeneity, it is necessary to characterize the
structural and functional properties of the different mutant PAH forms. Liver biopsies from
PKU/HPA patients are usually not available, but in vitro expression in eukaryotic cells has
been used for the characterization of certain mutant forms. However, it has been a problem
to express stable forms of PAH in such in vitro systems 1, and therefore only a few mutant
forms have been further characterized so far. This led us to clone and express the normal
and several mutated forms of human PAH eDNA into the pET-plasmid system of E. coli
(Novagen). Here we report the expression of both normal and seven disease associated forms
of PAH, i.e. mutations in exon 7 and 8 of the PAH gene.

METHODS

Subcloning of human PAH eDNA into pETlld

To obtain an appropriate distance between the Shine-Dalgamo sequence and the start
codon, ATG, a Nco! restriction site was created by PCR-based mutagenesis in PAH. This
was performed by amplification of PAH eDNA, using the mismatch primer, A-N col, and
the normal primer B3. The PCR product was treated with proteinase K2, digested with
Ncoi/BamHI, and the resulting DNA fragment corresponding to PAH eDNA position 222-
1036 was ligated into the Ncoi/BamHI digested plasmid pET11d. Then, the remaining 1393
bp fragment of PAH eDNA corresponding to position 1036-2429 was cut out of phPAH247 3

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 59
with BamBI and EcoRI, and ligated into the recombinant plasmid (also digested with
BamHI/EcoRI) from the first cloning step.

Site specific mutagenesis of PAH

The Ncoi site introduced in the initial PCR cloning step altered the second codon in
PAH from serine to alanine. In order to restore the wild type PAH codon 2 and introduce
the mutations in exon 7 and 8 of the normal PAH, a PCR-based procedure4 was used. The
introduction of each mutation was verified by DNA sequencing. The oligonucleotides used
for PCR mutagenesis are shown in Table 1.

Production of recombinant forms of human PAH in E. coli

Bacterial cultures, HMS 174 DE3, were grown at 37 °C in LB-medium containing 50

pg/ml of ampicillin. The T7 RNA polymerase was induced at an A590 nm of 0.6 with 1 mM
IPTG. After 2 h of induction the bacteria were harvested. The bacteria were disrupted by
passage through a French press at 69 MPa and diluted in 20 mM Tris/maleate containing
1 mM PMSF, 1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol, 10 mM benzamidine, and 2
pg/mlleupeptin, pH 7.5. This fraction was then centrifuged at 100 000 x g at 4 oc for 10
min and the supernatant was referred to as crude extract.

Assay of P AH activity

PAH activity was assayed (t = 5-30 min) at 25 oc with 0.05 M K-phosphate pH 6.8,
5 mM L-Phe, 0.6 mg/ml catalase, 100 pM Fe(II), 5 mM DTT and 6-methyltetrahydropterin
(6-MPH4) or (6R)-tetrahydrobiopterin (BH4). The reaction was stopped by adding 1 % (v/v)
acetic acid in ethanol, and after centrifugation tyrosine was measured by HPLC and
fluorimetric detection 5•

Table 1. Oligonucleotides used for PCR mutagenesis

Primer Sense eDNA position Sequence (5'-3')

A-Ncol Forward 208-226 CCCGGGGAGCCACCATGG£

A2S Forward 217-229 GAAGGAGATATACCATGTCC
A243X Forward 932-942 GCACTGGTTTCCGCCTCTGA
A252W Forward 959-978 CTGGCCTGCTTTCCTCTTQ
A259T Forward 980-1000 ATTTCTTGGGTGGCCTGACC
A259V Forward 981-1001 TTTCTTGGGTGGCCTGGTCT
A261Q Forward 987-1006 GGGTGGCCTGGCCTTCCAA
A280K Forward 1043-1063 AGCCCATGTATACCCCCAAA
A299C Forward 1101-1121 GTTTTCAGATCGCAGCTGTG
A2 Forward 674-694 ATCCTGTGTGTACCGTGCAAGC
T7 Forward pET-promotor TAATACGACTCACTATAGGG
B2 Inverse none CTGCCCATTCCTCATGTAGA
B3 Inverse 1063-1083 CAGCTCATGGCAGATGTCAG
B40I Inverse 1078-1098 CTGCCCATTCCTCATGTAGA-
GGCACATGTCCCAACAGCTC
B40II Inverse 1523-1543 CTGCCCATTCCTCATGTAGA-
TTTCACTGTTAATGGAATCA

The underlined sequences correspond to PAR eDNA.

Primer B2 is identical to the 20 first nucleoli des of primer B40I and B40II6•

60
RESULTS AND DISCUSSION

The wild type recombinant PAH, expressed with high yield (about 10 % of total
soluble cellular protein, revealed high catalytic activity, and was immunoreactive in Western
blot analysis using affinity purified polyclonal rabbit anti-rat PAH antibodies. The mutations
introduced and expressed in the pET-system were situated in the eDNA area corresponding
to exon 7 and 8, and represent mutations occurring in patients with PKU/HPA. The mutated
enzymes were recovered with similar yields and had catalytic activities (homospecific
activities) ranging from zero to 47 % (6-MPH4 as the cofactor) or 32 % (BH4 as the
cofactor) of the wild type recombinant PAH activity (Table 2).

Table 2. In vitro activity of wild type (wtPAH) and mutant PAH proteins

Specific activity %Activity

nmol tyr/min/mg PAir

Sample 6-MPH4 BH4 6-MPH4 BH4 COS cellsb

(0.5 mM) (75 pM)

wtPAH 4221 1842 100 100 100

R243X 0 ND 0 ND <16
R252W 23 ND 0.5 ND 07
A259V 9 8.5 0.2 0.4 ND
A259T 13 15 0.3 0.8 ND
R261Q 2090 591 47 32 30'·1
E280K 41 12.5 0.9 0.7 <31
F299C 0 0 0 0 <38

•The amount of PAH protein in crude extracts was measured by densitometric scanning of Western blots, using
purified rat PAH as standar<f. The activities represent the average of at least three experiments (SD < 10 %).
~iterature values.
'The R261Q-protein of COS cells is reported to give a specific activity close to the wild type
protein, but the amount of immunoreactive material was only about 30 % of that observed for the
wilde type PAH7•
ND = not determined

Western blot analysis of the crude extracts with anti-rat PAH antibodies gave two
major bands of slightly different Mr-values, the main band at about 50 kDa (shown in Fig.
1 for wild type PAH). The lower M. forms are most likely due to intracellular proteolysis,
and partial purification of the two main immunoreactive proteins by DEAE-chromatography
revealed no significant difference in their specific enzymatic activity. High level of
expression was also found for the mutant forms (PAH protein was 5-10 % of total soluble
protein).
The high recovery of the recombinant forms of PAH allowed precise activity
measurements, even for the mutant forms with extreme low activity (Table 2). Homo specific
activities lower than 0.3 % of that of wild type could reproducibly be measured (i.e. for the
mutations A259V/A259T). This finding explains the problem faced with in the reliable assay
of PAH activity in liver biopsies from PKU patients 10 •

61
 l 2

Figure 1. Expression of PAH in E. coli. Western blot of 0.7 Jig purified rat PAJI9 (lane 1) and crude extract
of induced bacteria containing the pETPAH (wild type) plasmid (lane 2), using affinity purified polyclonal
rabbit anti-rat PAH antibodies.

A comparison between published data on eukaryotic COS cell PAH expression and our
results from the prokaryote system is made in Table 2. In the COS cell system no activity
could be measured for the mutations R252W and E280K. The measurements were carried
out with very small amounts of gene product in the COS cells 1• With the pET system,
however, the low activity detected for these mutants can be directly related to low-functional
gene products since the activities are measured with large amounts of expressed protein. It
has been reported that the homospecific activity of the mutant R261Q is similar to that of
the wild type when both are expressed in COS cells 1, but the amount of functional protein
detected was 70 % lower for the mutant than for the wild type. Thus, the differences in
activity were related to instability of the R261Q PAH protein. In the pET/E. coli expression
system, in which we have a similar degree of expression for both this mutant and the wild
type form, we have been able to measure a homospecific activity of 32 % of that of wild
type (Table 2), indicating that this mutation in fact affects the catalytic function of the
protein. Due to the high level of expression, the pETPAH-system thus represents an
important source to obtain both normal and mutated forms of PAH for further studies on the
relation between structure and function of this enzyme.

Acknowledgements-We are grateful to Professor S.L.C. Woo for supplying the PAH eDNA
clone. This study was supported in part by the Research Council of Norway.

REFERENCES
1. Y. Okano, R.C. Eisensmith, F. Guttier et a!., New Eng. f. Med. 324:1232 (1991).
2. J.S Crowe, H.J. Cooper, M.A. Smith et al., Nucleic Acid Res. 19: 184 (1991).
3. S.C.M. Kwok, F.D. Ledley, A.G. DiLella et al., Biochemistry 24:377 (1985).
4. R.M. Nelson and G.L. Long, Anal. Biochem. 180:147 (1 989).
5. A.P. D¢skeland, S.O. D(llskeland, D. 0greid eta!. , J. Bioi. Chem. 259:11242 (1984).
6. T. Wang, Y. Okano, R.C. Eisensmith et al., Somal. Cell. Mol. Genet. 16:85 (1990)
7. Y. Okano, T. Wang, R.C. Eisensmith et al., Genomics 9:96 (1991).
8. R .C. Eisensmith and S.L.C. Woo, Hum. Mut. 1:13 (1992).
9. R. Shiman, D.W. Gray, and A. Pater, J. Bioi. Chem. 254:11300 (1979).
10. P.A. Friedman, D.B. Fisher, E.S. Kang et al., Proc. Nat/. Acad. Sci. USA 70:552 (1973).
62
A RE-EXAMINATION OF THE METAL REQUIREMENT OF
CHROMOBACTERIUM VIOLACEUM PHENYLALANINE HYDROXYLASE

Robert T. Carr and Stephen J. Benkovic

Department of Chemistry
The Pennsylvania State University
University Park, PA 16802

Phenylalanine hydroxylase (PAH) is the best characterized member of the family of

pterin- dependent mono-oxygenases that includes tyrosine hydroxylase and tryptophan
hydroxylase. All of the mammalian forms of these enzymes have a non-heme iron required
for activity 1,Z,3 • Though no direct experimental evidence ascribes a mechanistic role to the
metal, it is thought that the metal ion is required to be in its lower oxidation state for
activity4 •5•6.
To address the mechanistic question of metal ion function in pterin-requiring
hydroxylases we have focused on the copper center of the PAH from ChrortWbacterium
violaceum (CVPAH). Pember et al. 4 classified this hydroxylase as a copper metalloenzyme
based on a 1:1 Cu/enzyme stoichiometery and its inhibition by various chelating agents.
Further studies based on pulsed EPR 7, and x-ray absorption8 demonstrated that two histidines
in the enzyme were important copper ligands. ESR spectra with 5- 15N-6-
methyltetrahydropterin (6-MPH4) implicated the pterin cofactor as a ligand9.
The present study was initiated to answer questions concerning the redox potential and
binding affinity of the copper center; however, the results of a variety of experiments implied
that the classification of CVPAH as a copper metalloenzyme might have been premature. To
the contrary, the results presented here do not support a catalytic role for the copper center.

Materials and Methods

Recombinant CVPAH expressed in E. coli was purified with the inclusion of 0.6 mM
copper as described previously10. Protein was assayed by the BCA method (Pierce) using
albumin standards. The activity of CVPAH was assayed as described except a molar
extinction of 2.6 mM- 1 cm- 1 was used instead of 1.7 mM- 1 cm-1 which was originally
determined with 6MPH 4 in phosphate buffer at pH 6.8 for rat liver PAH (RLPAH). The
difference is that under these conditions the DTT induces formation of 7,8 dihydro 6,7
dimethylpterin (DMPH2) from quinonoid DMPH 2, which contributes to the change in
absorbance at 275 nm. The pterin oxidation assay of Ayling et al. 13 was used for measuring
activity in the absence of DTT. Reverse phase HPLC with isocratic elution with water was
used to directly quantitate the tyrosine produced in the reaction. The 4a-hydroxypterin product
was quantitated in 8 mM TRIS buffer (pH 8.45) by extrapolating the initial rate of formation to
the point where the reaction was quenched with SDS for tyrosine assay. A ~e for 6-MPH 4

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 63
 Table 1. Activity of"Copper-Free" CVPAH

Sample Cu/Enzymei Specific Activity (units/mg.)2

Control3 1.18 I2.2

I O.OI4 I1.3
2 0.010 II.O
3 0.021 I2.4

I Copper determined by AA spectrometry. 2 Standard assay conditions (see methods).

3 Enzyme taken through all extraction steps omitting DTI.

to 4a-OH-6MPH 4 of 7050 M-Icm-I at 257 nM was calculated from standard spectra (Lazarus
et al., 1983; 15). This wavelength is isosbestic for the background reactions of pterin and DTI
oxidation.
Copper was removed from the enzyme by extraction with DTI. Routinely 20-30 mg of
enzyme in 15 ml HEPES buffer (pH 7.4) was made 0.1 M in DTI and incubated for 10 min.
This solution was then concentrated by a factor of 20 using a Centriprep 10 (Amicon)
centrifugal ultrafilter, and then rediluted with fresh 0.1M DTI to make up the original15 ml
volume. The concentration and redilution was repeated two more times. To remove the DTI,
the final concentrate (-1 ml) was then applied to a 15 em Sephadex G-25 (Pharmacia) column
which had been washed extensively with "metal-free" 50 mM HEPES buffer.
The copper content of the enzyme was routinely determined by atomic absorption, and
was periodically double-checked colorimetrically using bathocuproine disulfonate (e 483 =
12,250) in 50 mM ascorbate. Atomic absorption was also used to detect the presence of iron,
cobalt, nickel, chromium, and zinc.
Results
The first indication that the copper in CVPAH might not be required for activity
occurred when we found that DTT readily removed the copper from the enzyme, even though
previous observations had shown that prolonged incubation with DTI caused no loss in
activity. The extraction procedure gave "copper-free" enzyme with residual copper between 40
and 100 ppb, or an average Cu/enzyme ratio of 0.015 (Table 1). The overall yield of "copper-
free" enzyme is about 80%.
The specific activity of the recombinant CVPAH is generally 10-19 units per mg 10.
The specific activity of the "copper-free" preparations averaged ca. 11 units/mg (Table 1),
which is approximately 85% of a control taken through the Cu extraction steps but omitting the
DTI chelator. Side by side comparison of"copper-free" and copper complexed CVPAH gave
identical Michaelis constants for both phenylalanine and pterin substrates.
The ratio of the total amount of copper in the entire reaction mixture to the moles of
enzyme used amounted to only 0.09, whereas the activity was 85% of that of the copper-
complexed control. The addition of the copper chelators bathocuproine and neocuproine had
no inhibitory effect on the activity of either "metal-free" or copper complexed CVPAH up to a
concentration of 20 11M chelator, beyond which the high background absorbance at 275 nm
interferes with the assay. Pember et al.I5 noted a moderate inhibition by these chelators at
higher concentrations but this may have been due to an interference with the assay. Cyanide

Table 2. Reaction stoichiometery

Phenylalanine Oxygen Tyrosine 4a-hydroxypterin/tyrosine

IOOJ.l.M 103J.1.M 10I J.l.M 1.05

Phenylalanine was limiting. Oxygen was determined by oxygen electrode,

tyrosine by HPLC, and 4a-hydroxypterin spectrophotometrically (see methods).

64
 Table 3. Activity of CVPAH Under Non-reducing Conditions

Sample Specific Activity (units/mg.)1

in HEPES (pH 7.4) in Imidazole (pH 7.5)

Cu-complexed CVPAH 0.2 3.3

"Copper-free" CVPAH 4.1 3.4

1 Assay by the pterin oxidation method (see methods).

also had no inhibitory effect at millimolar concentrations. EOTA, listed by Pember et al.l5 as
an inhibitor of CVPAH, must inhibit the enzyme by a mechanism other than copper removal
since we fmd no activity restored by addition of excess copper over EOTA.
Table 2 shows the tight coupling between the substrates and products of the reaction
with "copper-free" enzyme. For each phenylalanine molecule consumed one oxygen molecule
is consumed and one tyrosine molecule is formed. Also under conditions where the 4a-
hydroxypterin can be measured the ratio of its formation to tyrosine produced is unity.
Pember et al. 4 reported that the copper center of CVPAH must be reduced for activity,
requiring a relatively strong reducing agent such as dithionite or a thiol. OTT, which is used in
the normal assay mixture, provided an additional activation that increased the rate of
hydroxylation about tenfold over enzyme pre-reduced with dithionite.
If "copper-free" enzyme is indeed active without the need for copper then a copper
reducing agent should not be necessary. In fact, the specific activity of "copper-free" CVPAH
in the pterin oxidation assay, which does not contain OTT, is -4 units/mg (Table 3). This is
more than 20 times the activity of eu2+-complexed enzyme measured in the same assay.
When copper is added back to the "metal-free" enzyme the activity decreases proportionately.
When the assay is run in 20 mM imidazole buffer the rate is the same for both "Cu-free" and
Cu complexed CVPAH (Table 3). These rates are slower than those measured by the 275 nm
assay that includes OTT by a factor of 2-3. The reason for this is not clear but might involve
inhibition by residual metal or aggregation of CVPAH in the absence of OTT.
The titration of "Cu-free" CVPAH with Cu2+ was monitored fluorometrically and
produced a binding curve that follows simple saturation behavior (Fig. 1). At pH 7.4
computer fit gives a Kd value of 0.5 IJ.M.

Discussion
The copper Kd reported here indicates a rather modest affinity compared to most copper
enzymes ( "blue" copper proteins for example have affinities greater than can be determined by
standard techniques).
The results here in fact do not support a requirement for copper. The main evidence is
that CVPAH with a Cu/enzyme ratio ofO.Ol is nearly as active (at least 85%) as enzyme with a
Cu/enzyme ratio of 1.2. The possibility that "metal-free" CVPAH is being reconstituted by
copper in the assay solutions is disproved by the finding that the ratio of total copper to
enzyme in the assay mixture was only 0.07, whereas the activity was within 85% of a fully Cu
constituted control.
Copper then, instead of being a requirement for activity, should be thought of as an
inhibitor that is removed by the thiol for enzyme activation. The hysteretic product
accumulation seen when the assay is initiated by OTT was thought to be due to reductive
activation of the copper center (5). It now appears that the lag in product accumulation is
related to the activation of the enzyme by the ability of OTT to remove and sequester the
copper from the enzyme. Binding of the substrates blocks this site causing a much slower
activation when the thiol is added after the substrates. This hypothesis fits very well with the
observation that the "metal-free" enzyme is active without a thiol or other reducing agent
whereas the copper complexed form is not, and is further confirmed by the finding that
imidazole, a non-reducing metal ligand, is capable of activating copper inhibited enzyme.
If CVPAH is not a copper enzyme, is it in fact a metalloenzyme at all? Iron, nickel and
cobalt, the most likely metals to perform an electron transfer-type role proposed originally for

65
 4000

3500
Kd=0.5 j..LM
3000
g
Q)

Q)
() 2500
"'
~
0
::s 2000
~
1500

1000

500
0 1 2 3 4 5
j..LMCopper
Figure 1. Copper (II) Kd at pH 7.4

the copper in CVPAH, were not found in significant proportions in the "metal-free" CVPAH
(less than 3 mole%), and all are inhibitory. The simplest conclusion then is that this enzyme
can perform the hydroxylation reaction without the need of any redox active metal. This
immediately excludes any oxygen-metal intermediate including a hypervalent "cupryl" ion
(Cu=O)+ or a metal-pterin peroxo species. For this enzyme then, a pterin hydroperoxide
remains as the most likely activated oxygen intermediate.
The relevance of this study to the mammalian pterin dependent hydroxy lases is founded
on the structural and functional similarity between CVPAH and its mammalian counterpart,
RLPAH. Structurally the bacterial enzyme is smaller and less complex but has a sequence
homology that would indicate that the two enzymes share important structural elements. There
is, for example, a conserved region that could be the metal binding site 10. Functionally both
enzymes perform the same reaction on the same substrates with only slight differences in
binding affinities. The enzymatic rates are nearly the same when the two are compared on a
subunit basis, though the regulation of RLPAH is more complex4. Taken together then, it is
likely that these enzymes share a common mechanism, and our data would question the
intermediacy of iron-oxygen species.

REFERENCES
1 Gottschall, D. W., Dietrich, R. F., Benkovic, S. J., & Shiman, R. (1982) 1. Bioi. Chem. 257, 845-849.
2 Dix, T. A., Bollag, G. E., Domanico, P., & Benkovic, S. J. (1985) Biochemistry 24, 2955-2958.
3 Kuhn, D. M., & Lovenberg, W. (1985) in Folates and Pterins (Blakely, R. L., & Benkovic, S. J., Eds.) Vol. 2,
pp 353-382, Wiley-Interscience, New York.
4 Pember, S. 0., Villafranca, J. J., & Benkovic, S. J. (1986) Biochemistry 25, 6611-6619.
5 Wallick, D. E., Bloom, L. M., Gaffny, B. J., & Benkovic, S. J. (1984) Biochemistry 23, 1295-1302.
6 Marota, J. J. A., & Shiman, R. (1984) Biochemistry 23, 1303-1311. Academic Press, New York.
7 Pember, S. 0., Benkovic, S. J., Villafranca, J. J., Pasenkiewicz-Gierula, M., & Antholine, W. E. (1987a)
Biochemistry 26, 4477-4483.
8 McCracken, J., Pember, S. 0., Benkovic, S. J., Villafranca, J. J., Miller, R. J., & Peisach, J. (1988)
J. Am. Chem. Soc. 110, 1069-1074.
9 Blackburn, N. J., Strange, R. W., Carr, R. T., & Benkovic, S. J. (1992) Biochemistry 31, 5298-5303.
10 Onishi, A., Liotta, L. J., & Benkovic, S. J. (1991) J. Bioi. Chem. 266, 18454-18459.
11 Miller, M. R., McClure, D., and Shiman, R.(1975) J. Bioi. Chem. 250, 1132
12 Ayling, J., Pirson, R., Pirson, W., & Boehm, G. (1973) Analytical Biochemistry 51, 80-90.
13 Lazarus, R. A., Dietrich, R. F., Wallick, D. E., & Benkovic, S. J. (1981) Biochemistry 20, 6834-6841.
14 Dix, T. A., & Benkovic, S. J. (1985) Biochemistry 24, 5839-5846.
15 Pember, S. 0., Villafranca, J. J., & Benkovic, S. J. (1987b) Methods Enzymol. 142, 50-56.

66
HISTIDINES 138 AND 143 ARE COPPER BINDING LIGANDS IN
CHROMOBACTERIUM VIOLACEUM PHENYLALANINE HYDROXYLASE

Shankar Balasubramanian,§ Robert T. Carr,§ Christopher J. Bender,t

Jack Peisach, t and Stephen J. Benkovic§
§Department of Chemistry, The Pennsylvania State University, University
Park, Pennsylvania 16802.
tDepartment of Molecular Pharmacology, Albert Einstein College of
Medicine of Yeshiva University, Bronx, New York 10461

INTRODUCTION
Phenylalanine hydroxylase (PAH) from Chromobacterium violaceum (CV) is known
to bind an equivalent of divalent copper.1 Studies using pulsed EPR spectroscopy2 have
suggested that there are two equatorial imidazoles coordinated to Cu(II) of CV PAH. This
observation has been supported by copper-histidine model complexes of the active site3 and
more recently by X-ray absorption spectroscopy of the copper containing enzyme.4 We
have used a combination of site directed mutagenesis and pulsed EPR spectroscopy to
probe the copper binding site of CV PAH and identify the Cu(II) ligands.

THE HISTIDINE MUTANTS

The gene for CV PAH has been cloned and overexpressed in Escherichia coli.5 The
amino acid sequence of CV PAH contains 17 histidine residues, making the initial choice
of the potential Cu(II) ligand difficult. Alignment of the amino acid sequence of CV PAH
with those of rat liver and human PAHs, both of which bind iron, shows a region of high
sequence homology (Figure 1) containing two conserved histidine residues. Histidines 138
and 143 (as numbered in CV PAH) are the only conserved histidines in the entire amino
acid sequence and were thus considered to be strong candidates for the Cu(II) ligands.
Histidine to serine single mutants were generated from the E. coli expression systemS using
the PCR overlap extension method, 6 and were purified by the published protocol. 7

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 67
 HIS 138 HIS 143

!!
73 (numbering in C. violaceum PAH)

l •• • • ** * * * * * * * *

• denotes homology with eukaryotic tyrosine and tryptophan hydroxylases

Figure l. Alignment of amino acid sequences for CV, rat, and human PAHs showing highly conserved
region.

RESULTS
Catalytic Activity of Mutants
The two mutant PAH enzymes, Hl38S and Hl43S, were assayed for catalytic activity
by two independent methods:a the standard 275 nm UV assay to detect the fotmation of
tyrosine7 and by a more sensitive HPLC assay of the phenylisothiocyanate (PIT) derivative
of any tyrosine produced. We were unable to detect any phenylalanine turnover by either
mutant under conditions that would have detected 0.01 % of the corresponding turnover by
wild type PAH. The mutants remained inactive both with and without the presence of
Cu(II). We were still unable to detect any activity when supplementing the buffer with 30
mM imidazole.

Ability to Bind Copper

All bound copper was first removed from wild type and mutant enzymes by multiple
extractions with 0.1 M DTI followed by removal of residual DTI by gel filtration. The
level of Cu(ll) retained by each protein, after this procedure, was determined to be less than
3 % on the basis of atomic absorption measurements. The intrinsic tryptophan
fluorescence of CV PAH is efficiently quenched by bound divalent copper. The Cu(ll) Kd
values were determined for the mutant and wild type enzymes by fluorescence titration
measurements and are given in Table 1.

Table 1.
Cu(ll) Binding Affinities for CV PAH
Kd {Cu(ll)} I mM

Wild Type 0.5

H138S 0.1
Hl43S 6.0

a Assays were carried out at 25 °C, pH 7.4 (HEPES, 80 mM), and included 4.5 mM CV PAH, 1 mM L-
phenylalanine, 180 mM 6-methyltetrahydropterin, 6 mM DTT and 1 mg/ml catalase.

68
Pulsed EPR Experiments

The Cu(II)-bound mutants H138S and H143S, and the Cu(m-bound wild type enzyme,
were studied using pulsed EPR spectroscopy. Electron spin-echo modulation (ESEEM)
measurements were taken essentially as previously described by McCracken et af.2 to
elucidate any apparent changes in the ligand to Cu(II) interactions due to the histidine
mutations. The insert of Figure 2 shows the ESEEM data for wild type protein (A), and
mutants H143S and H138S (B and C respectively). The resultant Fourier transformed
spectra are presented in the main figure. The key features shared by all three Fourier
transfonned spectra are the three sharp lines near 0.55, 1.00 and 1.55 MHz and a broader
line near 4 MHz. These spectral features are characteristic of the coupling between the
electron spin of the Cu(II) with the remote 14N of coordinated histidyl imidazole.2 The
lower intensity spectral lines at 2.1, 2.6 and 3.2 MHz in the spectrum of wild type protein
(indicated by arrowheads in Figure 2) are combination lines that arise as a consequence of
two or more nuclei being coupled to the electron spin. These combination lines are absent
in the spectra of both mutants H143S and H138S suggesting the presence of only one,
rather than two copper-coordinating histidine.

2
FREQUENCY
(MHz )

Figure 2. ESEEM traces (insert) and Fourier transformed spectra (main figure) of Cu(II) bound to wild type
(A), H143S (B) and Hl38S (C) CV PAHs.

69
DISCUSSION
The most straightforward interpretation of the electron spin echo experiments is that
the Cu(II) of CV PAH is indeed equatorially coordinated by two histidine imidazoles as
previously predicted,2,3 and that histidines 138 and 143 can be assigned as these ligands.
This is the first piece of structural data to be obtained for CV PAH. Clearly the single
mutations were insufficient to remove the protein's ability to bind Cu(II), with the Kd
values not falling by 2-3 orders of magnitude, as might be predicted on loss of a ligand.
The fact that the Kd{Cu(II) } is actually slightly reduced for the mutant H138S suggests
that there are protein ligands, other than histidine, capable of coordinating Cu(II). We
cannot exclude the possibility that the serine amino acid replacement itself may coordinate
the Cu(II). The complete loss of catalytic activity for both mutants can be explained by one
of three possibilities. First, a catalytically essential role for the Cu(II) may have been
disrupted by the removal of a natural ligand. In the light of recent experiments by Carr and
Benkovic8, it now appears that the Cu(II) is not required for enzyme activity, making this
possibility unlikely. Another possibility is that both histidines 138 and 143 themselves play
an essential role in the catalytic mechanism. If this is so, it is currently unclear how the
histidines may be involved. A third possibility is that the integrity of the active site
structure was not preserved by the mutations. We have tested this possibility by carrying
out binding assays for wild type and mutant proteins using radiolabelled L-phenylalanine
and ultrafiltration. The three Kd(phenylalanine) values estimated by this method are all
within a factor of 3 of each other, suggesting that the substrate binding site structure has not
been significantly altered by the mutations. Finally, it is interesting to note that the
corresponding histidine mutants (by sequence alignment) of the rat liver PAH were also
found to show no catalytic activity.9 Given that this pair of histidines are also homologous
to histidines in human PAH and the pterin dependent eukaryotic tryptophan and tyrosine
hydroxylases, it is probable that they play some important, but as yet undetermined, role.

ACKNOWLEDGMENTS
We thank SERC I NATO for a Postdoctoral Fellowship for S.B. and the NSF for
supporting this work.

REFERENCES
1. S. 0. Pember, J. J. Villafranca, and S. J. Benkovic, Biochemistry, 25, 6611-6619,(1986).
2. J. McCracken, S. 0. Pember, S. J. Benkovic, J. J. Villafranca, R. J. Miller, and J. Peisach, J. Am. Chem.
Soc.,llO, 1069-1074 (1988).
3. T. Kohzuma, H. Masuda,andO. Yamauchi,]. Am. Chem. Soc., Ill, 3431-3433 (1989).
4. N.J. Blackburn, R. W. Strange, R. T. Carr, and S. J. Benkovic, Biochemistry, 31, 5298-5303 (1992).
5. A. Onishi, L. J. Liotta, and S. J. Benkovic, J. Bioi. Chem., 266, 18454-18459 (1991).
6. S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease, Gene, 77, 51-59 (1989).
7. S. 0. Pember, J. J. Villafranca, and S. J. Benkovic, Methods Enzymol., 142,50-56 (1987).
8. R. T. Carr and S. J. Benkovic, manuscript in preparation; R. T. Carr and S. J. Benkovic, paper presented
at this symposium.
9. B. S. Gibbs and S. J. Benkovic, J. Bioi. Chem., in press (1993).

70
CHARACTERIZATION OF THE IRON ENVIRONMENT IN RECOMBINANT
HUMAN TYROSINE HYDROXYLASE, USING MOSSBAUER AND EPR-
SPECTROSCOPY

Jan Haavik1, Eckhard Bilf, Marek Lengen 2, Aurora Martinez1, Torgeir

Flatmark1 and Alfred X. Trautwein2

1Department of Biochemistry and Molecular Biology

University of Bergen
N-5009 Bergen, Norway
2Institut ftir Physik

Medizinische Universitat LUbeck

D-23538 LUbeck 1, Germany

INTRODUCTION

Tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2, TH) is a

tetrahydropterin-dependent enzyme which catalyses the rate-limiting reaction in the
biosynthesis of catecholamines 1• The enzyme isolated either from bovine adrenals or rat
pheochromocytoma cells contains approximately one atom of tightly bound non-heme
iron/subunif· 3 • However, the ligands to the iron or its catalytic function is not known. An
important question has been the redox state of the iron atom, including possible changes in
its redox state during the catalytic cycle. Previous studies on the related enzyme phenylalanine
hydroxylase have indicated that the iron is in the ferrous state during catalytic turnover, but
that a small fraction of the iron is oxidised to Fe(III) during the reaction4 • It has been shown
that the tetrahydropterin cofactor can reduce this Fe(III) back to Fe(II), in addition to its
function as an electron donor during the catalytic turnove2.
Human THis present as four isoforms (hTH1-hTH4), generated by alternative splicing
of pre-mRNN-8 • In order to study the function of the metal center, all TH isoforms have been
expressed in E. coli and purified to homogeneity as the apoenzymes (metal free). We have
recently shown that the apoenzymes are selectively activated by added Fe(II) and that the
kinetics and stoichiometry of metal incorporation can be studied by equilibrium binding
assays, as well as by intrinsic tryptophan fluorescence and 1H-NMR paramagnetic relaxation
spectroscopl' 10•
In the present study, the pure hTH apoenzymes have been reconstituted with 57Fe(II)
and the iron environment has been studied by EPR and Mossbauer spectroscopy. Iron was
incorporated as high-spin Fe(II) and the protein was found to stabilize the ferrous state, even
in the presence of dioxygen, implying a relatively high redox potential for the enzyme-bound
iron.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 71
MATERIALS AND METHODS

57Fe in the metallic form was from Amersham Buchler (Darmstadt, Germany).
Fe(II)Cl2 was prepared by dissolving the metal in 1 M HCl under argon (99.99 %), using
platina as catalyst and drying the product under nitrogen (99.996 %). The concentration and
redox state of the product was verified by UV-visible absorption spectra of their 1,10-
phenanthroline complexes, in the presence and absence of ascorbic acid. The solid 57Fe(II)Cl2
was dissolved in oxygen-free water to a concentration of approx. 5 mM and added to the
apoenzyme in 20 mM Na-Hepes and 150 mM NaCl, pH 7.5010 •
All four isoforms of TH were expressed in E. coli and purified as previously
described9 • A typical yield was 80-100 mg of enzyme with a purity > 95 %, starting from 50
g of cell paste. The purified preparations of hTH1, hTH2 and hTH4 used in the present study
contained 0.02 ± 0.01, 0.02 ± O.Ql and 0.09 ± 0.02 (mean ± S.D., n = 3-4) atoms of
iron/subunit, respectively, as determined by atomic absorption spectrometry 10 • The
concentration of purified enzyme was measured using an extinction coefficient at 280 nm of
1.04 cm· 1 for 1 mg/ml2 • The enzyme activity was measured as described 1\
The EPR spectrometer was a conventional X-band apparatus (Bruker, model ER200D),
equipped with a helium flow cryostat (Oxford Instrument ESR910), which provided
temperatures down to 1.8 K. The Mossbauer spectrometer was of conventional type with
constant acceleration 12 and 1.856 GBq 57 Co in 6 pm Rh-matrix as y-source (Amersham
Buchler). Standard line width of a calibration absorber was 0.24 mm s· 1. Isomer shifts are
given relative to a-iron at ambient temperature.

RESULTS AND DISCUSSION

As shown in Fig. 1, the activity of hTH1 is stimulated about 40-fold by the presence
of Fe(II) and this effect is similar for both 57Fe(II) and 56Fe(II). Previous experiments have
demonstrated that the iron is rapidly bound in stoichiometric quantities under these
conditions 10 , indicating that the added iron is "correctly" inserted into the enzyme.

1000
~

I
CJl
E
800
~

I
c
E 600
0
E
c 400

"'
-
~

> 200
u
0
0
I
1-
0 10 20 30 40 50
[Fe(ll)] (I'-M)

Figure 1. The effect of added 5'Fe(II) and 57Fe(II) on the activity of tyrosine hydroxylase (hTHl ). The
enzyme activity was assayed for 1 min at pH 7.0, in the presence of either 5'Fe(II)S04 (•) or 57Fe(II)Cl2 (•)-

72
Mossbauer spectra of 57Fe(II) reconstituted tyrosine hydroxylase

The 57Fe Mossbauer spectra of hTHl, hTH2 and hTH4 in the temperature range 1.8
to 77 K in zero and weak applied magnetic fields are dominated by intense quadropole
o
doublets (solid line in Fig. 2) with isomer shifts (1.8-77 K) = 1.26 - 1.24 mm s·1 and
temperature independent quadropole splittings Ll£0 = 2.68 mm s·1 (typical errors for both
parameters: ±0.02 mm s·1). These values are characteristic of high-spin Fe(Il) in 6-
o,
coordination with oxygen- or nitrogen ligands. The isomer shift, which measures charge
density p(O) of s-electrons at the 57Fe nuclear site, is a sensitive parameter for the coordination
state of the Mossbauer atom, especially in the high-spin ferrous state. Shorter bond lengths
in 4-coordination with respect to 6-coordination, as well as the presence of covalent ligands
like sulfur in contrast to ionic ligands like oxygen, increase the s-density at the 57Fe nucleus
and hence reduce the isomer shift. Thus, for 4-coordination with oxygen or nitrogen ligands
we would expect isomer shifts for high-spin Fe(II) < 1 mm s· 1 at these temperatures and,
alternatively, 6-coordination would yield isomer shifts > 1.20 mm s·113 • Substitution of one
oxygen or nitrogen by one sulfur ligand would reduce the isomer shifts by about 0.1 mm s· 1•14 •
It is therefore very likely that the high-spin Fe(II) in hTH is exclusively coordinated to
oxygen and nitrogen ligands.
In addition to the dominant ferrous species, the spectra of hTH exhibited minor
contributions of ferric iron (dashed line in Fig. 2), which were preparation-dependent, but
never exceeded relative intensities of 5%. It is interesting to note that this ferric spectrum
appears to be distinct from the ferric species obtained after addition of dopamine, which is
a magnetically split spectrum (see below). The protein was found to stabilize the Fe(II)-state,
even in the presence of dioxygen, implying a relatively high redox-potential for the iron.

1.000

1:
0
'iii
.!!! .995
E
(/)
1:
0
.....
l-
Cll
.::: .990
0
Cll
0:::

.985
-10 -5 0 5 10
Velocity [mm s-'J

Figure 2. Mossbauer spectrum of 57Fe-tyrosine hydroxylase (hTHl) at 4.2 K. The apoenzyme (0.52 mM
subunit concentration) was incubated with 0.5 mM 57Fe(II)CI2 for 1 min at 20 •c before freezing in liquid
nitrogen. The solid line and the dashed line represent a least squares fit to the data, showing the major Fe(II)
species and a minor Fe(III) species, respectively.

73
The effect of added catecholamines on the Mossbauer- and EPR-spectra of tyrosine
hydroxylase

Natural catecholamines such as dopamine, noradrenaline and adrenaline are potent

inhibitors of TH, probably due to their tight binding to iron(III) at the active site of the
enzym~·3 •9 • 10 • 15 • We have recently shown by 1H-NMR and EPR spectroscopy that the addition
of stoichiometric amounts of dopamine to hTH results in a rapid oxidation of the iron to
Fe(Ill) 10 • Thus, Mossbauer spectroscopy was employed in order to characterize the
catecholamine coordinated Fe(III) form of the enzyme (Fig. 3). The spectra recorded in the
temperature range 1.8 K to 77 K in weak applied fields (20 mT) exhibit a magnetically split

1.001

r:::: 1.000
-~
II)

.E
II)

~ .999
...0
1-
Q)
>
~
..2 .998
Q)
a::

.997
-10 -5 0 5 10
Velocity [mm s·'J

Figure 3. Mossbauer spectrum of 57Fe-reconstituted hTH4 in the presence of stoichiometric amounts of

dopamine. The apoenzyme (0.43 mM subunit concentration) was incubated with 0.42 mM 57Fe(II)Cl2 and 0.43
mM dopamine for 1 min at 20 oc before freezing in liquid nitrogen. The spectrum was obtained at
4.2 K with a 20 mT magnetic field applied perpendicular to they-ray.

hyperfine pattern due to internal magnetic fields, originating from S = 5/2. Spin relaxation is
in the slow or intermediate range, with respect to the Mossbauer time window("' 10·7 s), even
at elevated temperatures (77 K). This is not unusual for Fe(III) in biological systems, in which
a large separation of the paramagnetic sites in the protein moiety results in slow spin-spin
relaxation. Furthermore, in such systems weak spin-orbit coupling between the 6S ground-state
and an excited spin quartet of Fe(III) results in a small spin-lattice relaxation. Detailed spin-
Hamiltonian analyses of Mossbauer spectra are in progress, in conjunction with
complementary EPR studies.

74
 As recently shown 10, the oxidation of enzyme-bound Fe(II) and formation of the
characteristic catecholamine-iron-enzyme complex occurs rapidly upon addition of
stoichiometric amounts of catecholamines. However, based on visible spectroscopic studies
on the formation of this complex, the second order rate constant (k = 1.3'103 M-Ls- 1 at 20 oc
and pH 7.5) for the formation of the dopamine complex with hTHl, is still at least 500-fold
smaller than the rate of iron incorporation into the apoenzyme (data not shown). The
dopamine complexes of hTHl and hTH4 have also been studied by EPR spectroscopy. The

g-va lues
20 10 6.0 4 .0 3.0 2.0 1.5

QK" \
dB __ _j \

0 100 200 300 400 500

B (mT )

Figure 4. EPR-spectra of iron-reconstituted tyrosine hydroxylase and the effect of stoichiometric amounts of
added dopamine. The samples were prepared as described in the legend to Fig. 3 and the spectra were
recorded at 2.8 K, using 2 mW microwave power (upper trace) and 20 11W (lower trace), 1 mT field
modulation, 9.43 GHz microwave frequency and 100kHz modulation frequency . The lower trace represents
the spectrum of the Fe(II)-enzyme before addition of dopamine.

iron-reconstituted tyrosine hydroxylase isozymes were found to be EPR-silent, but after

addition of a stoichiometric amount of dopamine, the EPR-spectra revealed mainly an axial
high-spin (S = 5!2) Fe(Ill) form, with g-values at 7.3, 5.0 and 2 (lowest Kramers doublets),
and a feature at g = 5.8 (middle Kramers doublet), corresponding to E/D-values in the range
0.05-0.1 and to a significant rhombic distortion in the ligand field (Fig. 4, upper trace?· 10•12 •
The catecholamine-enzyme complexes appear to represent inactive forms of the enzyme which
possibly may be formed in vivo2•15 • However, the physiological role or biological advantage
of such complexes remains to be settled.

75
Acknowledgments-We are grateful to Dr. Beatrice Le Bourdelles and professor Jacques
Mallet at CNRS, Gif-sur-Yvette, France for supplying the bacterial strains expressing human
tyrosine hydroxylase. Sidsel E. Riise and Ali J. Sepulveda at Department of Biochemistry and
Molecular Biology, University of Bergen are thanked for expert technical assistance. This
study was supported by the Research Council of Norway, Rebergs legat and Medizinische
Universitiit Dilbeck.

REFERENCES
1. M. Levitt, S. Spector, A. Sjoerdsma, and S. Udenfriend, J. Pharmacal. Exp. Ther. 148:1 (1965).
2. J. Haavik, K.K. Andersson, L. Petersson, and T. Flatmark, Biochim. Biophys. Acta 953:142 (1988).
3. K.K. Andersson, C. Vassort, B. Brennan, L. Que Jr., J. Haavik, T. Flatmark, F. Gros, and J. Thibault,
Biochem. J. 284:695 (1992).
4. T.A. Dix and S.J. Benkovic, Biochemistry 24:5839 (1985).
5. D.E. Wallick, L.M. Bloom, B.J. Gaffney, and S.J. Benkovic, Biochemistry, 23:1295 (1984).
6. B. Grima, A. Lamouroux, C. Boni, J.-F. Julien, F. Javoy-Agid, and J. Mallet, Nature 326:707 (1987).
7. N. Kaneda, K. Kobayashi, H. Ichinose, F. Kishi, A. Nakazawa, Y. Kurosawa, K. Fujita, and T. Nagatsu,
Biochem. Biophys. Res. Commun. 146:971 (1988).
8. K.L. O'Malley, M. Anhalt, B.M. Martin, J.R. Kelsoe, S.L. Winfield, and E.I. Ginns, Biochemistry
26:6910 (1987).
9. J. Haavik, B., Le Bourdelles, A. Martinez, T. Flatmark, and J. Mallet, Eur. J. Biochem. 199:371 (1991).
10. J. Haavik, A. Mardnez, S. Olafsdottir, J. Mallet, and T. Flatmark, Eur. J. Biochem. 210:23 (1992).
11. J.F. Reinhard Jr., G.K. Smith, and C. A. Nichol, Life Sci. 39:2185 (1986).
12. A.X. Trautwein, E. Bill, E.L. Bominaar, and H. Winkler, in: Structure and Bonding 78, pp.1-95, Springer
Verlag, Berlin (1991).
13. R. Reschke, A.X. Trautwein, and J.P. Desclaux, J. Phys. Chern. Solids 38:837 (1977).
14. E. Bill, C. Haas, X.-Q. Ding, W. Maret, H. Winkler, A.X. Trautwein, and M. Zeppezauer, Eur. J.
Biochem. 180:111 (1989).
15. K.K. Andersson, D.D. Cox, L. Que Jr., T. Flatmark, and J. Haavik, J. Bioi. Chern. 263:18621 (1988).

76
INTERACTION OF SUBSTRATE AND PTERIN COFACTOR WITH THE METAL
OF HUMAN TYROSINE HYDROXYLASE AS DETERMINED BY 1H-NMR

Aurora Martinez1, Chitrananda Abeygunawardana 2, Jan Haavik 1,

Torgeir Flatmark1, and Albert S. Mildvan 2

1Department of Biochemistry and Molecular Biology, University of Bergen

N-5009 Bergen, Norway
2Department of Biological Chemistry, The Johns Hopkins University School

of Medicine, Baltimore, Maryland 21205, USA

INTRODUCTION

Tyrosine hydroxylase (EC 1.14.16.2, TH) catalyses the rate-limiting step in the
biosynthesis of catecholamines. Human TH exists as four different isofmms (hTH1 to hTH4)
which have been expressed in E. colt The purified apoenzymes are rapidly activated (up
to 40-fold) by the incorporation of stoichiometric amounts of Fe++ 2 • All isozymes are
competitively inhibited by other divalent metal ions, e.g. zn++, Co++ and Ni++ which bind
with similar affinity and stoichiometty as Fe++ 2 •3.
Several mechanisms have been proposed for the reaction catalyzed by TH4 , but the
actual catalytic mechanism is not clear. Little is also known about the specific interactions
of substrates and inhibitors with the enzyme. The present paper reports the conformation of
the substrate (i.e. phenylalanine, Phe) and the pterin cofactor (i.e. 6-methyltetrahydropterin,
6-MPH4) bound to hTHl. These conformations were determined based on the metal-
substrate and metal-pterin distances measured by the paramagnetic probe-T 1 method 5.

THE BINDING OF PHENYLALANINE TO hTHl

As forTH isolated from other mammalian sources, Phe is a good substrate for hTHl
which hydroxylates Phe both to tyrosine (Tyr) and DOPA, with a ~<u, = 85 ± 4.9 11M and
a Vmax (for the hydroxylation to both products)= 461 ± 89 nmol/min/mg (n = 3) at pH 7.0
with tetrahydrobioptetin (BH4 ) as the cofactor. The low affinity of the enzyme for Phe is
in the ideal range in order to get fast exchange between Phe in the bound and the free states
in our NMR experiments. In contrast, Tyr has too high affinity for the enzyme (Km = 11 ±
1.7 11M) and very low solubility.
The 600-MHz 1H-NMR spectmm of Phe in the presence of hTH1 apoenzyme and the
assignment of proton resonances is shown in Figure lA. The addition of Co++ results in a
paramagnetic line broadening of the Phe signals and detailed titrations adding CoC12 (from

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 77
0 to 50 pM) to samples containing 5 mM Phe and 0.21 mM hTHl subunit were performed
measuring the paramagnetic effects of Co++ on the l!f1- and l!f2-values of Phe protons at
600-MHz. Because of the high affinity binding of co++ to hTHl (K0 "' 1 pMi·3 it was
assumed that the added metal was properly inserted at the active site forming a hTHl-Co++·
Phe complex. Based on a ~ value of 85 pM for Phe, the enzyme was assumed to be
saturated with Phe in calculating the normalized paramagnetic contributions to the
longitudinal relaxation rates, l/fT1/ 6 (Table 1). Moreover, the 1/tf2p-values exceeded all the
l//f1p-values by several orders of magnitude (not shown), indicating that the l/fT1p-values
are not limited by the exchange rate of Phe into the paramagnetic environment.

IIllS ()

I ~:tt.:l ,
114
IIJ.S I 112.6 IIN:xll
1/(,

I ,N~N ~
\ I
I 117S
111H
II
llu 1

r·L
1171!

---' "---'
___..il
7.5 7.0 4.0 J .! J.O 2.! J.S J.O l.S I.S 1.0
ppm ppm

Figure 1. 1H-NMR spectra ofPhe and 6-MPH4 taken in the presence of hTHI at 20 °C. A) 600-MHz 'H-NMR
spectrum of 5 mM Phe and 0.21 mM subunit hTHl, prepared in D20 containing 4 mM NaHepes (pH' = 7.5)
and 0.2 M NaCI. Peaks a, band care signals from Hepes. B) 400-MHz 1H-NMR spectrum of 4.6 mM 6-MPH4
and 22 pM subunit hTHl prepared in 0.1 M deuterated K-phosphate buffer, 0.1 M NaCJ (pH' = 7.45).

The correlation time ('tc) for the enzyme-substrate complex was calculated from the
frequency dependence of l/fT1p of the Hex proton at 250 and 600 MHz5•6• From the ratio
/f1p(600 MHz)/fT1p(250 MHz) of 1.38 ± 0.13, a 'tc of 1.8 ± 0.1 ps was calculated. This 'tc-
value, which represents the longitudinal electron spin relaxation time ('t8) of co++5, was used
to calculate the distances from the Phe protons to the Co++ ion (Table 1).
Based on the proposed ordered kinetic mechanisms for TH, with the order of binding
being pterin cofactor, oxygen and Tyr (or Phe)4•7 •8, a different conformation for Phe might
exist when it binds to the enzyme-pterin complex, than when it binds to the enzyme alone.
Since some activity is detected for the apoenzyme as isolated2, the conformation of Phe
could not be studied in the presence of BH4 • L-erythro-1 ,8-dihydrobiopterin (BH2) inhibits
hTHl competitively vs. BH4 with a Ki of 0.07 mM and it was used as a BH4 analogue (0.5
mM BH2 for 0.2 mM hTHl subunit). As found by the paramagnetic probe-T1 method, when
Phe binds to hTHl-Co++ (pterin absent) the aromatic H4 proton is slightly closer to the metal
(6.56 ± 1.18 A) (Table 1) than when it binds to the hTHl-Co++· BH2 complex (7.03 ± 1.25
A), other distances not being significantly different from those shown in Table 1.

78
 Table 1. Distances from Co++ to protons of Phe in the hTH1-Co++, Phe complex

nucleus o• (ppm)
H3,5 7.39 31.7 ± 1.5 6.80 ± 1.20c
H4 7.35 38.4 ± 3.2 6.56 ± 1.18
H2,6 7.31 31.0 ± 1.6 6.80 ± 1.21c
Ha 3.97 22.3 ± 2.1 7.18 ± 1.28
HBS 3.25 19.5 ± 2.5 7.35 ± 1.31
HBR 3.10 ::;17 "?.7.52

'From external TSP.

bErrors in r include contributions from errors in l/ff1p and tc (::; 5 % error) and from the g value for high-spin
co•• (4 ± 2) used in the calculations of r, which contributes to 15 % error in the absolute distances 5• Since
this g tensor should be the same for the interaction with all protons in Phe, the errors in the relative distances
would be approximately 2 %.
<Distances calculated assuming that both protons of the degenerate pairs H3,H5 and H2,H6 experiences the
paramagnetic effect of co•• (symmetric limiting case).

THE BINDING OF 6-METHYLTETRAHYDROPTERIN TO hTHl

In order to determine the conformation of pterin cofactors bound to hTH1,

measurements were done in anaerobic solutions and anaerobic NMR cells to avoid the rapid
oxidation of these compounds. The natural cofactor, BH4 , has too high affinity for hTH1 (~
= 4.5-19 J.1M?· 9 and, moreover, 1H-NMR signals from the Ha and HB protons of the side-
chain at C6, and the H6 and H7S protons of the pterin ring are not completely resolved. The
BH4 analogue 6-MPH4 has a lower affinity for the enzyme(~= 61-103 J.1M) 8•9, it is a very
good cofactor, the activity ofTH being higher with 6-MPH4 than with BH4 , and its 1H-NMR
spectrum at neutral pH* shows completely resolved resonances even at 400 MHz (Fig. lB).
In the presence of hTHl, addition of Co++ also results in paramagnetic broadening of the
proton resonances of 6-MPH4 • The 1ljT1p-values of 6-MPH4 protons (Table 2) were
calculated from titration of samples containing 5 mM 6-MP~ and 20 J.1M hTH1 subunit
with co++ (from 0 to 6 pM CoC12 ) at 400-MHz. By using the same 'tc-value for the hTH1-
co++· 6-MP~ complex as that calculated for the hTH1-Co++· Phe complex ('tc of 1.8 ±
0.1 ps), the distances from the 6-MPH4 protons to the Co++ ion were calculated (Table 2).

Table 2. Distances from Co++ to protons of 6-MPH4 in the hTHl-Co++· 6-MPH4 complex

nucleus o• (ppm) 1iffJP (s. 1) r(A)b

H7S 3.42 308.8 ± 29 4.55 ± 0.80

H6 3.16 869.5 ± 40 3.79 ± 0.65
H7R 2.99 380.7 ± 10 4.38 ± 0.75
CH3 1.18 121.2 ±7 5.33 ± 0.90
a,bSee Table 1.

79
CONCLUSIONS

The conformations of Phe and of 6-MPH4 bound to hTHl that we have determined
from the experimental distances obtained by the paramagnetic probe-T1 method have
mechanistic implications. Thus, the distances from the aromatic protons of Phe to Co++, both
in the presence and the absence of BH2, place the aromatic ring in the second coordination
sphere of the metal (Table 1). Then, the metal could contribute to the correct orientation of
the substrate but no group from the aliphatic chain of Phe (i.e. the carboxyl group) could
coordinate to the metal-ion (Fe++ in the catalytic form of the enzyme). For 6-MPH4 ,
however, proton-metal distances are shorter than for Phe (Table 2) and a direct coordination
to the metal may be presumed, both the carbonyl group at C4 or the NS being candidates
to directly coordinate to Fe++. Moreover, the calculated distances from the metal to the
aromatic ring of the substrate seem to be adequate for a Fe++-bound oxy or peroxy species,
acting as an hydroxylating intermediate, to approach molecular contact with C3/C4, which
are the positions in which Phe is hydroxylated by TH. Such a highly reactive oxygen
containing intermediate could be an activated enzyme bound iron-oxy, a peroxy adduct or
a 4a-peroxytetrahydropterin-iron species 4 ' 8, the latter being in agreement with the distances
from the metal to 6-MPH4 here reported. The final and unambiguous determination of the
conformation of both substrate and pterin cofactor when bound to the enzyme awaits the
determination of structures satisfying both the metal-proton distances calculated by the
paramagnetic probe-T 1 method and the intramolecular distances obtained by NOESY studies.
In the case of Phe, both sets of data are mutually consistene 0 , and in the case of 6-MPH4
experiments are in progress.

Acknowledgments

We are very grateful to Dr. Beatrice Le Bourdelles and professor Jacques Mallet,
Centre National de la Reserche Scientifique, Gif-sur-Yvette, France, for the supply of the
bacterial strains expressing human tyrosine hydroxylase isozymes. This work was supported
by grants from the Norwegian Research Council For Science and the Humanities and the
National Institutes of Health Grant DK-28616.

REFERENCES
1. B. Le Bourdelles, P. Horellou, J.-P. Le Caer, P. Denefle, M. Latta, J. Haavik, B. Guibert, J.-F. Mayaux,
and J. Mallet,]. Bioi. Chern 266:17124 (1991).
2. J. Haavik, B. Le Bourdelles, A. Martinez, T. Flatmark, and J. Mallet, Eur. J. Biochem 199:371 (1991).
3. J. Haavik, A. Martinez, S. Olafsdottir, J. Mallet, and T. Flatmark, Eur. J. Biochem. 210:23 (1992).
4. T.A. Dix and S.J. Benkovic, Ace. Chern. Res. 21:101 (1988).
5. A.S. Mi1dvan, J. Granat, G.M. Smith, and M. Liebman, Adv. Inorg. Biochem. 2:211 (1980).
6. E.H. Serpersu, D.W. Hibler, J.A. Gerlt, and A.S. Mildvan, Biochemistry 28:1539 (1989).
7. P.F. Fitzpatrick, Biochemistry 30:3658 (1991).
8. S. Kaufman and E.S. Kaufman, in: "Folates and Pterines" (R.L. Blakley and S.J. Benkovic, eds., Vol. 2,
pp 251-352, John Wiley & Sons, New York (1985).
9. T.C. Williams and C.B. Storm, Biochemistry 24:458 (1985).
10. A. Martinez, C. Abeygunawardana, J. Haavik, T. Flatmark, and A.S. Mildvan, Biophys. J. 64:A368 (1993).

80
MECHANISTIC STUDIES OF TYROSINE HYDROXYLASE

Paul F. Fitzpatrick
Departments of Biochemistry and Biophysics and Chemistry
Texas A&M University
College Station, TX 77843

INTRODUCTION
Tyrosine hydroxylase catalyzes the rate-limiting step in the biosynthesis of thecate-
cholamine neurotransmitters, the hydroxylation of tyrosine to dihydroxyphenylalaninel.
The other substrates for the reaction are molecular oxygen and a tetrahydropterin. In addi-
tion, tyrosine hydroxylase requires one atom of ferrous iron per active site for activity2,3;
the role of the iron atom is unknown. While the central position of tyrosine hydroxylase in
the function of the central nervous system has resulted in a great deal of interest in the
enzyme over the years, very little is known about the actual mechanism of catalysis. This
report describes recent studies towards elucidating the mechanism of this important
monooxygenase.

RESULTS AND DISCUSSION

Preparation of enzyme
For many mechanistic studies it is necessary to have readily available large amounts of
purified enzyme. This is especially true in the case of tyrosine hydroxylase, where the ki-
netic and regulatory properties have been reported to vary depending upon the degree of
purification. Our approach has been to express rat tyrosine hydroxylase in a heterologous
system. Two different expression systems have been used. The enzyme is expressed at
about 5% of the total protein when expressed using a baculovirus expression vector4 in
Spodopterafrugiperda cells. At these levels, purification is straightforward, requiring only
an ammonium sulfate fractionation and a single heparin-Sepharose column to yield 10 mg
of enzyme per liter of tissue culture. More recently, the T7 polymerase based expression
system of Studier et a1.5 has been used to express rat tyrosine hydroxylase in E. coli6.
While the levels of expression are slightly lower (2-3% of the total protein), no changes in
the purification protocol are required. Both sources provide enzyme of very high specific
activity. Recent preparations routinely have specific activities of 2-3 IJ.moles/min-mg at 30
oc.
Steady state kinetic mechanism
With sufficient enzyme in hand, the steady state kinetic mechanism of tyrosine hy-
droxylase was determined. While tetrahydrobiopterin is the physiological substrate, strong
substrate inhibition is seen with tyrosine with this substrate. Consequently, 6-methylte-
trahydropterin was used for the kinetic studies. The kinetic mechanism was determined at
pH 6.5 and 30 °C. The complete steady state kinetic mechanism for a three substrate reac-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 81
tion is given in Equation 1. To evaluate whether all eight terms are present in the rate equa-
tion, the method of Rudolph and Fromm7 was used. Rather than vary all three substrates
independently, the concentrations of pairs of substrates are kept at constant ratios close to
the ratios of their KM values. The presence of individual terms in the rate equation can be
determined from the shapes of the slope and intercept replots using such an analysis, since
terms containing the concentrations of the two substrates in fixed ratio will contain squared
terms, generating nonlinear replots. The replots established that the values of both the
Ko 2N and coef(02) terms in the steady state rate equation for tyrosine hydroxylase are
zero, while all of the other terms are present. Therefore, the steady state kinetic equation for
rat tyrosine hydroxylase is given by Equation 2. The kinetic parameters obtained from the
data are summarized in Table 1.

v = V[tyr][02][6MPI4]/([tyr][02][6MPJ4] + Ktyr[02][6MPJ4]
+ Koz[tyr][6MPJ4] + KMP14[tyr][02] + coef(tyr)[tyr] (1)
+ coef(6MPJ4)[6MPJ4] + coef(02)[02] +constant)

v= V[tyr][02][6-MePJ4]/([tyr][02][6-MePl4] + Ktyr[<>l][6-MePJ4] (2)

+ KMPJ4[tyr][D2] + coef(tyr)[tyr] + coef(MePf4)[6-MePJ4] +constant)

Table 1. Steady state kinetic parameters of tyrosine hydroxylase at pH 6.5, 30 oc.

parameter Vmax coef(tyr) constant
value 92 min-1 1710 M2 8.7xlo4 M3

Inhibition patterns with competitive inhibitors (6-methyl-7 ,8-dihydropterin, nore-

pinephrine, 3-I-tyrosine) were consistent with ordered addition of 6-MePI-4 before tyro-
sine. At concentrations of tyrosine sufficiently high that the only effect is inhibitory, tyro-
sine is a competitive inhibitor versus 6-MePI-4 (Figure 1). All of the kinetic data are
consistent with the mechanism of Scheme 1 for rat tyrosine hydroxylase.

k16-MePH4
E E·6-MePH4 E

k10 n 2
kgty:
E·tyr
Scheme 1

82
 0.08

c
'E• 0.04
~

0
0 0.05 0.1 0.15
1/[6-Me-PH,J, J.1M' 1

Figure 1 Substrate inhibition by tyrosine. Initial rates were measured as a func-

tion of 6-methyltetrahydropterin concentration at (A) 200, (e) 400, and (0) 600
IJ.M tyrosine. Conditions: 14m 8-mercaptoethanol, 75 J.Lg/ml catalase, 10 11M fer-
rous ammonium sulfate, 0.25 mM oxygen, 50 mM MES, 50 mM sodium acetate,
100 mM Tris-HCI, pH 6.5, 30 °C.

Burst kinetics

To determine if the rate limiting step in the enzyme reaction occurs after oxygen trans-
fer to tyrosine, the rate of DOPA formation during the first few enzyme turnovers was de-
termined. At saturating levels of 6-MePH4 and oxygen and high levels of tyrosine, there
was no detectable burst of DOPA formation (Figure 2). This result establishes that no slow
step occurs after oxygen transfer to tyrosine, so that product release must be rapid.
Consequently, the rate-limiting step in catalysis is a chemical step.

2
c..
Q)

20 t 40 60
•s

Figure 2. Rate of DOPA formation during the fD'st turnover. The rate of
DOPA production was determined at pH 7 at 2 oc with 150 1J.M tyrosine, 500 IJ.M
6-methyltetrahydropterin, and 440 IJ.M oxygen. p/e is the number of molecules of
DOPA formed per enzyme monomer.

83
Kinetic isotope effects
To determine if cleavage of the tyrosine CH bond occurs in a slow step, kinetic isotope
effects were determined with [3,5-2H2]tyrosine as substrate. The measured isotope effects
on the Vmax and V/K!Yr values were 0.98±o.06 and 1.02±o.06, respectively. Neither is
significantly different from one. Since no significant isotope effect was seen on the VIKtyr
value, carbon-hydrogen bond cleavage either occurs after the first irreversible step or 1s
much faster than another step preceding or including the first irreversible step. The lack of
an observed isotope effect on the Vmax value indicates that carbon-hydrogen bond cleavage
is much faster than another chemical step, since product release is rapid.
Transition state analogs as tests of an electrophilic mechanism
A plausible mechanism for hydroxylation by tyrosine hydroxylase involves attack of the
aromatic ring of the substrate on an electrophilic pterin peroxide (Scheme 2); this would be
assisted by the electron donating ability of the phenolic group8,9. Carbon-hydrogen bond
cleavage would occur during the tautomerization of the hydroxycyclohexadienone-like
intermediate II; such a step is expected to be quite rapid, so that no isotope effect would be
expected. As a test of this mechanism, several compounds similar to the proposed inter-
mediates were tested as inhibitors (Scheme 3). 4-Pyridylalanine N-oxide (III) was tested as
a mimic of the proposed phenoxide. The preferred tautomers of both IV and V are the keto
specieslO, so they were tested as analogs ofii. With all three proposed inhibitors, only very
weak or no inhibition was seen, and the inhibition was more characteristic of catechols than
amino acids. Also, no time-dependent inhibition was seen with IV or V, either alone or
when S-deaza-6-MePH4 was added.

Scheme 2. Mechanism for hydroxylation by tyrosine hydroxylase by electrophilic aromatic substitution.

m
Scheme 3. Transition state analogs for tyrosine hydroxylase based on the mechanism of Scheme 2.

The lack of any inhibition by the phenoxide analog III suggests that phenoxide formation is
not obligatory for catalysis. The weak inhibition with IV and V is consistent with a lack of
enzymatic catalysis of the formation of a hydroxycyclohexadienone intermediate in the
hydroxylation of tyrosine by tyrosine hydroxylase.
Evidence for rate-limiting oxygen activation
The effect of changing the reactivity of the amino acid substrate upon the rate of catalysis
was determined. The total rate of tetrahydrobiopterin oxidation was measured, to ensure

84
that the total flux through the catalytic cycle was detected, using a coupled reaction with
dihydropterin reductase. Steady state kinetic parameters were determined with a number of
tyrosine analogs, varying both the amino acid and tetrahydrobiopterin. The KM values for
tetrahydrobiopterin were about 20 J..LM with all the substrates tested. Also, no
tetrahydrobiopterin oxidation was detected in the absence of an amino acid. The Vmax and
Kaa values are summarized in Table 2. Surprisingly, the Vmax values are essentially inde-
pendent of the reactivity of the amino acid substrate, with an average value of about 100
min-1. This clearly establishes that the rate-limiting step in catalysis by tyrosine hydroxy-
lase does not involve the amino acid substrate, although the amino acid must be bound to
the enzyme for catalysis to occur. Further, 4-CH30-phenylalanine is efficiently hydroxy-
lated by tyrosine hydroxylase, establishing that there is no requirement for removal of the
phenolic proton as depicted in Scheme 2.
Table 2
Steady State Kinetic Parameters for Alternate Substrates of Tyrosine Hydroxylasea

substrate Vmax Kaa

(min-1) (J..LM)
tyrosine 92.4±10.7 10.8±4.4
4-NH2-phenylalanine 115±5.8 36.8±10.2
4-CH30-phenylalanine 65±6.1 460±84
phenylalanine 111±6.6 49.3±10.9
4-F-phenylalanine 83±4.8 11.8±2.2
3-HO-phenylalanine 83.9±7.4 219±41
3-F-phenylalanine 118±17 23.5±8
alnitial velocities were determined as the rate of dihydrobiopterin production in 5 J.1.M ferrous am-
monium sulfate, 75 J.Lg/ml catalase, 200 J.1.M NADH, 0.45 units/ml sheep dihydropterin reductase, 50
mM sodium acetate, 50 mM MES, 0.1 MTris-HCI, pH 6.5, at 30 °C.

Among the possibilities for the identity of the hydroxylating intermediate, the one which is
most frequently proposed is a 4a-peroxytetrahydropterin (Scheme 2). Studies by Eberlein et
al.11 are consistent with the mechanism of Scheme 4 for the reaction of molecular oxygen
with tetrahydropterins. In the slow first step single electron transfer from the reduced pterin
to 02 forms superoxide and the pterin radical cation. These two radicals rapidly recombine
in a second step to form the peroxytetrahydropterin.

Scheme4

Obviously, no primary kinetic isotope effect is expected if single electron transfer is rate-
limiting. In addition, no solvent isotope effect should be seen, since no exchangeable
proton is in flight in the rate-limiting transition state. Consistent with such a prediction, the
Vmax and VIKtyr values are unchanged when the reaction is run in D20 instead of water,
when the values at the pH optima are compared.
Conclusions

The ready availability of significant amounts of purified rat tyrosine hydroxylase has
allowed us to begin investigating the mechanism of hydroxylation by this enzyme. The
steady state kinetic mechanism has been determined. Further, all results to date are consis-

85
tent with the rate-limiting step in catalysis being fonnation of the hydroxylating intermedi-
ate.
Acknowledgments
The support of the National Science Foundation, the National Institutes of Health, and
the American Heart Association during the course of this work is gratefully acknowledged.
P.F.F. is an Established Investigator of the American Heart Association.

REFERENCES
1. S. Kaufman and E.E. Kaufman. Tyrosine hydroxylase, in: "Folates and Pterins, Vol. 2", R.L. Blakley et
a!., eds., John Wiley & Sons, New York (1985).
2. P.F. Fitzpatrick. The metal requirement of rat tyrosine hydroxylase, Biochem. Biophys. Res. Comm.
161:211 (1989).
3. J. Haavik, B. LeBourdelles, A. Martinez, T. Flatmark, and J. Mallet Recombinant human tyrosine
hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions, Eur. J.
Biochem. 19:371 (1991).
4. P.F. Fitzpatrick, L.J. Chlumsky, S.C. Daubner, and K.L. O'Malley. Expression of rat tyrosine hydroxylase
in insect tissue culture cells and purification and characterization of the cloned enzyme, J. Bioi. Chem.
265:2042 (1990).
5. F.W. Studier, A.H. Rosenberg, J J. Dunn, and J.W. Dubendorff. Use of T7 RNA polymerase to direct
expression of cloned genes, Methods Enzymol. 185:60 (1990).
6. S.C. Daubner, C. Lauriano, J.W. Haycock, and P.F. Fitzpatrick. Site-directed mutagenesis of serine 40 of
rat tyrosine hydroxylase effects of dopamine and cAMP-dependent phosphorylation on enzyme activity,
J. Bioi. Chem. 267:12639 (1992).
7. F.B. Rudolph and HJ. Fromm. Plotting methods for analyzing enzyme rate data, Methods Enzymoi.
63:138 (1979).
8. K. Detmer and V. Massey. Effect of substrate and pH on the oxidative half-reaction of phenol
hydroxylase, J. Bioi. Chem. 260:5998 (1985).
9. G. Guroff, J.W. Daly, D.M. Jerina, J. Renson, B. Witkop, and S. Udenfriend. Hydroxylation-induced
migration: The NIH shift, Science 157:1524 (1967).
10. A.R. Katritzky and J.M. Lagowski. "Chemistry of the Heterocyclic N-Oxides", Academic Press, New
York (1971).
11. G. Eberlein, T.C. Bruice, R.A. Lazarus, R. Henrie, and S.J. Benkovic. The interconversion of the 5,6,7 ,8-
tetrahydro-, 7,8-dihydro-, and radical forms of 6,6,7 ,7-tetramethyldihydropterin. A model for the
biopterin center of aromatic amino acid mixed function oxidases, J. Am. Chem. Soc. 106:7916 (1984).

86
ALLEVIATION OF CATECHOLAMINE INHIBITION OF TYROSINE
HYDROXYLASE BY PHOSPHORYLATION AT SERINE40

S. Colette Daubner and Paul F. Fitzpatrick

Department of Biochemistry and Biophysics

Texas A&M University
College Station, TX 77843

INTRODUCTION
Tyrosine hydroxylase (TYH) catalyzes the rate-determining step in the synthesis of
catecholamine neurotransmitters, the hydroxylation of tyrosine to L-dihydroxyphenylala-
nine with the oxidation of tetrahydrobiopterin to dihydrobiopterin 1. The activity of TYH is
highly regulated. Tyrosine hydroxylase is inhibited by catecholamines and high levels of
tyrosine, and activated by anions, polyanions, phospholipids and phosphorylationl,2.
cAMP-Dependent protein kinase phosphorylates serine40; calmodulin-dependent protein
kinase II phosphorylates serinel9 preferentially, but also phosphorylates serine40; and ser-
ine31 is also phosphorylated2. Phosphorylation at serine40 has been studied more thor-
oughly than phosphorylation at the other serines. The reported effects have varied widely2.
At pH 6, phosphorylation of the rat enzyme decreases the KM value for 6-methyltetrahy-
dropterin four to eight-fold, and increases the V max value up to threefold. Much less work
has been done with the physiological substrate tetrahydrobiopterin at the physiological pH
of 7. Under these assay conditions with partially or fully purified enzyme, the effect on the
KBH4 value is reported to be about threefold, while the increase in the Vmax value is up to
tenfold. This variability may be due to different preparations containing variable amounts
of phosphate at each of the serine residues. In addition, TYH purified from PC12 cells or
bovine adrenal medulla contains substoichiometric amounts of bound catecholamines3;
several labs have noted a relationship between phosphorylation and feedback inhibition by
catecholamines4.
We recently reported the expression and isolation of large quantities of rat TYH5.
This paper reports work done with the enzyme expressed in E. coli. We focused initially on
the effect of phosphorylation of serine40, producing a S40A mutant of TYH. With the pro-
duction of large quantities of unphosphorylated and catecholamine-free TYH, we have
been able to study the relationship between the two modes of regulation of TYH.

METHODS
Preparation of recombinant DNA molecules
The eDNA for rat TYH was initially cloned into a baculovirus expression vectorS. It
was removed from this vector by Bamm digest and inserted into the Bamm site of
pTZ18R, giving plasmid pTH5. This vector was used as the substrate for oligo-directed
mutagenesis6. Best expression results from pET3b (a T7 RNA polymerase promoter-con-
taining vector?) if the insert is placed between the Ndei and the Bamffi sites; accordingly, a
new Ndel restriction site was required at the start codon of the TYH eDNA.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 87
 EB EP EBNEP

NIB digest,
isolation of
UJ
band with TYH
NIB digest, eDNA
$10 band
promotor isolation
_ _ _ _ _ _...,.mix DNA
fragments
ligate

$10 N Jp

pETOHl
6410 bp

Figure 1. Construction of the plasmid pETOHl, allowing overexpression of rat tyrosine hydroxylase in E.
coli. The $10 promoter is a T7 RNA polymerase promoter (8). Restriction enzyme sites are abbreviated: E
refers to EcoRI; N, Ndel; B, Bamlll; P, Pstl; and H, Hindiii. Ampr refers to the 8-lactamase gene.

88
 (loss of
EBNEP Hinfl site)

E
E

N/B digest,
zo... isolation of TYH eDNA
N/B digest,
portion of plasmid
isolation of
pET3b
portion of
-!:P:::Ia:::s~m;,;.:id:-..----t~~ mix DNA

pETOH1
fragments
6410 bp ligate

~.P10
! S40A
(loss of
Hinfl site)
promotor

pETOH1S40A
6410 bp

Figure 2. Construction of the vector coding for tyrosine hydroxylase with alanine substituted for serine at po-
sition 40. Notations for restriction enzyme sites are the same as for Figure 1.

89
Plasmid pTH5 was subjected to mutagenesis, giving plasmid pTH6. Plasmids pET3b and
pTH6 were digested with Ndel and BamHI; both the opened pET3b vector and the eDNA-
containing fragment from pTH6 were purified from agarose gels and exposed to T4 DNA
ligase. Possible recombinants were screened by restriction enzyme analysis. The plasmid
which contained the TYH eDNA inserted between the Ndel and BamHI sites of pET3b was
named pETOHl. These DNA manipulations are pictured in Figure 1.
To substitute alanine for serine40, oligo-directed mutagenesis was performed on plas-
mid pTH6 to generate plasmid pTH6S40A. Introduction of the mutation into pETOHl was
performed by exchanging the Ndei-BamHI fragment of pTH6S40A for that of pETOHl, by
digesting the plasmids with the enzymes, isolating the correct fragments from agarose gels,
and ligating with T4 DNA ligase. Digestion with Hinjl provided the screen for S40A muta-
tion, and one positive plasmid was sequenced to insure that it did contain the mutation.
This vector was designated pETOH1S40A. These steps are shown in Figure 2.
Enzyme purification
A major advantage of isolation of TYH from E. coli is that enzyme is obtained in fully
unphosphorylated form without bound catecholamine; the S40A mutant serves also as non-
phosphorylatable enzyme. For purification of the enzyme from bacteria, competent cells of
the E. coli strain BL2l(DE3) pLysE were transformed with pETOHl or pETOH1S40A.
Luria-Bertani medium plus carbenicillin and chloramphenicol was inoculated with an
overnight culture. When the cells were well into logarithmic growth expression was in-
duced by addition of IPTG. After 2 hours cells were pelletted by centrifugation. Pellets
were lysed by sonication and TYH was purified using a protocol consisting of centrifuga-
tion, polyethyleneimine precipitation, ammonium sulfate precipitation, and heparin-
Sepharose chromatography. Tyrosine hydroxylase purified from PC12 cells was a gift from
Dr. R. Roskoski of the Louisiana State University. Tyrosine hydroxylase was assayed as
previously describeds with an assay which measures tritium release from 3,5-[3H]-tyrosine.
Effects of phosphorylation and catecholamine inhibition
To study the effects of phosphorylation two parameters had to be measured, the extent
of phosphorylation and its impact on activity. For incorporation of 32p into TYH by cAMP-
dependent protein kinase, the method of Roskoski et al. 8 was used. To determine the effect
of phosphorylation on enzyme activity, TYH was incubated with cAMP-dependent protein
kinase catalytic subunit (60-80 Jlg/ml final concentration), ATP (0.1 mM), and MgCb (10
mM) for various times before an aliquot (10-40 J,l.l) was added to the TYH assay mix.
To determine the effect of dopamine on the enzyme activity, TYH (6-10 J.!M) was in-
cubated for varying times in 50 mM HEPES-TEA, pH 7, at 30 °C in the presence or ab-
sence of an excess of dopamine. A 10 J,1.1 aliquot of this reaction mixture was then added to
assay mix containing 20 J.!M tyrosine and tetrahydrobiopterin. Controls established that the
small amounts of dopamine carried over into the assay mix (-50 nM) had no effect on the
observed initial rates.
To ensure that excess dopamine was not interfering with TYH activity after preincu-
bations of enzyme plus dopamine, the enzyme was separated from free dopamine on a col-
umn of Sephadex G-50. This chromatography was carried out in 50 mM Tris-HCl, 10%
glycerol, pH 7.0. Tyrosine hydroxylase (2-6 nmoles in 250 Jll) was incubated for 20 min-
utes at 20 °C with an excess of dopamine and applied to the Sephadex G-50 column.
Fractions were assayed for dopamine and for protein. Enzyme eluting from this column
was used in steady-state kinetic experiments after further incubation with cAMP-dependent
protein kinase with or without MgATP as described above.

RESULTS AND DISCUSSION

Recombinant tyrosine hydroxylase was purified approximately 40-fold with a final
specific activity of 1.8 JlmoVmin-mg. The S40A mutant protein was purified using the
protocol developed for the wild-type enzyme. The V max values and the KM values for the
physiological substrates tetrahydrobiopterin and tyrosine and for the analog 6-methylte-
trahydropterin were determined at pH 7 for wild-type TYH and the S40A mutant. The re-

90
sults are shown in Table 1. Both preparations exhibited the kinetic parameters expected of
phosphorylated enzyme. Remarkably, the S40A enzyme, serving as totally unphosphory-
lated enzyme, had a slightly lower KBH4 value (rather than the higher value predicted from
the literature) and a slightly lower Vmax value (Table 1). The KtYl: values were unaffected
by the mutation or by the expression system. No phosphate could be detected by direct
phosphate assay of enzymes expressed in E. coli. Wild-type TYH incorporated 0.9±o.2
moles [32P]-phosphate per mole monomer. No [32P]-phosphate was taken up by the S40A
enzyme exposed to the same phosphorylating conditions.

Table 1. Steady State Kinetic Parameters of Recombinant Tyrosine Hydroxylase at pH 7a

wild type 101±15.7 16.5±3 8.6±2.8 41±4.0

S40A 78+4.4 10.5±0.6 8.0±1.7 23+3.8
llConditions: 14 mM 6-mercaptoethanol, 10 1J.M ferrous ammonium sulfate, 75 f.J.g/ml catalase, 50 mM
HEPES-TEAOH, pH 7, 30 oc.
bDetermined at 35 11M tyrosine.
~Determined at 500 11M tetrahydrobiopterin.
Determined at 100 11M tyrosine.
Tyrosine hydroxylase was incubated with the catalytic subunit of cAMP-dependent
protein kinase and MgATP under the conditions which resulted in stoichiometric incorpo-
ration of phosphate. Phosphorylation effected a twofold decrease in the KBH4 value of the
wild-type enzyme but no change for the S40A mutant (Table 2). No significant increase in
the Vmax value was found for either enzyme.
Table 2. Effect of Phosphorylation by cAMP-Dependent Protein Kinase on the Activity of
Recombinant Tyrosine Hydroxylase
control protein kinase treated
enzyme KBH4 Ymax
(JJ.M) %
wild-type 19±2.1 1oo±3.7 8.3±1.0 109±3.3
S40A 12±0.6 1oo±l.4 12±0.4 111±1.4
aPercent untreated control.
Andersson et al. have shown that TYH from bovine adrenal glands and PC12 cells
contains bound catecholamines3. We examined the effect of added dopamine on there-
combinant enzyme. Wild-type TYH was incubated with a stoichiometric amount of
dopamine at 30 °C and pH 7. Under these conditions, there was a time dependent loss of
activity with a second order rate constant of about 8000 M-1s-1. The loss of activity leveled
off at about 10% of the initial value under these assay conditions. In contrast, no loss of ac-
tivity was detected under these conditions with pure nonrecombinant enzyme from PC12
cells.
To establish if the dopamine was tightly bound, the recombinant enzyme was incu-
bated with an excess of tritiated dopamine for 10 minutes at 20 °C followed by chromatog-
raphy on Sephadex G-50. This resulted in two well-separated peaks of radioactivity, with
all of the protein in the first peak. Determination of the number of moles of dopamine and
of TYH in this peak gave a stoichiometry of 0.55 dopamine molecules per monomer.
The steady state kinetic parameters of TYH treated with unlabeled dopamine and purl-
fied on Sephadex G-50 were determined. The decrease in activity was due to a combination
of a twofold increase in the KBH4 and a 17-fold decrease in the Vmax value (Table 3).
Similar results were obtained with the S40A mutant.
Subsequent phosphorylation of the dopamine-inhibited wild-type enzyme activated it
by increasing the Vmax value tenfold and decreasing the KBH4 value twofold (Table 3). The
kinetic parameters of the S40A enzyme were unaffected by incubation with cAMP depen-
dent protein kinase, establishing that the release from inhibition was due in full to phospho-
rylation at serine 40.

91
Table 3. Steady-State Kinetic Parameters of Recombinant and Nonrecombinant Tyrosine
Hydroxylase

KBH4UJM) Ymax (min- 1)

enzyme control kinase treated control kinase treated
wild type 25±4 13±3 53±2 59±2
Dopamine-bound wild type 58±17 29±3.7 2.9±0.3 31±1
PC12 56±15 31±7 2.3±0.2 24±1.5
S40A 11.8±0.6 12.1±0.3 34±1.7 31±0.3
dopamine-bound S40A 69±18.2 69±15 2.9±0.3 2.9±0.3

The rates and stoichiometry of incorporation of 32p from ["t32P]ATP by cAMP-de-

pendent protein kinase were determined for the different enzyme preparations. Dopamine-
modified TYH was labeled more slowly than native enzyme at 30 oc, but both enzymes
could be stoichiometrically labeled in 10 minutes. In contrast, no label was incorporated
into the S40A protein.
These results establish that the effect of phosphorylation of serine 40 of TYH depends
upon the starting form of the enzyme. For the free enzyme, phosphorylation results in a de-
crease in the KM value for tetrahydrobiopterin at pH 7. Phosphorylation of the cate-
cholamine-bound enzyme, in contrast, increases the Ymax value 10-fold in addition to de-
creasing the KBH4 value twofold. Presumably, these two modes of regulation of the activity
of TYH, namely, phosphorylation and time-dependent feedback inhibition, act in concert in
determining levels of catecholamine synthesis in vivo. This is in addition to the competitive
inhibition of TYH seen with catecholamines, which is not time-dependent and which would
be freely reversible1.
These experiments utilizing purified components thus provide insight into the regula-
tion of TYH and dramatically demonstrate the dynamic responsiveness of the physiological
system. In addition, the ability of the S40A enzyme to mimic the unphosphorylated enzyme
will be invaluable in investigating the roles of the other phosphorylated serine residues in
regulation.
Acknowledgments
The support of the National Science Foundation, the National Institutes of Health, and
the American Heart Association during the course of this work is gratefully acknowledged.
P.F.F. is an Established Investigator of the American Heart Association.

REFERENCES
1. S. Kaufman and E. E. Kaufman, in Folates and Pterins, Vol. 2, Blakley, R. L. and Benkovic, S. ]., eds.,
John Wiley & Sons, New York (1985)
2. R. E. Zigmond, M.A. Schwarzschild, and A. R. Rittenhouse, Ann. Rev. Neurosci. 12:415 (1989)
3. K. K. Andersson, J. Haavik, L. Que, T. Flatmark, J. Thibault, and L. Petersson,J./norg. Biochem. 36:323
(1989)
4. J. Haavik, A. Martinez and T. Flatmark, FEBS Letts. 262:363 (1990)
5. P. F. Fitzpatrick, L. J. Chlumsky, S.C. Daubner, and K. L. O'Malley, J. Bioi. Chern. 265:2042 (1990)
6. T. A. Kunkel, J.D. Roberts, and R. A. Zakour, Methods Enzymol. 154:367 (1987)
7. F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, Methods Enzymol. 185:60 (1990)
8. R. Roskoski, Jr., P.R. Vulliet, and D. B. Glass, J. Neurochem. 48:840 (1987)

92
GL YCERYL ETHER MONOOXYGENASE [EC 1.14.16.5]:
STOICHIOMETRY AND INHIBITION

Behjat Kosar-Hashemi*, Hiroyasu Taguchit and Wilfred L.F.Armarego*

* Division of Biochemistry and Molecular Biology, John Curtin School of Medical

Research, Australian National University, GPOBox 334, Canberra, ACT 2601, Australia,
t Department of Natural Science, Kyoto Women's University, 35 Kitahiyoshi-cho,
Imakumano, Higashiyama-ku, Kyoto 605, Japan

Glyceryl ether monooxygenase is a microsomal enzyme which hydroxylates the a-

carbon atom of the fatty side-chain of glyceryl ethers of long-chain fatty alcohols. The
reaction requires oxygen and a tetrahydropterin cofactor. It is a mixed-function oxidase
and by analogy with phenylalanine hydroxylase the reaction shown in Scheme 1 was
postulated. I We investigated aspects of the stoichiometry of the oxygenase because two
earlier reports were not entirely satisfactory. Tietz, Lindberg and Kennedy1 had shown
that the amount of fatty aldehyde produced, in a non regenerating system, was 40-50% of
tetrahydropterin oxidised and attributed this to the fact that the pterin was a DL mixture.
This statement is incorrect in view of later work2 and because we have found that the
monooxygenase activity with the two separate enantiomers S(-) and R(+) 6-methyl-
5,6,7,8-tetrahydropterin as cofactors is the same. The second report was by Kotting,
Unger and Eibl3 who determined the stoichiometry of the reaction in a coupled reaction
with DHPR and NADH, and showed that the amount of NADH oxidised after one minute
was equivalent to the total amount offatty aldehyde, alcohol and acid produced.3 Since
the fatty aldehyde, alcohol and acid are produced by several reactions following enzymic
hydroxylation of the ether substrate the conclusion was dubious.
RCH20CH2CHOHCH20H
+ 6-MePH 4 monooxygenase
... RCH(OH)OCH2CHOHCH20H
+ q-6-MePH2

Scheme 1

We investigated two aspects of the stoichiometry of the reaction viz: (i) the determination
of the ratio of the amount of 6-MePR4 oxidised to q-6-MePH2 in the direct assay with the
amount of NADH oxidised in the coupled reaction using DHPR and (ii) the determination
of the ratio of the amount of 6-MePR4 oxidised to q-6-MePH2 to the amount of batyl
alcohol consumed.
(i) For this study, 6-MePR4 oxidation was followed by the direct assay (Scheme
1) in order to obtain the initial rates of oxidation, and compared these with the initial rates
of conversion of NADH to NAD in a separate coupled reaction (Scheme 2). Catalase was
added to the reaction mixtures to minimise autoxidation of 6-MePR4. The rates are
summarised in Table 1. The data shows that at concentrations of 6-MePR4 below 0.1
mM, the stoichiometry of NADH consumed per 6-MePR4 oxidised was- 1 as expected
Abbreviations: DHPR = dihydropteridine reductase [EC 1.6.99.7]; 6-MePH4 =RS-6-methyl-5,6,7,8-
tetrahydropterin; q-6-MePHz = RS-6-methyl-7,8(6H)dihydropterin; Mega-10 = decanoyl N-methyl-N-glycamide;
lyso-P AF = 3-1' -hexadecyloxy-2-hydroxypropane-1-phosphocholine; DCIP = dichloroindophenol

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 93
 OH OH

',~OH ',~OH
"7
MONOOXYGENASE
o-...,.CH, ~ o- £~
02
.a-
6-MePH 4 q-6-MePH 2

NAD NADH

Scheme 2

from Scheme 2. It should be noted that the Km for 6-MePR4 is 138 J.l.M, and the Km for
batyl alcohol was 25 J.l.M.4 At concentrations of 6-MePR4 above 0.1 mM the ratio
decreases. One possibility for these results could be that DHPR in the coupled assay is
inhibited by the higher concentrations of 6-MePR4. This was tested by determining the

Table 1 Initial rates of monooxygenase reaction. a

6-MePf4 q-6-MePH2 formedb NADH consumedc NADH/q-6MePH2

(mM) (nmoles/rnin) (nmoles/min)

0.48 12.3 10.2 0.83

0.24 9.9 8.4 0.85
0.10 6.0 5.7 0.95
0.05 3.4 3.5 1.01

a In 0.1 M Tris-HCl buffer pH 7.5 and 25°C; RS-Batyl alcohol at 0.1 mM, per 0.5 mg of microsomal
protein. b Direct assay (Scheme 1) c Coupled assay with NADH at 100 J1.M (Scheme 2).

Table 2 Inhibition of DHPR activity in Tris-HCl buffer pH 7.5 at 25oca

6-MePR4 added (mM) Initial rates of NADH oxidation (.1. absorbance/min)
DCIP (0.037 mM) K3Fe(CN)6 (0.075 mM)

0.07 (0.033 mM excess) 0.0804 (100%) 0.128 (100%)

0.46 (0.423 mM excess) 0.0771 (96%) 0.127 (99%)
0.92 (0.883 mM excess) 0.0657 (82%) 0.104 (81%)

a NADH at 100 J1.M

nitial rates of oxidation of NADH in the DHPR reaction using DCIP (two electron oxidant)
and K3Fe(CN)6 (one electron oxidant) in which increasing excesses of 6-MePR4 were
present in solution. Inhibition of DHPR was observed (Table 2) and may partly account
for the apparent decoupling of the reaction in Scheme 2. Another contributor to the
apparent decoupling at high concentrations of 6-MePR4 could be due to theE value for 6-
MePR4 used for determining the reaction extinction coefficient. In Table 1 (column 1)

94
 the initial rates in the direct assay at 0.48 mM of
6-MePI4 gave a ratio of 0.83 when the E value of
3440 M-lcm-1 at pH 2.5 (4 mM HCl) was used.
If an E value of 3705 M-1cm-1 is used the ratio
becomes 0.89. The possibility of uncoupling
makes the assays that use NADH consumption in
a coupled reaction as a measure of
monooxygenase activity for comparing the
activity of cofactors or substrates unreliable (cfref
3 when 6-MePH4 was compared with 6,7-
DiMePf4).
(ii) In the second study the amounts of 6-
4 6 10 12 MePH4 oxidised in the direct assay at various
Time(min) time intervals were determined. The direct
Figure 1. Stoichiometry of 6-MePH4
reaction was then repeated using the same
oxidised and batyl alcohol consumed. conditions but with a-14C-batyl alcohol (3-
1'[14C]octadecyloxypropane-1,2-diol) as the
substrate. Aliquots were withdrawn from the mixture at time intervals, the reaction was
stopped with formic acid, and the solution was lyophilised. The residues were extracted
with CH2Ci2 and the lipids were separated on tlc plates [silica gel, with n-hexane, ethyl
ether, acetic acid (80:30:1) as eluant], and the Rpvalues of the radioactive lipid bands were
visualised by autoradiography. The various bands were collected, extracted with ethanol
and the radioactivity was determined by scintillation counting. The combined results are in
Figure 1. This Figure shows that during the first two minutes of the reaction the
stoichiometry of 6-MePI4 oxidised to the batyl alcohol consumed is one and confirms that
initial rates measured during the first two minutes are reliable for kinetic studies.

INHIBITION OF GLYCERYL ETHER MONOOXYGENASE

The kinetics of hydroxylation of several ether lipid substrates by the
monooxygenase using 6-MePJ4 as cofactor were studied by the direct assay and apparent
Km and Vmax values were evaluated.4,5 Most of the ether lipids were insoluble in water,
but were readily solubilised in the presence of 0.08% of the non-ionic detergent Mega-10.
The concentration of Mega-10 in the assay mixture was 0.08% (2.3 mM) which is below
the critical micellar concentration (7 mM)6 of the detergent in water. Monooxygenase
activity at various concentrations of Mega-10 (with 0.5 mM 6-MePI4 and 0.1 mM batyl
alcohol) was steady up to 0.2% of detergent but decreased when the concentration
increased further (Figure 2). Because of this decrease in activity we studied the inhibition
of activity further (cf ref 4 for effect of other detergents). The activity at various
concentrations of batyl alcohol and of Mega-10 (at 0.5 mM 6-MePI4) revealed that
inhibition was of the "noncompetitive type" type [ki = 1.74 ± 0.37 mM] (Figure 3). We
also found that at constant concentrations of Mega-10 (0.08%) octadecanol inhibited
monooxygenase activity. The kinetics revealed that inhibition was of the "competitive
type" [ki = 765 ±80 j.!M] (Figure 4).
RS-lyso-PAF is water soluble and its kinetic parameters in the absence of detergent
were determined (app Km 173 J.LM, and app Vmax 34 nmoles of6-MePf4oxidised/min.
mg of protein). Addition of Mega-10 (at 0.08%) inhibited enzyme activity completely.
Comparison of the activity in the presence of 0.005% of Mega-10 showed that the
inhibition was of the "noncompetitive type" [ki = 141 ± 15 J.LM] (Figure 5).
The ether substrates are not unlike detergents and most likely form mixed micelles.
Inhibition of activity could be due to a variety of effects, e.g. the detergent may decrease
the rates by binding at or close to the active site of the enzyme, or decrease the effective
concentration of the substrate by forming mixed micelles, or vary the critical micellar
concentration. Since reasonable kinetics of inhibition could be obtained with this system
further study is warranted in order to better understand the processes involved.

95
 0.05

0.04
~:?
015 0.03
~i:l
~o
~'""
=to
=·- o.oz
=~
=::
~~
0.01

0.00 +-T"""T.....,.....,..-T"""''.....,.....,..-T""'I-r....
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.5 1.0
Mega-10 (%) Batyl alcohol (11 S) )lM-1

Figure 2 Effect of Mega-10 on mono-

Figure 3 Monooxygenase inhibition by
oxygenase actitvity. 6-MePf4 =0.5 mM,
Mega-10: A=2.3 mM; B=7.0 mM;
batyl alcohol =0.1 mM C=9.8mM

0.20

f
2
~

0.15

~ 0.10
=
~
Q 0.05
~
::;
0.00 0.05 0.10 0.15
0.0 0.1 0.2 0.3
Batyl alcohol (liS) !lM-1
lyso PAF (1/S) J.l.M-1
Figure 4 Monooxygenase inhibition by Figure 5 Monooxygenase inhibition
octadecanol: A= 0 mM, B = 0.10 mM, by Mega-10: A= 0 mM, B = 0.14 mM
C=0.30mM

REFERENCES

1. Teitz, A., Lindberg, M. & Kennedy, E.P. (1964) J.Biol.Chem. 239, 4081-4090
2. Armarego, W.L.F. & Kosar-Hashemi, B. (1990) Chemistry and Biology of Pteridines (Curtius,
H.Ch., Gisla, S. & Blau, N, eds) Walter de Gruyter pp. 620-623
3. Kotting, J., Unger, C. & Eibl, H. (1987) Lipids 22, 824-830
4. Armarego, W.L.F. & Kosar-Hashemi, B. (1992) Pteridines 3, 95-96
5. Kosar-Hashemi, B. & Armarego, W.L.F. (1993) Hoppe-Seyler, (in press)
6. Neugebauer, J.M.. (1990) Methods Enzymol. 182, 239-253

96
THE ISOLATION AND CHARACTERIZATION OF CLONES OF
4a-HYDROXYTETRAHYDROBIOPTERIN DEHYDRATASE

Seymour Kaufman, Bruce A. Citron, Michael Davis and Sheldon Milstien

Laboratory of Neurochemistry, National Institute of Mental Health
Betheada, MD 20892

The phenylalanine hydroxylating system is complex, consisting of three enzymes

and two coenzymes 1•2•3. One of the coenzymes, NADH, plays a near-ubiquitous role in
intermediary metabolism. By contrast, the other cofactor, tetrahydrobiopterin (BH 4),
discovered during our early studies on the characterization of the phenylalanine
hydroxylating system, 1 plays a unique role as the essential coenzyme for certain
hydroxylases such as the enzymes that catal~ze the hydroxylation of the aromatic amino
acids, phenylalanine, tyrosine and tryptophan .
The scheme shown in Figure 1, summarizes the reactions catalyzed by each of the
three enzymes. Phenylalanine hydroxylase catalyzes a coupled reaction in which
phenylalanine is oxidized to tyrosine and the tetrahydropterin (BH4 or an active analogue
such as 6-methyltetrahydropterin) is converted to the carbinolamine, 4a-
hydroxytetrahydropterin. Although this compound is unsta~le, breaking down
nonenzymatically to the corresponding quinonoid dihydropterin, the reaction is also
catalyzed by an enzyme, 4a-hydroxytetrahydrobiopterin dehydratase, also called 4a-

NAD+ ~ DHPR 0 DEHYDRATASE I

~ II ~20
NADH+H+ N~1<~
HN)l~)
2 N N
H

Figure 1. The phenylalanine hydroxylating system.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 97
 carbinolamine dehydratase 3.4·5. Finally, the cycle is completed by the reduction of the
dihydropterin back to the tetrahydro level, a reaction catalyzed by dihydropteridine
reductase (DHPR), which utilizes NADH (NADPH is less active) as the electron donor6•
Since this communication deals principally with the dehydratase, it may be useful to
review some of its characteristics, as well as those of the reaction that it catalyzes. Unlike
the other components of the hydroxylation system, whose general roles were rather quickly
sorted out once they were separated from each other, the participation of still another
enzyme in the hydroxylation reaction remained concealed for about 20 years. The reason
for this is that under our usual assay conditions, which are carried out at neutral pH, the
reaction catalyzed by this cryptic enzyme occurs fast enough nonenzymatically so that it
does not limit the rate of phenylalanine hydroxylation. In the pH range of 8.2 to 8.4,
however, the nonenzymatic rate is slow enough to severely limit the rate of the
hydroxylation reaction .
In the early 1970's, we purified a protein from rat liver, that we called phenylalanine
hydrox~lase stimulator (PHS), which could stimulate the hydroxylation reaction at pH 8.2
to 8.45• • Furthermore, in experiments carried out with BH 4 under these conditions, a
pterin accumulated that we showed was a precursor of quinonoid dihydrobiopterin, i.e., it
was an intermediate between BH 4 and the dihydro derivative. We also showed that PHS
was an enzyme that catalyzed the conversion of this intermediate to the dihydropterin.
Based on the properties of this pterin, we concluded that it was the corresponding 4a-
hydroxytetrahydropterin Fd that PHS is an enzyme that catalyzes its conversion to the
quinonoid dihydropterin2• . These conclusions were subsequently substantiated4.
With the demonstration that the phenylalanine hydroxylating system consists of
multiple components, it was predicted that variant forms of phenylketonuria exist, each one
caused by the lack of one of the essential components of the system, phenylalanine
hydroxylase, DHPR and BH/. Because the reaction catalyzed by the dehydratase, as
mentioned, can occur nonenzymatically, it was also predicted that a complete lack of this
enzyme would be expected to lead to only mild hyperphenylalaninemia (HPA) 10•
Recently, several patients have been described with a mild form of HPA, who have
elevated urinary levels of an isomer of BH 4, 2-amino-4-hydroxy-7-[1,2-dihydroxypropyl)-
(L-erythro)-5,6,7 ,8-tetrahydropteridine] ("7-tetrahydrobiopterin"or7-BH 4 ), * and
decreased levels of BH 411 • It has been suggested that the accumulation of abnormal
amounts of 7-BH 4 might be the result of an alteration of the gene coding for 4a-
carbinolamine dehydratase 12•13 . A possible causal connection between excretion of7-BH4
and a dehydratase deficiency was suggested by the observation that in addition to its rapid
nonenzymatic dehydrati~n in vitro, a small percentage of the 4a-carbinolamine rearranges
to form the 7-isomer 12. 1 • Furthermore, under conditions where the carbinolamine and 7-
BH4 are being generated during the course of the phenylalanine hydroxylase-catalyzed
hydroxylation of phenylalanine, the addition of the dehydratase markedly inhibits the rate
of formation of the 7-biopterin derivative, presum~bly by diverting a greater fraction of the
4a-carbinolamine to the quinonoid dihydropterin 1 •
Although the precise mechanism by which a dehydratase deficiency would lead to
mild HPA is not known, there are several plausible possibilities. First, in the absence of the
dehydratase, the dehydration of the 4a-carbinolamine may become rate-limiting for the
hydroxylation of phenylalanine. Alternatively, 7-BH4 itself may impair the phenylalanine
hydroxylation reaction. One mechanism for such impairment was suggested by the
finding that although 7-BH4 is active as a coenzyme with phenylalanine hydroxylase, its
oxidation by Jhe hydroxylase is largely uncoupled from the hydroxylation of
phenylalanine 1 . Furthermore, 7-BH4 is also a potent inhibitor of the hydroxylase 1 .
Thus, the dehydratase plays a dual role of catalyzing the dehydration of the carbinolamine
and preventing the isomerization of the pterin cofactor to an inhibitory analogue.
A direct test of the hypothesis that HPA patients who excrete large amounts of 7-
BH4 suffer from a dehydratase deficiency would be to assay for the enzyme in a suitable
tissue sample from an affected patient. This approach was precluded, however, because the
tissues that contain an abundance of the dehydratase, such as liver and kidney, 16 are not
readily available for this purpose. Furthermore, neither dehydratase activity nor Western-
positive material could be dFtected in primary human fibroblasts or in human blood cells 16•
Accordingly, we necided to clone and sequence the dehydratase. After isolating
and sequencing tryptic peptides isolated from the pure rat liver enzyme, we used PCR
*Due to the fluorescence properties of oxidized pterins, urine samples are oxidized prior to analysis by
HPLC. For this reason, the actual compound that is detected in the urine of these patients by this method
is 7-biopterin, which is almost certainly derived from 7-BH4
98
primers based on these peptides to generate a putative dehydratase-specific fragment. After
ligation to a plasmid vector, two inserts containing the primers straddling an identical76-
base-pair segment were isolated and sequenced. The correspondence between the DNA
and peptide sequence confmned the identification of our subclone as dehydratase eDNA 17•
A comparison of our DNA sequence for the dehydratase with the sequence data
bases indicated that it is identical with the reported coding sequence for a nuclear protein,
DCoH, which is the dimerization cofactor for the homeodomain protein, hepatocyte nuclear
factor-Ill ~1-ll). DCoH is required for maximal transcription activity mediated by
HNF1-Il 1 • Furthermore, the molecular mass of DCoH (12kDa) and its amino acid
composition are essentially identical to those previously reported for the dehydra\\se 5• In
addition, the tissue distribution of dehydratase activity and DCoH are very similar1 •16•
To determine whether the dehydratase activity and HNFl-ll homodimerization
activity are indeed present within the same polypeptide, the dehydratase activity of purified,
recombinant DCoH protein18 was examined by two different methods. First, dehydratase
activity was determined indirectly by measurement of the stimulation of phenylalanine
hydroxylase activity under c9.nditions where the breakdown of the 4a-carbinolamine limits
the rate of hydroxylation 2• •8•19. Recombinant DCoH and glutathione S-transferase-
DCoH (GST-DCoH) fusion protein had.yssentially the same dehydratase specific activity
as pure rat liver dehydratase (Fig. 2A) 1 . A direct assay for the dehydratase activity is
based on monitoring the loss of the 245-nm absorbance of the 4a-carbinolamine that is
formed duri% the pterin-dependent hydroxylation of phenylalanine by phenylalanine
hydroxylase · (Fig. 2B). Initially, there is a rapid increase in a~sorbance due to the
enzymatic conversion of tetrahydrobiopterin to the 4a-carbinolamine ·8. Upon addition of
dehydratase (arrow, Fig.2B), the concentration of the 4a-carbinolamine decreased as
indicated by the disappearance of the characteristic absorbance at 245 nm. The addition of
cloned DCoH resulted in a rapid decline in 245-nm absorbance (Fig. 2B), indicating not
only that DCoH has dehydratase activity, but that the specific activities of the two proteins
are similar. Dehydratase activity was also found in cell extracts of Chinese hamster ovary
cells transfected with DCoH or DCoH plus HNFl-ll but was absent in transfectants
containing only the parent vector or HNFl-ll alone 17.
In addition to the dehydratase and DCoH having essentially the same specific
enzyme activity, DCoH and its fusion protein react with an antibody raised against pure rat
liver dehydratase (Fig. 3).

0.3

J
'tl
.!! 0.2
()

...l!!
0
0
0.1

2 4 6 8 10
Time (min)
Figure 2A. 4a-Carbinolamine dehydratase activity. Stimulation of phenylalanine hydroxylase-catalyzed
hydroxylation of phenylalanine with the addition (at arrow) of rat liver dehydratase, cloned DCoH, or the
GST-DCoH fusion protein. Neither the addition of glutathione alkyltransferase nor glutathione
alkyltransferase fusion protein with FK506-binding protein 12 stimulated this activity (data not shown).

99
 0.3

0
}
0.2

0.1 +----...------.-~-----.-----,-----J
0 100 200 300 400
Time (sec)

Figure 28. 4a-Carbmolamme dehydratase acuvlty Effect of the DCoH (added at ~~w) on the UV
absorbance changes dunng OXIdation of tetrahydrob10ptenn by phenylalarune hydroxylase .

0
~
~ k<J
~ ~
c; c; cY (g
A B
- 50

- 33
- 28

20- -19
14-
6.1- -
Figure 3. Gel electrophoresis and 1mmunoblot analySIS of 4a-tetrahydrob1optenn dehydratase (CDH),
dlmenzauon cofactor (DCoH), and GST-DCoH fus1on protem. (A) Punfied dehydratase (3 Jl.g) stamed
w1th Coomass1e blue R-250. (B) lmmunoblots (16) of 7 IJ.g of dehydratase or DCoH and 20 IJ.g of GST-
DCoH fus10n protem. Dashes mdlcate the pos1Uons of molecular mass (kDa) markers. The molecular
mass of the dehydratase/DCoH 1s 12 kDa, and that of the fus10n protem IS 38 kDa.

100
 The final test of the hypothesis that mild HPA patients who are characterized by the
excretion of elevated amounts of 7-biopterin suffer from a defective 4a-carbinolamine
dehydratase depends on the demonstration that such a patient does, in fact, have a defect in
the gene coding for the dehydratase.
We have recently isolated genomic DNA from such a patient, as well as from both
of his parents. An examination of the two alleles of the dehydratase revealed that both
alleles in the patient are defective; one is a premature termination mutation and the other is a
substitution mutation. The father had the wild-type and termination alleles and the mother
carried the wild-type and the substitution mutation (B. Citron, S.Kaufman, S. Milstien,
E.W. Naylor, C. Green and M.Davis, submitted for publication).
These findings confirm the hypothesis that a defect in the 4a-carbinolamine
dehydratase gene impairs the ability of BH 4 to function as a coenzyme for phenylalanine
hydroxylase and thereby leads to mild hyperphenylalaninemia. Together with previously
identified disorders in phenylalanine hydroxylase and dihydropteridine reductase, there are
now identified genetic deficiences in the three enzymes involved in the phenylalanine
hydroxylation system.
It is of interest that the patient that we have studied shows no signs of impaired
catecholamine or serotonin metabolism, a result that is coherent with the finding that these
patien<V' have been reported to have normal levels of biogenic amines in their cerebral spinal
fluid2 . These findings are consistent with results of our studies of the tissue distribution
of the dehydratase in the rat, which showed that except for the pineal gland, the level of
dehydratase is low in tissues that contain high levels of tyrosine and tryptophan
hydroxylase activity. Based on these results, we concluded that if the tissue distribution of
the dehydratase in humans is the same as in the rat, then this enzyme may not play an
important role in the regulation of the synthesis of those neurotransmitters that are derived
from the hydroxylated amino acids 16•
Finally, the demonstration that genetic defects in the dehydratase gene exisf when
considered together with our finding that this enzyme is the same as DCoH, 1 which
together with HNFl- a. is involved in the regulation of transcription, raises questions about
whether any biochemical and physiological processes regulated by the DCoHIHNFl-a.
complex are impaired in these patients.
Table llists some of the genes that are regulated by the DCoH/HNFl-a. system.
As can be seen, some of these regulated gene products function in such diverse processes
as blood clotting, glycolysis and gluconeogenesis. Our results, therefore, should serve to
focus attention on these physiological functions in this subset of HPA patients to ascertain
whether they are within the normal range during the neonatal period and remain in this
range during development. If these processes prove to be normal in our
hyperphenylalaninemic patients, it would suggest that the mutant dehydratase with an
amino acid substitution may still be sufficiently active in its HNF-1 a. binding role, even
though it is devoid of dehydratase activity.
Although it is conceivable that the dimerization cofactor activity of the dehydratase
involves its dehydratase enzymatic activity, it is probably more likely that this represents
another example of two disparate activities being carried on the same polypeptide chain.

Table 1. Some of the genes regulated by HNFl-a.

Albumin ~-Fibrinogen
Aldolase B Glutathione-S-Transferase Ya subunit
Alcohol Dehydrogenase 1 Hepatitis B virus preSl
a.l-Antitrypsin Phosphoenolpyruvate carboxykinase
Aminopeptidase N Pyruvate kinase
Apolipoprotein B a. 2,6-Sialyl-Transferase
Apolipoprotein A-ll Transthyretin
a.-Fetoprotein Tyrosine Aminotransferase
a.-Fibrinogen Urate Oxidase
A2 Vitellogenin

101
REFERENCES
1. S. Kaufman, Proc. Natl. Acad. Sci. USA 50:1085-1093 (1963).
2. S. Kaufman."Chemistry and Biology ofPteridines," Walter de Gruyter, Berlin (1975) pp. 291-304.
3. S. Kaufman, and D.B. Fisher. "Molecular Mechanisms of Oxygen Activation," Academic Press, New
York (1974) pp. 285-369.
4. R.A. Lazarus, S.J. Benkovic, and S. Kaufman, J. Bioi. Chem. 258:10960-10962 (1983).
5. C.Y. Huang, E. E. Max, and S. Kaufman, J. Bioi. Chem. 248:4235-4241 (1973).
6. J. E. Craine, E. S. Hall, and S. Kaufman, J. Bioi. Chem. 247:6082-6091 (1972).
7. S. Kaufman,!. Bioi. Chem. 245:4751-4759 (1970).
8. S. Kaufman. "Iron and Copper Proteins, Advances in Experimental Medicine and Biology," Plenum
Press, New York (1976) pp. 91-102.
9. S. Kaufman. "Phenylketonuria and Allied Metabolic Diseases," U.S. Govt. Printing Office, Washington
D.C. (1967) pp. 205-213.
10. S. Kaufman. "Advances in Human Genetics," Plenum Press, New York (1983) pp. 217-297.
11. N. Blau, H-Ch. Curtius, T. Kuster, A. Matasovic, G. Schoedon, JL. Dhondt, T. Guibaud and M..
Blascovics, J. Inherited. Metab. Dis. 12:335-338 (1989).
12. H-Ch. Curtius, C. Adler, I. Rebrin, C. Heizmann, and S. Ghisla, Biochem. Biophys. Res. Commun.
172:1060-1066 (1990).
13. M.D. Davis, S. Kaufman, and S. Milstien, Proc. Natl. Acad. Sci. USA 88:385-389 (1991).
14. M.D. Davis, and S. Kaufman, FEBS Lett. 285:17-20 (1991).
15. M.D. Davis, P. Ribeiro, J. Tipper, and S. Kaufman, Proc. Natl. Acad. Sci. USA 89:10109-10113
(1992).
16. M.D. Davis, S. Kaufman, and S. Milstien, FEBS Lett. 302:73-76 (1992)
17. B. A. Citron, M.D. Davis, S. Milstien, J. Gutierrez, D. B. Mendel, G. R. Crabttee, and S. Kaufman,
Proc. Natl. Acad. Sci. USA 89:11891-11894 (1992).
18. D. B. Mendel, P.A. Khavari, P. B. Conley, M. K. Graves, L. P. Hansen, A. Admon, and G.R. Crabttee,
Science 254:1762-1767 (1991).
19. C. Y. Huang, and S. Kaufman, J. Bioi. Chem. 248:4242-4251 (1973).
20. N. Blau, L. Kierat, H-Ch. Curtius, M. Blaskovics, and T. Giudici, !.Inherit. Metab. Dis. 15:409-442
(1992).

102
MOLECULAR CLONING AND RECOMBINANT EXPRESSION OF THE
HUMAN LIVER PHENYLALANINE HYDROXYLASE STIMULATING FACTOR
REVEALED STRUCTURAL AND FUNCTIONAL IDENTITY TO THE
DIMERIZATION COFACTOR FOR THE NUCLEAR TRANSCRIPTION
FACTOR HNF-la

Beat Thi:iny, Frank Neuheiser, Charles R. Hauer and Claus W. Heizmann

Division of Clinical Chemistry

University Children's Hospital
CH-8032 Zurich, Switzerland

INTRODUCTION

Conversion of phenylalanine to tyrosine by the cytosolic liver enzyme phenylalanine

hydroxylase (PAH) requires phenylalanine, molecular Oz and the obligatory cofactor
tetrahydrobiopterin (BH4)l.The phenylalanine hydroxylation produces stoichiometrically
an oxidized BH4 intermediate, the pterin-4a-carbinolamine (4a-CA). This unstable
intermediate is proposed to be dehydrated to the quinonoid dihydrobiopterin (q-BHz) by
the phenylalanine hydroxylase stimulating factor (PHS; also termed pterin-4a-
carbinolamine dehydratase)2. The presence of the dihydropteridine reductase (DHPR)
together with NADH completes the reductive recycling of the BR4 cofactor. A deficiency
in PAH, PHS or DHPR leads to different forms of hyperphenylalaninemia3.
We were interested in understanding the molecular nature of primapterinuria, a
variant form of hyperphenylalaninemia, characterized by the high level of blood
phenylalanine concomitant with the excretion of ?-substituted pterins in the patients'
urine4,5. Primapterinuria was proposed to be due to a block in the dehydration of the 4a-
CA that leads to the non-enzymatic conversion of ?-substituted pterins. To better define
the role of PHS in the BR4 cycle, and as a means to later characterize the gene from
primapterinuric patients, we cloned its eDNA from human liver and expressed the
recombinant protein in E. coli. In this report we summarize recent work which revealed
that the PHS is structurally and functionally identical to a regulatory cofactor (DCoH) that
dimerizes the homeodomain nuclear transcription factor HNF-la responsible for the
expression of liver specific genes6. (Part of this work has recently been published by our
group7 and others8.)

MATERIALS AND METHODS

Standard PCR was performed using rat liver eDNA as a template and the
oligonucleotides PCDH4 (5'-CGGAA TTCGGC(TCGA)GT(TCGA)GG(TCGA)TGGAA
(TC)GA-3') and PCDH5 (5'-CGGGATCCTT(AG)TA(TCGA)AC(AG)TT(AG)AACCA
(TC)TC-3') to amplify the intervening region between amino acid 21 and 70 of the PHS
sequence (Fig. 3 in ref. 7). The resulting fragment of 150 bp was confirmed to be coding

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 103
for the intervening sequence and was then used as a probe to screen a A.gt11-phage library
(Clontech) using standard hybridization conditions (62°C)9. Oligonucleotides used to
amplify the eDNA encoding the entire PHS/DCoH were: PCDH15 (5'-CGGAATTCATA
TGGCTGGCAAAGCACACAG-3') containing two restriction sites (EcoRI, Ndel) for
cloning and the codons for the first 7 amino acids, and PCDH16 (5'-CGGGATCCTATGT
CATGGACACGGTAC-3') containing one restriction site (BamHI) plus the codons for the
7 COOH-terminal amino acids and a stop codon. The template for standard PCR was one
of the positive A.gt11-clones from filter screening. The amplified DNA fragment coded for
a protein of 104 amino acids starting with methionine. The PCR fragment was first cloned
into pUC18 using the EcoRI/BamHI sites, and subsequently cut out with Ndei!BamHI to
ligate the resulting 0.3 kb fragment into pGEMEX-2 to generate pHDH5. This plasmid
contains the gene for PHS/DCoH fused with the start codon of the phage T7 gene 10.
Overexpression in E. coli BL21 (LysS) containing pHDH5 or pGEMEX was achieved in
2.5 hrs by inducing the cells with 0.4 mM IPTG at an OD of 600 nm of 0.6. A crude
protein extract was prepared according to a standard lysis protocol. PHS/DCoH activity
was determined by the ability to stimulate 5 mU of PAH (6.25 U/mg protein) at pH 8.4 in
the presence of 5 1..1M Blf4_ in a volume of 50 1!1, according to a described methodlO. Assays
for 4a-CA and q-BH2 were performed in a cuvette at 25oC in a volume of 1 ml containing
50 mM Tris-HCl, pH 8.4, 0.5 mM NADH, 2 mM L-phenylalanine, and 50 mU of
preincubated PAH. At time zero 15 J..LM BH4 was added.

RESULTS AND DISCUSSION

We have determined the complete amino acid sequence for the purified rat and
human liver PHS by using mass spectrometry (liquid secondary ionization mass
spectrometry/tandem quadrupole mass spectrometry and electrospray ionization-mass
spectrometry) in combination with Edman microsequencing analysis?. The primary
sequences of rat and human were found to be absolutely identical. The mature PHS
polypeptide consists of 103 amino acids with an acetylated alanine at its N-terminal end. A
protein molecular mass of 11,909 Da was obtained by using electrospray ionization-mass
spectrometry. This number coincides with the calculated mass of the protein assembled
from the amino acid sequencing data.
In parallel, we isolated a eDNA fragment coding for about 50% of the rat PHS gene
(150 bp). This was done by performing PCR with two degenerate oligonucleotides
(PCDH4 and PCDH5) and rat liver eDNA as a template. Using this eDNA as a
hybridization probe, we found at least 10 positive clones after filter-screening of 500,000
A.-phages from a A.gt11-derived human fetal liver library. Subcloning and DNA sequence
analysis of one of the candidate clones revealed an open reading frame coding for 104
amino acids, confirming the determined 103-residue of the protein primary sequence. The
5'-end of the eDNA encoded a starting methionine which appears to be cleaved off, since
the protein isolated from liver was found to bear an N-terminally acetylated alanine.
By searching the EMBL sequence library (release 30) we discovered about the same
time that three deposited nucleotide sequences were essentially identical to the liver PHS:
the rat, mouse and human dimerization cofactor referred to as DCoH, isolated from liver
cell nuclei6 (nucleotide sequence accession numbers: rat M83740, mouse M83741, human
M83740). DCoH was reported to be essential for the dimerization of the hepatocyte
nuclear transcription factor-1a (HNF-1a), a homeodomain-containing protein that is
functional only when in dimer form. For functional proof that a transcriptional
dimerization cofactor should bear in addition a dehydratase activity, we expressed and
examined the recombinant protein which we termed PHS/ DCoH.
A DNA fragment encoding the human liver PHS/DCoH was amplified by PCR using
two specific oligonucleotides (PCDH15 and PCDH16). The template used was the isolated
A.gt11 phage described above, which carried the complete coding sequence for PHS/DCoH.
The DNA fragment was inserted into an IPTG-inducible prokaryotic expression vector to
generate pHDH5. Induction of E. coli cells harboring the eDNA for PHS/DCoH, yielded
the 11.9 kDa protein in large quantities (Fig. lA). A crude protein extract prepared from
the overproducing E.coli strain showed an up to five-fold stimulation of PAH activity
when this was assayed in a standard tyrosine producing reaction (Fig. lB). The saturating
activity of the recombinant protein assayed in the crude extract was comparable to the

104
 "(:> "(:>
-$. -$ ~
\ ' \X 0
~'-> ~'-> ~c;
~~~ ~~~ q,x;~' M kD
A

'"'"" 120
B PHS/DCoH

......
-..
0 IK)
E
2:
Q)
&J

..
66 c:
(ii
45
31
...>-
0 40

--
t-

-
21.5 a>
......
pGEMEX
14.4
6.5 0
0 a:xl 400 roo eoo 1000 1200

2 3 4 Protein (ng)

Figure 1. Recombinant expression of PHS/DCoH and stimulation of tyrosine production by the addition of
PHS/DCoH to PAH. (A) Silver-stained SDS-polyacrylamide gel containing 400 ng crude extract from E. coli
cells harboring the plasmid pHDH5 not induced with IPTG (lane I) or IPTG-induced (lane 2), and 300 ng
purified PHS/DCoH from human liver (lane 3). Lane 4 contains the marker proteins. The dash indicates the
11.9 kDa PHS/DCoH. (B) The activity of the purified human liver PHS/DCoH is compared to the crude
protein extracts from E. coil cells harboring the expression vector with the human eDNA (pHDH5) or the
expression vector alone (pGE.MEX).

purified human liver enzyme. The reaction was strictly dependent on the addition of the
cofactor BI4 and the DHPR, the enzyme that reduces the q-BH2 to BI4 (data not shown).
In a more direct assay, we followed the formation of 4a-CA and q-BH2 during
phenylalanine hydroxylation in the presence of the purified PHS/DCoH compared to the
crude E. coli extract containing the recombinantly expressed protein (Fig. 2). At distinct
wavelengths, we followed directly the educt 4a-CA (at 244 nm) and the product q-BH2 (at
334 nm). Control experiments showed that, depending on the amount of PHS/DCoH
added, part of the accumulated 4a-CA is converted immediately, as reflected by the
decrease in absorption. At the same time, the product q-BH2 increases. This effect can be
observed when using either the purified human liver protein, or the recombinant
PHS/DCoH expressed in E. coli.
Thus, because the enzyme produced in E. coli has the same characteristics as those
described for the authentic human liver enzyme, the recombinant protein appears to be
identical and does not need any specific accessory functions. In addition, these results
clearly demonstrate that the nuclear protein required for dimerization of HNF-la, DCoH,
is identical to the cytosolic stimulatory factor for PAH. Whether primapterinuric patients
harbor a defect in PHS/DCoH, i.e. in 4a-CA dehydratase activity, as it is postulated,
remains to be clarified.

ACKNOWLEDGMENTS
We are grateful to Dr. I. Rebrin for the gift of purified PHS/DCoH and PAH. This
work was supported by a grant from the Swiss National Science Foundation (No. 31-
33897 .92), and in part by the Helmut Horten Stiftung, Stiftung fiir wissenschaftliche
Forschung an der Universitat Zurich, and Ciba-Geigy-Jubilaums-Stiftung.

105
 0.08

0.12
A B PHS/DCoH

E E
c c
~ 0.08
v
C')
C\1

...
C') 0.04
1ii 1ii
0 0.3j.lg 3119 0
0 0 .04 0
PHS/DCoH

0 0
0 20 40 80 80 100 120 0 20 40 60 80 100 120
Time (seconds) Time (seconds)

....... ........
0.4 0.08
c pGEMEX
D

E E
c c 35#lg Fr. I pHDHS
v pHDHS v
v C')
C\1 0.2 C') 0.04
a; 1ii
0 0 pGEMEX
0 0

35#lg Fr.!
0 0
0 20 40 60 80 100 120 0 20 40 60 80 100 120

Time (seconds) Time (seconds)

Figure 2. Formation of 4a-CA and q-BH2 during phenylalanine hydroxylation in the presence of
PHS/DCoH. Parallel detection of 4a-CA (A, C) and q-BH2 (B, D) was performed by following the
absorption at 244 nm and 334 nm, respectively. At the indicated time points (arrows) PHS/DCoH was added
either as purified human liver enzyme (A, B; dotted line: no protein added; dashed line: 0.3 Jlg PHS/DCoH
added; solid line: 3 Jlg PHS/DCoH added), or as a crude protein fraction (35 Jlg Fr. I) from E. coli (C, D;
dotted line: crude fraction harboring pGEMEX alone; solid line: crude fraction harboring pHDH5). The
addition of E. coli crude protein fraction itself in panel C resulted in a nonspecific increase of absorption at
244 nm.

REFERENCES

1. C.R. Scriver, S. Kaufman and S.L.C. Woo, The hyperphenylalaninemias, in: "The Metabolic Basis of
Inherited Disease," C.R. Scriver, A.L. Beaudet, W.S . Sly and D. Valle, eds., McGraw-Hill, New York
(1989).
2. R.A. Lazarus, S.J. Benkovic and S. Kaufman, J. Bioi. Chem. 258:10960 (1983).
3. F.K. Trefz, U. Lichter-Konecki and D. Konecki, Current Opinion in Pediatrics. 1:421 (1989).
4. N. Blau, H.-Ch. Curtius, T. Kuster, A. Matasovic, G.Schoedon, L. Dhondt, P. Guibaud, T. Giudici and M.
Blaskovics, J. lnher. Metab. Dis. 2:335 (1989).
5. N. Blau, L. Kierat, H.-Ch. Curtius, M. Blaskovics and T. Giudici, J. Inher. Metab. Dis. 15:409 (1992).
6. D.B. Mendel, P.A. Kahavari, P.B. Conley, M.K. Graves, L.P. Hansen, A. Admon and G. Crabtree,
Science. 254:1762 (1991).
7. C.R. Hauer, I. Rebrin, B. ThOny, F. Neuheiser, H.-Ch. Curtius, P. Hunziker, N. Blau, S. Ghisla and C.W.
Heizmann, J. Bioi. Chem. 268:4828 (1993).
8. B.A. Citron, M.D.Davis, S. Milstien,J. Gutierrez, D.B. Mendel, G.R. Crabtree and S. Kaufman, Proc.
Nat/. Acad. Sci. USA. 89:11891 (1992).
9. J. Sambrook, E.F. Fritsch and T. Maniatis. "Molecular Cloning: A Laboratory Manual," Cold Spring
Harbor, New York (1989)
10. C.Y. Huang, E.E. Max and S. Kaufman, J. Bioi. Chem. 248:4235 (1973).

106
PROGRESS IN THE STUDY OF BIOSYNTHESIS AND ROLE OF 7-SUBSTITUTED
PTERINS: FUNCTION OF PTERIN-4a-CARBINOLAMINE DEHYDRATASE

Hans-Christoph Curtius 1 , Sandro Ghisla 2 , Hiroyuki Hasegawa 3 ,

Nenad Blau 1 and Igor Rebrin 4

1 Division of Clinical Chemistry, Department of Pediatrics, University of

Zurich, Switzerland
2 Faculty of Biology, University of Konstanz, Federal Republic of
Germany
3 Department of Bioscience, Nishi-Tokyo University, Uenohara,
Yamanashi,
Japan
4 Department of Human Genetics, University of Greifswald, Federal
Republic of Germany

INTRODUCTION

A new form of atypical phenylkeketonuria (PKU) was discovered in 1988.

Characteristic for this transient hyperphenylalaninemia is the excretion of 7-substituted
pterins in patients' urine, i.e. L-primapterin (7-isomer of L-biopterin), D- or L-ana-
pterin (7-isomer of D- or L- neopterin) and 6-oxo-L- or D-primapterin (7-isomer of
7-oxo-L- or D-biopterin) (1,2,3).
Meanwhile, the formation of these 7-substituted pterins could be reproduced by
in vitro incubation experiments using pterin-4a-carbinolamine dehydratase (PCDH)-free
phenylalanine hydroxylase (PAH). From these results a dehydratase defect (PCDH) was
postulated for these patients which provokes the conversion of the 6-substituted
pterins to their 7-substituted isomers (4,5).

DHPR
Tetrahydrobiopterin • I quinonoid -Dihydrobiopterin
Phe, 02 ~ NADH PC~ H:P
rvr ·4~:c~~bi~~i~~~~~·
~ t
~ t
Spiro-Compound
~ rearrangement

~
Primapterin
(L-7-Biopterin)

Fig. 1 Proposed reaction sequence for the formation of 7-pterins

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 107
 Until now 9 patients have been detected so far. All of them exhibit a light
hyperphenylalaninemia and most of them were detected by repetition of the new-born
screening and/or investion of family members. Hyperphenylalaninemia tends to develop
a little later in comparison to other PKU patients and was corrected with a low protein
diet and with administration of BH4.

MATERIALS AND METHODS

Materials: Pterin standards were purchased from Dr. B. Schircks Laboratory

(Jona, Switzerland). Human tyrosine hydroxylase isoenzyme 1 (THl) was a gift from
Dr. J. Haavik, Bergen Norway. Dihydropteridine reductase (DHPR) was purchased from
Sigma (St. Louis, U.S.A.). Catalase and NADH were purchased from Boehringer
(Mannheim FRG).
Methods: Tryptophan hydroxylase (TPH) was extracted and purified to homo-
genity from mouse mastoma cells (P-815 and FMA3) following the procedure of H.
Hasegawa et a!. (7). Phenylalanine hydroxylase (PAH) and PCDH were purified to
homo-genity from human livers of kidney transplantation donors by the method
reported in (6).
Tryptophan hydroxylase assay: the standard reaction mixture contained 0.1 M
tris/HCl,pH 8.4 200 )JM L-tryptophan, 0.3 mM NADH, 15 )JM Fe (NH4)2(S04)2, 30
).lg/ml DHPR, 1 mg/ml of catalase, 50 )JM BH4, and 1-2 ).lg of activated tryptophan
hydroxylase per 150 ).11 of reaction mixture. Reaction was performed at 30°C for 5 min
and stopped with HCl04. 5-hydroxy-tryptophan (5HTP) was determined by HPLC with
fluorescence detection. The PHA, tyrosine hydroxylase (TH), DHPR and dihydrofolate
reductase (DHFR) and PCDH activity were measured, described in (6) and (9).

RESULTS AND DISCUSSIONS

It appears that the rearrangment of 6- to 7-pterin is independent of the

structure of the pterin side-chain. Thus, tetra-hydro-neopterin (NH4) and 6-
hydroxymethylpterin are also converted to the corresponding 7-substituted isomers (6)
and Davis et a!. have demonstrated that this is also the case for 6-methyltetrahydro-
pterin (5). The quantity and activity of PAH and PCDH in different tissues are shown
in the following two tables.

TABLE 1
Activity of phenylalanine hydroxylase and pterin-4a-carbinolamine dehydratase (in ).lmol
tyrosine/min/ g tissue).

Tissue PAH PCDH

Rat liver 2.11 6.18

Rat kidney 0.74 1.08
Human liver 2.60 5.31
Human kidney 0.52

Activity of PAH and PCDH were measured as described in (6) and (9).

108
 TABLE 2
Quantity of phenylalanine hydroxylase and pterin-4a-carbinoamnine dehydratase (in
mg/ g tissue).

Tissue PAH PCDH

Rat liver 0.45 0.09

0.42a O.llb
Rat kidney 0.14 0.04
Human liver 0.43 0.08
0.41c

a Data from S. Kaufman, in "Methods in Enzymology" (S. Kaufman, ed.), p.3,

Academic Press, Orlando, 1987.
b Data from C.Y. Huang, E.E. Max and S. Kaufmann (1973) J. Bioi. Chern. 248,
4235.
c Data from J.P. Abita et a!., in "Methods in Enzymology" (S. Kaufman, ed.)
p.27, Academic Press, Orlando, 1987.

The two tables show, that the PCDH content of the tissues is about one fourth
of the amount of PAH. But when the enzyme activities are compared, they are twice as
high for PCDH compared to PAH. These findings allow the hypothesis, that PCDH is
not only involved in the aromatic amino acid hydroxylation but also acts in the cata-
lysis or regulation of other biochemical processes. The findings of Davis et a!. (9) that
the distribution of PCDH in rat tissues is not related to the distribution of TH and
TPH is in favour of this hypothesis. Most importantly, it has been shown by Hauer et
a!. (10) as well as by Citron et a!. (11) that PCDH is identical with a dimerization
cofactor of hepatocyte nuclear transcription factor HNF -1 a.

Formation of ?-substituted pterins from their 6-isomers occurs in vitro also

with TH and TPH. The experiments were performed with recombinant human TH (6);
TPH was purified to homogenity from mouse mastoma cells by Dr. H. Hasegawa. To
measure the TH activity we have used the method described in (6) using Phe as
substrate and measuring dopa and tyrosine as product. Measuring parallely the
formation of L-tyrosine/L-dopa and L-primapterin during the TH reaction in absence
of PCDH a parallel increase of all reaction products was observed. The ratio of for-
mation of L-tyrosine plus L-dopa as compared to L-primapterin is about 1000 turn-
over events, indicating that 1 mol L-primapterin is formed for about 1000 turnover
events leading to L-tyrosine/L-dopa production.
Formation of L-primapterin during in vitro incubation of L-BH4 with PCDH-
free TPH also occurs. As with PAH and TH (6) it was not possible to investigate
primapterin formation after short incubation times (before 1 h of incubation) because
low concentrations of primapterin appear as a shoulder on the HPLC chromatograms of
biopterin.
The ratio of the formation of 5-hydroxytryptophan (5HTP) as compared to
primapterin is approx. 3 to 1, but this result can not be compared with the results for
TH or PAH where the ratios are about 600 to 1 and 1000 to 1 respectively. The TPH
reaction is only linear for about 5 minutes (7), (8) and after this time the 5HTP
formed is probably very low. In this context it is of special interest to know whether
tetrahydroprimapterin inhibits also in vivo PAH, TH and TPH leading to HP and
reduced dopa and serotonin formation. Our group (6) as well as Davis et a!. (5) could
clearly show that 7-BH4 is a strong competitive inhibitor of 6-BH4 (Ki~8 )JM) in the
PAH reaction but only under saturating conditions with Phe. In contrast, no apparent
inhibition of DHPR was found. We do not know whether the inhibition of PAH by 7-
BH4 is a sufficient explanation for the HP in these patients. As reported by Kaufman

109
the effect of 7-BH4 on the other two mamalian aromatic amino acid hydroxylases
indicated that 7-BH4 is a weak competitive inhibitor and a poor substitute for the
natural cofactor in both hydroxylation reactions.
We have investigated this question using purified TPH isolated from mouse
mastoma cells. We found a Ki value of approx. 225 ).lM compared to Davis et a!. (5)
who found a Ki value about 15 times higher (3,3 mM) in the crude extract from rabit
brain. In contrast to the experiments of Davis et a!., we have used purified enzyme
from a different source, using Hasegawa's procedure.
As previously mentioned, we have also found that NH4 is converted to the
corresponding 7-isomer and serves as cofactor in the PAH reaction in vitro. The de-
tection of 7-N in the urine of patients and controls requires that NH2 is reduced in
vivo to its tetrahydroform by DHFR and subsequently acts as cofactor of the pteridine
dependent hydroxylases. It could be shown by our group and others that in vitro NH4
can act as cofactor for the PAH reaction, however, the co-factor activity is only 20%
compared to that of BH4.
The concentration of 7-iso-N in urine is very low but on the other hand NH4
is very unstable due to cleavage of the side chain which is not the case with NH2 or
neopterin (N).
Thus, upon in vitro incubation of D-NH2 with DHFR and NADPH at pH 7.4
the formation of D-NH4 was clearly demonstrated.
This result and the finding of ?-substituted N in patients with atypical PKU but
also in normal individuals, is an indirect but clearcut evidence that D-NH4 acts as
cofactor of pterin dependent aromatic amino acid hydroxylases also in vivo. In our
opinion this is the only possible rationalization for the finding of 7-N both in the
patients and in normal controls. It is very unlikely that in vivo the formation of 7-N
is based on an other yet unknown mechanism.
Attempts to detect NH4 in the body cells by HPLC with EC detection have not
been successful up to date.

REFERENCES

(1) Curtius, H.-Ch., Kuster, T., Matasovic, A., Blau, N. and Dhont, J.-L. (1988)
Biochem. Biophys. Res. Commun. 153, 715.
(2) Dhont, J.-L., Guibaud, P., Rolland, M.O., Dorche, C., Andre, S., Forzy, G.
and Hayte, J.M. (1988) Eur. J. Pediatr. 147, 153.
(3) Blaskovics, M. and Giudici, T.A. (1988) New Eng. J. Med. 319, 1611.
(4) Curtius, H.-Ch., et a!. (1990) Biochem. Biophys. Res. Commun. 172, 1060.
(5) Davis, M.D., Kaufmann, S. and Milstein S. (1991) Proc. Natl. Acad. Sci. 88,
385.
(6) Adler, C., Ghisla, S., Rebrin, J., Haavik, J., Heizmann, C.W., Blau, N.,
Kuster, T. and Curtius, H.-Ch. (1992) Eur. J. Biochem, 208, 139-144.
(7) Hasegawa, H. and Ichinyama, A. (1987) "Methods in Enzymology" vol. 142, ed.
S. Kaufmann, Academic Press, Orlando, pp. 88 - 93.
(8) Fujisawa, H. and Nakata, H. (1987) "Methods in Enzymology" vol. 142, ed.
S. Kaufmann, Academic Press, Orlando, pp. 93 - 96.
(9) Davis, M.D., Kaufmann, S. and Milstien, S. (1992) FEBS Lett. 302, 73- 76.
(10) Hauer, C.R., Rebrin, J., ThOny, B., Neuheiser, F., Curtius, H.-Ch., Hunziker,
P., Blau, N., Ghisla, S. and Heizmann, C.W. (1993) J. Bioi. Chern. 268.
( 11) Citron, B.A., Davis, M.D., Milstien, S., Gutierrez, J., Mendel, D.B., Crabtree,
G.R. and Kaufmann, S. (1992) Proc. Natl. Acad. Sci. U.S.A. 89: 11891.

110
SPECTROSCOPIC CHARACTERIZATION OF HUMAN LIVER PTERIN
4a-CARBINOLAMINE DEHYDRATASE

Igor Rebrin\ Hans-Ch. Curtius 2, Sandro Ghisla3, and Falko H.Herrmann1

1Instituteof Medical Genetics, Ernst-Moritz-Arndt University, Greifswald, Germany

2Division of Clinical Chemistry, University of Zurich, Switzerland
3Faculty of Biology, University of Constanz, Germany

INTRODUCTION

The hepatic hydroxylation of phenylalanine catalyzed by phenylalanine hydroxylase

(PAH), is dependent on tetrahydrobiopterin (BH4) and two additional enzymes, pterin 4a-
carbinolamine dehydratase (PCD) and dihydropterin reductase (1}. PCD, previously named
"phenylalanine hydroxylase stimulating protein", catalyzed the conversion of 4a-OH BH4, an
intermediate product in hydroxylation reaction, to quinonoid dihydrobiopterin and water (3).
It was proposed that PCD play a key role in preventing the formation of 7-pterins which are
characteristic for primapterinuria, a new variant form of hyperphenylalaniemia ( 4,5). The
PCD was purified from rat and human liver and partially characterized (2,6). The primary
structure of purified enzyme was determined and it was been found that PCD is identical
to a homeodomein-containing protein involved in regulation of transcription (7). These facts
suggest on a double role of the dehydratase in the cell. In present work we undertook the
further characterization of PCD by means of fluorescence spectroscopy.

MATERIALS AND METHODS

The P AH and PCD were purified to homogenity from human livers of kidney
transplantation donors bathe method reported (6). The 6(R)-BH4 was from Dr. B.Shirks
Laboratory (Jona, Switzerland). Fluorescence spectra were measured with a corrected
spectrofluorometer Shimadzu RF5001PC, slit width used was 3 nm for excitation and
emission. Molar concentrations of native enzymes were expressed in terms of the tetramer
(Mr 45000) for PCD and of monomer (Mr 50000) for PAH. All buffers were filtered,
degassed and saturated with nitrogen before use.

RESULTS

Corrected fluorescence spectra of tryptophanyl residues of PCD are shown in Figure 1.

Excitation of native dehydratase at 295 nm in 0.1 M sodium phosphate buffer, pH 6.8,

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 111
 w
()
zw
()
(/)
w
a:
0
:J
...J
LL
w
>
~
w
a:

300 350 400 450

WAVELENGTH (nm)
Figure 1. Fluorescence emission spectra of tryptophanyl residues of PCD. Corrected fluorescence spectra were
obtained at 25 C by exciting at 295 nm, concentration of the enzyme was 1.1 uM. Native dehydratase in 0.1 M
phosphate buffer in the absence (a) or after addition of 40,uM BH4 (b); native dehydratase in 0.1 M Tris/HCI
buffer, pH 8.4 in the absence (c) or in the presence (d) of 40 pM BH4 • The spectrum (e) was obtained in 6 M
guanidine hydrochloride.

results in a peak with emission maximum at 225 nm. When enzyme was incubated in 0.1 M
Tris/HCl buffer, pH 8.4, the maximum wavelength shifted toward longer wavelength by 9
nm, without change in the intensity. On the other hand the fluorescence intensity decreased
by 60% with the emission maximum wavelength at 334 nm, upon addition of 40 pM BH4 to
native enzyme (Figure 1). A comparison of two difference spectra calculated by substracting
the initial fluorescence and those after incubation with BH4 shows that the resulting two
difference spectra are similar, although at pH of 8.4, a larger change in intensity is observed
as at pH of 6.8. The titration of native PCD with BH4 exhibits positive cooperativity (nH
1.5), with half maximal effect at about 12 .uM. Excitation spectra of tryptophan monitored
at various emission wavelengths shows maximum intensity at 280-285 nm, which are identical
with wavelength of the maximum absorption of 282 nm (data not shown). The intensity ratio
of the excitation spectrum at 280 nm to that at 300 nm varied and decreased as the emission
wavelength monitored was changed from a shorter to a longer one (Figure 2), which
suggests that two tryptophans are in different environments. The values of the intensity ratio
decreased when dehydratase is pre incubated with BH4, which implies that the absorption
band with longer emission peak also shifted to longer wavelength upon incubation.

112
 8~----------------------------~

... ...
...

6
0
i= +
<!: + + +
a: ...
~ +
U) +
z ...
w ...
1- +
z 4
+
...
...

I I I I
300 320 340 360 380
EMISSION WAVELENGTH (nm)

Figure 2. Intensity ratios of excitation spectra at 280 nm to those at 300 nm. Intensity ratios were obtained from
excitation spectra of PCD in the absence ( +) and in the presence of 20 pM tetrahydrobiopterin (A). Corrected
excitation spectra were measured by changing the emission wavelength monitored : 310, 320, 330, 340, 350, 360,
370 and 380 nm.

We have inspect the effect of PCD on intrinsic fluorescence spectra of the PAH. We have
found that pre incubation of PAH with PCD at molar ratio of 10:1 is accompaned by a
change in the fluorescence emission spectrum of hydroxylase (spectra not shown). The
corrected emission spectrum of PAH upon incubation with PCD exhibits a three distinct
peaks, with maximum at 338 nm (major peak), at 325 nm and 350 nm (minor peaks or
shoulders) which have different relative fluorescence quantum yields (Table 1).

DISCUSSION

Our results provide the first direct evidence for different conformational states of PCD.
The red shift in the fluorescence emission spectrum of the dehydratase at high pH or in the
presence of BH4 demonstrates that buried tryptophan residues ( >-em=325 nm) in
hydrophobic interior become some exposed to solution. The single emission peak of native
enzyme at pH 8.4 with maximum at 334 nm can be due one tryptophan residue (.\em =330
nm) which buried in the protein interior (not exposed to solvent) and second (.\em =340 nm
) which is partially exposed to solvent (8). Measurements of fluorescence excitation spectra
confirmed that the two tZsPtophan residues exists in the different microenvironment. The
estimated Kd of about 10· M for binding of BH4 suggest on lower affinity of dehydratase
to cofactor in comparison with PAH (9).

113
Table 1. Relative fluorescence quantum yieldsa of PAH, effect of incubation with PCD.

tryptophan
fluorescence

excitation ( nm) emission ( nm) Relative yields

0.1 M phosphate, pH 6.8

PAH 295 320 1.00

PAH + PCD" 295 325 0,84
295 340 0.94
295 350 0.82

PAH + Phe 295 340 1.20

PAH + Phe + PCD 295 325 0.81
295 340 0.96
295 350 0.86

0.1 M Tris, pH 8.4

PAH 295 340 1.15

PAH + PCD 295 325 0.87
295 340 1.00
295 350 0.90

PAH + Phe 295 340 1.17

PAH + Phe + PCD 295 325 0.98
295 340 1.03
295 350 0.94

•Relative quantum yield of PAH to free L-tryptophan.

• Molar ratio of PAH to PCD was 10:1

The changes in fluorescence of PAH followed by activation with substrate or by high pH

were extensively described in past (9,10). In present work we have detect further changes
in the fluorescence spectra of the PAH induced by low concentration of PCD. We have
shown differences in the fluorescence spectra of the PAH pre incubated with PCD from
those of native P AH either with or without activation by phenylalanine. These facts suggests
on the interaction of both enzymes and ability of PCD to induce some alterations in tertiary
structure of PAH.

REFERENCES

1. S.Kaufman, in:"Amino acid in Health and Disease", S.Kaufman, ed., Alan R.Liss, New York (1987).
2. C.Y. Huang, E.E. Max, and S.Kaufman, J.Biol.Chem.248: 4235-4251 (1973).
3. RA. Lazarus, S.J. Bencovic, and S.Kaufman, J.Biol.Chem. 258: 10960 (1983).
4. H.Ch. Curtius, A. Matasovic, G.Schoedon, T.Kuster, P.Guibaud, T.Guidici, and N.Biau, J.Biol.Chem. 265:
3923 (1990).
5. H.Ch. Curtius, CAdler, I. Rebrin, C. Heizmann, and S. Ghisla, Biochem.Biophys.Res.Commun. 172: 1060
(1990).
6. I. Rebrin, L.Petruschka, H.Ch. Curtius, CAdler, and F.H.Herrman, Pteridines 3: 55 (1992).
7. C.R. Hauer, I.Rebrin, B.Thony, F.Neuheiser, H.Ch.Curtius, P.Hunziker, N.Blau, S.Ghisla,and C.W.Heizmann,
J.Biol.Chem. 268:(1993).
8. EA. Burstein, N.S. Vedenkina, and M.N. Irkova, Photochem.Photobiol. 18: 263(1983).
9. R.S. Philips, MA.Parniak, and S.Kaufman, Biochemistry 23: 3836 (1984).
10. MA. Parniak, M.D.Davis, and S.Kaufman, J.Bioi.Chem. 263: 1223 (1988).

114
IS DffiYDROPTERIDINE REDUCTASE AN ANOMALOUS DffiYDROFOLATE

REDUCTASE, A FLAVIN-LIKE ENZYME, OR A SHORT-CHAIN

DEHYDROGENASE?

John M. Whiteley,l Nguyen H. Xuong,2 and Kottayill. Varughese1,2

lThe Scripps Research Institute, La Jolla, CA 92037

2university of California at San Diego, La Jolla, CA 92093-0317

INTRODUCTION

Dihydropteridine reductase (DHPR, EC 1.6.99.10) and dihydrofolate reductase

(DHFR, EC 1.5.1.3) both employ a reduced dinucleotide cofactor to convert a dihydro
pteridine substrate to a tetrahydropteridine product. In the former case the substrate has
a quinonoid dihydro structure whereas in the latter the 7,8-dihydro form is the substrate.
The quinonoid form resembles a flavin molecule and the enzymatic mechanism of
reduction has common features to this latter class of compound. Additionally, DHPR
contains a specific Tyr XXX Lys motif in its sequence that allows comparison with a
class of short chain dehydrogenases.l The relationship of DHPR to these differing
enzymatic types is illustrated briefly in the following discussion.

RESULTS AND DISCUSSION

Dihydropteridine reductase converts quinonoid dihydrobiopterin (qBH2) to

tetrahydrobiopterin (BH 4) in an NADH-mediated reaction. Dihydrofolate reductase
generates tetrahydrofolate (FH4) from 7,8-dihydrofolate (FH2) in an NADPH-mediated
reaction. Both products have the 5,6,7,8 tetrahydropteridine structure but have differing
substituents at the 6-position; BH4 having a dihydroxypropyl group, whereas FH4 has a
para-aminobenzoylglutamate component (Figure 1).
There is clearly a superficial similarity insofar as each enzyme employs a reduced
dinucleotide cofactor to convert a dihydropteridine to its tetrahydro counterpart.
However, upon further examination this initial observation proves to be deceptive
(Table 1). For example, DHPR is a dimer of Mr - 51,000 daltons,2 whereas DHFR
usually exists as a monomer with Mr - 18 -- > 22,000 daltons, 3 and DHPR has a

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 115
 NADH
NH2"y.70~tH
I I H
--H-+--t~ HN N fH·fH·CH,
DHPR 0 H HOHOH

NH2Y?LI ~-4-H ft COOH NADPH NH2Y?LI ~+H -oft COOH

HN N~CH2·NH-o~ C-NH-{H --.4~ HN N,1-CH2·NH fj ~ C-NH-6H
0 - (7H2)2 DHFR
H
o
H H
- (7H2)2
I

COOH COOH
Figure 1: The biosynthetic reductive pathways converting qBH2 and 7,8-FH2 to BH4 and FH4
respectively.

clearly enhanced specificity for NADH4 which contrasts with the preference for NADPH
exhibited by DHFR. 3 The specificity for the dihydropteridine substrate also differs.
Although a quinonoid dihydrofolate can be generated that can be a substrate for DHPR,
the quinonoid form cannot be a substrate for DHFR nor can the 7,8-dihydroform be a
substrate for DHPR. This is really not surprising as the ground state distribution of
electron density around the two dihydropteridine rings is distinctly different (J. Gready,
personal communication) and would suggest a priori an altered receptor site might be
necessary for reactivity. Moreover, the specific activities of the two enzymes are ver~
different (approx. 300 and 25 units/mg protein for DHPR2 and DHFR,
respectively) suggesting both a different mechanism and site of reaction for the two
enzymes. This is further substantiated by the clear lack of affinity exhibited by DHPR

Table 1
Comparative Properties of Dihydropteridine
Reductase and Dihydrofolate Reductase

Property DHPR DHFR

Molecular Weight -51,000 -18-22,000
{dimer) (monomer)
Cofactor NADH NADPH
Substrate q-Dihydropteridine 7,8-Dihydropteridine
Specific Activity -300 units/mg -25 units/mg
Oxidised Dinucleotide No Affinity Affinity
pH Optimum One Two
Hydrophobicity High Low
Amino Acid Titration Low Specificity High Specificity
Sequence Correlation Very Low

116
for the oxidised dinucleotide product of reaction, NAn+, 6 which is a very different
situation to that of DHFR, where NADP+ has been identified as a participant in the
kinetic turnover of the enzyme7 and has also been used in the generation of ternary
complexes that have sufficient stability to be examined by X-ray crystallography. 8 Other
experimentally observed differences would include a single pH optimum for DHPR4
compared to the two for DHFR, 3 the hydrophobic nature of DHPR, exhibited by its
binding to phenyl sepharose, 9 and the lack of unique titratable amino acids which can
cause abrupt changes in specific activity .10 This contrasts markedly with the
characteristic enhancement of eukaryotic DHFR specific activity when this enzyme is
titrated with mercury salts. 3 Moreover, there is no sequence correlation between the two
enzymes.2,3

DHPR

DHFR

Figure 2: Comparison of the backbone folding of DHPR with DHFR. The a-helices are
represented as cylinders, and the B-sheets are represented as triangles.

Both enzymes have now been crystallised 11,8 with resolution - 2.0 A and
comparison of the two structures illustrated diagrammatically in Figure 2 shows a
distinct change in format between the two proteins. Each structure contains eight B-
sheets with seven parallel and one antiparallel, however, DHPR has a higher a-helical
content with helices E and F forming the interface of the dimeric active enzyme. Of
greater significance is the arrangement of the dinucleotide cofactor on the protein matrix.
With DHFR the nicotinamide component is close to the N-terminal with the adenine
sector buried in the body of the enzyme whereas with DHPR the reverse is true, as the
adenine is close to the N-terminal and the nicotinamide is closer to B-sheets E and F and
a-helix G where it is poised to participate at the enzymatic active site. Moreover, theN-

117
Figure 3: Stereoview of the active site of DHPR showing a possible binding mode for qBH 2
and amino acids pertinent to the binding site and mechanism of action.

terminal region of DHPR has a classic Rossman fold12 suggesting similarities between it
and lactate dehydrogenasel3 rather than the folate reductases. In addition DHFR differs
insofar as it contains two sites, one for dinucleotide and one for folate. In the case of the
Escherichia coli enzyme the amino acids Asp 27, Phe 31 and Arg 57 play major roles in
folate binding. However, with DHPR the active site for pteridine binding does not
appear to exist until NADH is bound to the protein. The site is then created from the
connecting regions between fin and fip and the crossover that occurs between fiE and
fia. Thus rather than having a separate binding domain for substrate such as is observed
with lactate dehydrogenase or folate reductase, DHPR uses a strategy of simply
elaborating connecting loops to facilitate substrate binding that is intimately connected to
a requirement for prior NADH interaction.

exocyclic qBH 2 endocyclic qBH 2

Figure 4: Differing possible NADH-mediated reductive routes for qBH2 when bound to DHPR.
(R = -CH-CH-CH3).
OHOH

118
 The generally accepted mechanism for the reduction of 7 ,8-dihydrofolate by
NADPH and DHFR necessitates the reduced nicotinamide ring be aligned and
sufficiently close to the C6 position of the folate molecule - 3 A) such that hydride
transfer might occur followed by a proton transfer from solvent to the N5 position to
give the fully reduced pteridine molecule. This concept has been supported by a wealth
of kinetic data3 and more recently by definitive molecular structures derived for ternary
complexes from high resolution crystallographic studies of DHFR. 8 The situation with
DHPR is somewhat different. The quinonoid structure of the substrate pteridine has
distinct similarities to that of the flavin molecule and thus mechanisms of reduction such

130 135 140 145 150

DHPR 125 G G L L T L A G A K A A L D G T P G M I G Y G M A K --

PGDH 130 G G I I I N M S S L A G L M P V A Q Q P V Y C A S K --

17,8DH 134 S G R V L V T G S V G G L M G L P F N D V Y C A S K --

DADH 131 G G I I C N I G S V T G F N A I Y Q V P V Y S G T K --

20,8DH 131 G G S I V N I S S A A G L M G L A L T S S Y G A S K --

Figure 5: Alignment of common selected regions of five short chain dehydrogenases. Strictly
conserved residues are outlined. Residue numbers at the start of each line refer to each sequence
and those above to the rat liver DHPR. Abbreviations are: DHPR, rat liver dihydropteridine
reductase; PGDH, human 15-hydroxyprostaglandin dehydrogenase; 176DH, human 176-
hydroxysteroid dehydrogenase; DADH, Drosophila melanogaster alcohol dehydrogenase and
206DH, Streptomyces hydrogenans 20 6-hydroxysteroid dehydrogenase.

as those described for the NADPH-mediated glutathione reductase16 and 12.-

hydroxybenzoate hydroxylase17 appear to be better analogies for understanding the
DHPR mechanism of action. In crystallographic analyses of glutathione reductase the
reduced nicotinamide ring is aligned parallel to the flavin such that a hydride transfer is
facilitiated to N5 of the flavin. A similar situation can be created using graphic
techniques with DHPR (Figure 3). In this figure, the reduced nicotinamide is oriented
such that transfer of the C4 hydride can readily occur to the N5 position of the pteridine.
Such a transfer has been suggested previously on structural grounds, 18 by the low
ground state electron density at N5 (J. Gready, yersonal communication), and by
analogy with flavin mediated reductive processes. 6 In this instance, however, the
transfer of hydride must again be followed by a proton transfer to achieve a fully
reduced pteridine. Several possibilities exist for the site of protonation 19 (Figure 4).
Experiments conducted with tritium-labeled NADH have indicated that hydride is
transferred to a solvent exchangeable position on the pteridine ring18 and mechanistically
this type of center also accommodates the proton, therefore labeling experiments cannot
define the reductive centers. Reports have also indicated little energy difference between

119
the exocyclic and endocyclic isomeric forms of the quinonoid dihydropteridines19
therefore a priori it is difficult to assign a certain course of reductive action.
Nevertheless, specific features of the DHPR active site containing a "docked" qBH2
suggest a course of reaction in which the Tyr 146 XXX Lys 150 motif participates in
proton donation. A structure can be realised in which the hydroxyl substituent of Tyr
146 is within 3 A of the 4-oxo group on the pteridine, hence direct protonation could
occur. However, more recent high resolution X-ray structures suggest a water molecule
is situated such that proton donation could be translocated to N3 of the pteridine. Both
the lysine E-amino groups and tyrosine hydroxyl groups have pKs - 10, thus it is
possible that a protonated Lys 150 influences Tyr 146 sufficiently so that it or the
adjacent water molecule is the source of the compensatory proton. Compelling
supportive evidence stems from the observation that a Lys 150 Gin mutant has a specific
activity of only 50 units/mg protein compared to - 300 units/mg for the natural enzyme
and that a Tyr 146 Phe mutant has virtually no activity, yet has an equally strong affinity
for NADH as the wild-type enzyme, and also responds to the rat DHPR polyclonal
antibody. However, Lys 150 is also close to the nicotinamide ribose hydroxyl groups
(Figure 3) therefore an additional or alternate function of this amino acid could be to aid
in the correct orientation of NADH relative to substrate. Such issues have yet to be
clarified.
It has been reported that the Tyr XXX Lys pattern is present throughout a diverse
group of enzymes classified as the short chain dehydrogenases1 (Figure 5).
Each has a binding domain for a dinucleotide cofactor and each has a second
domain containing the sequences outlined in the figure. In every case, and more than
twenty examples are now known, the Tyr XXX Lys motif appears essential for
enzymatic activity. One other crystal structure has been reported, that of 20J3DH20 and
here again it is suggested that the tyrosine participates in proton donation via linked
water molecules. 21

CONCLUSION

It can be stated that DHPR has superficial similarities to DHFR insofar as a

reduced dinucleotide participates in generating a tetrahydropteridine, however,
mechanistically there appears to be a greater similarity to the flavin reduction pathway
existing in enzymes such as glutathione reductase. Furthermore, when examined in finer
detail, DHPR also incorporates the general features of the short chain dehydrogenases
exemplified by the 20 13-hydroxysteroid dehydrogenase from S. hydrogenans.

ACKNOWLEDGEMENT

This investigation was supported by grants from NIH: CA11778 (TSRI), RR01644
(UCSD); NSF: DIR88-22385 (UCSD) and the Markey Foundation (UCSD).

120
REFERENCES

1. B. Persson, M. Krook and H. Jornvall, Eur. J. Biochem. 200:537-543 (1991).

2. M. Shahbaz, J. Hoch, K.A. Trach, J.A. Rural, S. Webber and J.M. Whiteley, J. Bioi.
Chern. 262:16412-16416 (1987).
3. R.L. Blakley, in: "Folates and Pterins", R.L. Blakley and S.J. Benkovic, eds., John Wiley
and Sons, New York (1984), pp. 191-253.
4. C.E. Grimshaw, D.A. Matthews, K.I. Varughese, M. Skinner, N.H. Xuong, T. Bray, J.
Hoch and J.M. Wniteley, J. Bioi. Chern. 267:15334-15339 (1992).
5. T.J. WilJiams, T.K. Lee and R.B. Dunlap, Arch. Biochem. Biophys. 181:569-579 (1977).
6. K.E. Lind, Eur. J. Biochem. 33:67-70 (1973).
7. C.A. Fierke, K.A. Johnson and S.J. Benkovic, Biochemistry 26:4085-4092 (1987).
8. C. Bystroff, S.J. Oatley and J. Kraut, Biochemistry, 29:3263-3277 (1990).
9. D.A. Matthews, S. Webber and J.M. Whiteley, J. Bioi. Chern., 261:3891-3893 (1986).
10. S. Webber and J.M. Whiteley, Arch. Biochem. Biophys. 206:145-152 (1981).
11. K.I. Varughese, M.M. Skinner, J.M. Whiteley, D.A. Matthews and N.H. Xuong, Proc.
Nat!. Acad. Sci. USA 89:6080-6084 (1992).
12. R.K. Wierenga, P. Terpstra and W.G. H. Hoi, J. Mol. Bioi. 187:101-107.
13. J.L. White, M.L. Hackert, M. Buehner, M.J. Adams, G.C. Ford, P.J. Lentz, Jr., I.E.
Smiley, S.J. Steindel and M.G. Rossman, J. Mol. Bioi. 102:759-779 (1976).
14. L.E. Webb, J. Hill and L.J. Banaszak, Biochemistry, 12:5101-5109 (1973).
15. H. Eklund, J.P. Samana, L. Wallen, C.I. Branden, A. Akeson and T.A. Jones, J. Mol.
Bioi., 146:561-587 (1981).
16. E.F. Pai and G.E. Schulz, J. Bioi. Chern. 258:1752-1757 (1983).
17. H.A. Schreuder, W.G.H. Hoi and J. Drenth, Biochemistry 29:3101-3108 (1990).
18. W.L.F. Armarego, Biochem. Biophys. Res. Commun. 89:246-249 (1979).
19. J.E. Gready, J. Amer. Chern. Soc., 107:6689-6695 (1985).
20. D. Ghosh, C.M. Weeks, P. Grochulski, W.L. Duax, M. Erman, R.L. Rimsay and J.C.
Orr, Proc. Nat!. Acad. Sci USA 88:10064-10068 (1991).
21. D. Ghosh, personal communication.

121
TWO CRYSTAL STRUCTURES OF RAT LIVER DYHYDROPTERIDINE
REDUCTASE

Kottayil I. Varughese 1•2, Ying Su 1, Matthew M. Skinner 1,

Nguyen-huu Xuong 1, David A. Matthews3, and John M. Whiteley 2

1 University of California, San Diego, La Jolla, CA 92093-0317

2 The Scripps Research Institute, La Jolla, CA 92037
3 Agouron Pharmaceuticals, Inc. San Diego, CA 92121

INTRODUCTION

Dihydropteridine reductase (DHPR EC 1.6.99.10) catalyzes the reduction of

quinonoid dihydrobiopterin(qBH 2) to tetrahydrobiopterin, using NADH as a cofactor.
Tetrahydrobiopterin is a cofactor for the hydroxylating enzymes - phenylalanine hydroxy-
lase, tyrosine hydroxylase and phenylalanine hydroxylase. The genetic errors in
phenylalanine hydroxylase, DHPR or in genes coding for enzymes on the biosynthetic
route from GTP to qBH2, the substrate for DHPR, together contribute to the inherited
disease phenylketonuria (PKU). Recently several of the errors occurring in DHPR have
been identified. The structure of DHPR complexed with its cofactor, NADH in an
orthorhombic form (C222 1) has been solved and refined with 2.0 A resolution data. In
addition we determined the crystal structure of a monoclinic form which has two dimers in
the asymmetric unit. The orthorhombic form has only one monomer per asymmetric unit.
Modelling studies suggested the involvment of Trp 86 and Tyr 146 in substrate binding.
The W86I mutant was therefore constmcted and was found to have diminished enzymatic
activity. The Y146F mutant was constructed and was found to have no activity at all.

MATERIALS AND METHODS

All the studies described here were carried out using rat liver enzyme which was
purified using the procedures reported earlier1. DHPR crystallizes in a monoclinic and an
orthorhombic form. The first form to be grown was monoclinic 2 and it was obtained using
PEG as a precipitant at pH 7.8 by hanging drop methods. The orthorhombic form was also
crystallized under the same conditions3 and the crystal data for the two forrhs are given
below.
Orthorhombic: a= 50.10 A, b = 139.13 A, c = 64.93 A, Space group C222 1.
Monoclinic: a= 222.2 A, b = 46.5 A, c = 94.3 A, p= 100.1°, Space group C2.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 123
 The orthorhombic form was solved3 using multiple isomorphous replacement tech-
niques using 4 heavy atom derivatives. The model was built using an IRIS work station
using the program FROD04 . The structure consists of all but 4 residues at the N-terminal
(5 to 240) and 136 water molecules and it was refined using 8-2.0 ft.. data to an R of 16.3 %
using the programs X-PLOR5 and PROLSQ6. Even though the asymmetric unit consists
only of a monomer, the structure exists as a dimer in the crystal lattice and it uses the crys-
tallographic two fold axis for the formation of the dimer. The monoclinic form was solved
by molecular replacement techniques using the program MERLOT7 . A Crowther rotation
function was calculated using 10-4 ft.. data with a Patterson radius cut off of 23 ft... The
rotation search resulted in two unique solutions conesponding to the two dimers in the
asymmetric unit. The translation search using Crowther and Blow's method8 also gave
unique solutions. The initial R for the oriented and positioned model was 37%. The struc-
ture was refined using restrained refinement techniques to an R of 17.6% with 8-2.6 ft.. data.
The model consists of two dimer of DHPR and 160 water molecules.

RESULTS AND DISCUSSION

The geometry of the DHPR molecule is the same in both the crystal fonns. It is an
alP protein with a central P-sheet and a-helices on either sides as is seen in figure 1.

Figure 1. A backbone drawing of the DHPR dimer. The N and C-terminals of one of the
monomers are marked by the residue numbers 5 and 240 respectively.

A number of hydrophobic residues from each protomer come together at the dimer inter-
face to form the hydrophobic core and these hydrophobic interactions are a major stabiliz-
ing force for the dimer. Even though the molecular geometry is very similar in both crystal
fonns, the crystal packing is different in the two forms. The orthorhombic form is more
closely packed and it appears that the active site is less accessible due to crystal contacts.
The packing of the two forms are depicted in Figure 2.

124
 a

Figure 2. The packing diagrams for the orthorhombic (a) and monoclinic (b) crystal
forms. A solvent channel is formed parallel to the b-axis in the monoclinic form and it
makes the active site more accessible for substrate diffusion. The active site of DHPR is
only a surface channel and not a deep pocket. Figure 3 shows the active site of the
molecule.

Figure 3. A stereoview of a model of the ternary complex. Residues Trp86, Tyr 146 and
Lys150 are numbered. These residues appear to play key roles in the enzyme mechan-
ism(9).

125
 ACKNOWLEDGEMENT

This investigation was supported by Grant RR01644 (UCSD) from the National Institutes
of Health, Grant DIR88-22385 from the National Science Foundation (UCSD), the Lucille
P. Markey Foundation (UCSD), and Grant CA11778 from NIH (TRSI).

REFERENCES
1. S. Webber and J. M. Whiteley, Chemistry and Biology of Pteridines (Kisliuk, R.L. and
Brown, G.M. eds) Elsevier/North Holland, Amsterdam pp.211-214 (1978)

2. D. A. Matthews, S. Webber, J. M. Whiteley, J. Bioi. Chern. 261:3891-3893 (1986).

3. K. I. Varughese, M. M. Skinner, J. M. Whiteley, D. A. Matthews, and N. H. Xuong.

Proc. Nat!. Acad. Sci. USA 89:6080-6084 (1992).

4. A. T. Jones, J. Appl. Cryst. 11:268-272 (1978).

5. A. T. Brunger, J. Kuriyan, and M. Karplus, Science 235:458-460 (1987).

6. J. H. Konnert, and W. A. Hendrickson, Acta Cryst. A36:344-350 (1980).

7. P.M. D. Fitzgerald, J. Appl. Cryst. 21:273-278 (1988).

8. R. A. Crowther, and D. A. Blow, D. M. Acta. Cryst. B25:544-548 (1967).

9. J. M. Whiteley, N.H. Xuong and K.I. Varughese (this volume)

126
NEW INHIBITORS OF
DIHYDROPTERIDINE REDUCTASE (HUMAN BRAIN)

David Randlesa, Hiroyasu Taguchib and Wilfred L.F. Annaregoc

a Therapeutic Goods Administration, Dept. of Health, Housing and Community Services,

POBox 100, Woden, ACT 2606, Australia
b Dept. of Natural Science, Kyoto Women's University, 35-Kitahiyoshi-cho, Imakumano,
Higashiyama-ku, Kyoto 605, Japan
c Div. of Biochemistry and Molecular Biology, JCSMR, ANU, GPO Box 334, Canberra,
ACT 2601, Australia

The dihydropteridine reductase (DHPR) gene from rat liver has recently been clonedl
and the protein that was expressed from the eDNA was crystallised as the binary DHPR-
NADH complex. 2 The X-ray structure of the enzyme was determined and although the exact
location of NADH in the enzyme was determined in the binary complex, the precise position
of the pteridine cofactor was not obtained and had to be deduced from known kinetic data of
various pteridine cofactor analogues. 2 In order to obtain the precise position of the pteridine
cofactor it is necessary to crystallise the ternary complex ofDHPR, NADH and an inhibitor
whose structure is so close to that of a viable cofactor (eg 1) that it would bind at the active
site in the same manner as the pterin.

H2N
J~NtM•N N
1 H
[1] [2a] R = SMe
[2b] R = SCHzC6Hs
[2c] R = SCHz C6H4COzH(p)
[2d] R = NH2
[2e] R = OH
[2f] R = Me

Several inhibitors of sheep, rat and beef dihydropteridine reductases (DHPR) were
described more than a decade ago. These include methotrexate,3 aminopterin,4
amethopterin,5 folic acid6 and 6,7-dimethylpterin4,6 which were studied because they
inhibited dihydrofolate reductase (DHFR) which catalyses an apparently similar reaction, i.e.
the reduction of a dihydropterin [7 ,8(3H)-dihydrofolic acid] using a reduced pyridine
nucleotide cofactor (NADPH). These inhibitors were believed to bind at the pteridine domain
of the reductase. Other inhibitors of DHPR were Blue Dextran, Cibacron Blue F3GA and p-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 127
aminobenzoylglutamic acid which were bound to the pyridine nucleotide domain ofDHPR.5
In looking for better pterin inhibitors we reasoned that compounds related to quinonoid 6-
methyl-7 ,8(6H)dihydropterin [1] but which were not substrates for DHPR, would be
potential inhibitors of the enzyme and could be used to reveal the pteridine binding domain in
crystals of human DHPR containing NADH. 6-Methyl-6-azapterins (2, 6-
aminopyrimido[ 4,5-e][l,2,4]triazin-8-ones) suit many of the desired criteria. Their
structures are very similar to the effective substrate [1] and are unlikely to be reduced by
DHPR. The active site of the enzyme is known to tolerate small and large substituents at C7
of structure [1] without loss of activity_7,8 We have prepared six derivatives [2 a-t] and all
inhibit the enzyme from human brain.

0 0
r Me 3 :rN-~.:.C1 =NH
A A
HN H2N-N-C =NH. HX HN
Br + 1 2 ! .. R HX
H2N N O [ 4a] R = SMe, X Br= HzN ~N O •
[4b] R = SCH2 Ph, X = Br
[3] [4c] R = SCH2C6H4·P· C02H [S]
X= Br
[4d] R = NH2, X= Br [2 a-d,f]
[4e] R=Me,X=CI

SCHEME 1

Pyrimido[4,5-e][l,2,4]triazin-8-ones are known compounds and many have been

prepared, notably by D. J. Brown, S. Matsuura, E. C.Taylor and F. Yoneda and
coworkers.9 Most of the known compounds however have an oxo group at C6 as well as C8
and many have aryl substituents on N2 of structure [2]. Sugimoto and MatsuuralO prepared
several 6-amino-8-oxo derivatives with amino and alkylthio groups at C3 but none had an
alkyl group at N2. The derivatives [2 a-f1 that we prepared were not previously known.
We applied the procedure of Sugimoto and Matsuura successfully to prepare the 2-
methylpyrimidotriazinones [2 a-f1.11 The syntheses involved the condensation of 5,5-
dibromo-2-aminopyrimidine-4,6-dione [3] with the appropriate aminoamidine salts [4 a-e]
(Scheme 1). The dioxotriazinone [2e] was obtained by hydrolysis of [2b].

Table 1 Ionizationa of Pyrimido[4,5-e][1,2,4]triazin-8-ones in H20 at 25°C

0 1 0

N~N;wM• HN~N:N>
HN
~N
2
.. N
~R
HzN
~N '?..
N 3 R

pKa Species 'Anmb pKaC Species

3-SMe [2a] 4.26 (+) 274 3-SEt 6.72 (-)
3-SCH2Ph [2b] 4.72 (+) 272 3-SCH2Ph 6.72 (-)

3-NH2 [2d] 4.87 (+) 240 3-NH2 3.91 (+)

3-0H [2e] 1.43 (+) 240
6.11 (-) 370
3-Me [2f1 4.32 (+) 270
a Measured by standard procedures, cfThe Determination of Ionization Constants by A. Albert and
E. P. Serjeant, third edition, Chapman and Hall, London 1984; (+)for basic pKa and(-) for acidic pKa
b Analytical wavelength. c Taken from Ref. 10.

128
At first we thought that the products of the reaction were the intermediates [5] but ionization
data (Table 1), the stability of the free bases, 13C nmr chemical shifts and uv spectrall
confirmed that the products of these reactions are pyrimido[4,5-e][l,2,4]triazin-8-ones [2].
The ionization data are consistent with resonance stabilised cations (Scheme 2) but more

SCHEME2

important show that at the pH of the enzymic reaction (7 .4) these compounds are non-ionic
except for the dioxo compound [2e]. The open structures [5] would have pKa values above
9 in order to be consistent with an amidinium cation.12

Table 2. Inhibition ofDihydropteridine Reductase (human brain)a in 0.1 M Tris-HCl

(pH - 7.4 and 250C)

Inhibitor Procedure NADH Ki (and Kis) J..LM Inhibition Type

(J..LM)
3-SMe [2a] K3Fe(CN)6b 110 77 (±13) Competitive
{134 (±17), 160 {Mixed}
(±24)}
3-SCHzPh [2b] Peroxidase/HzOzb 132 17 (±2) Competitive
K3Fe(CN)6b 120 24 (±3) Competitive
3-SCHzC6f4COzH[2c] K3Fe(CN)6c 120 10.2 (±1.4) Competitive
3-NHz [2d] K3Fe(CN)6C 120 36.2 (±8.2) Competitive
3-0H [2e] K3Fe(CN)6b 112 307 (±30) Non-
competitive
{144 (±42)} {Competitive}
3-Me [2e] K3Fe(CN)6b 120 163 (±20) Competitive

a The substrate was quinonoid 6-methyl-7,8-dihydro(6H)pterin [1], values in chain brackets are from
alternative computations. b Using a single beam spectrophotometer. c Using a double beam
spectrophotometer.

129
All the pyrimidotriazinones inhibit human DHPR and the inhibition constants were
determined by measuring the rates at various concentrations of quinonoid 6-methyl-
7,8(6H)dihydopterin [1] and various concentrations of the triazinones [2 a-f]. The rate data
were computed for the different types of inhibition and the constants in Table 2 were those
which gave inhibition constants with the smallest error values. All the data fitted reasonably
well for competitive inhibition as we suspected from the close similarity of the structures of
the inhibitors [2] and substrate [1].

The sulphur derivatives [2a], [2b] and [2c] hydrolysed slowly at neutral pH to the
same dioxo derivative [2e] and would not be very good candidates for the ternary complex.
Also the sulphur compounds could be affected by X-ray radiation. The triazinedione [2e] is
not a very satisfactory example because it is predominantly in the anionic form at pH 7.4 and
because the presence of the 3-oxo group may allow the compound to bind at the active site
differently from the substrate [1], ie the 3-oxo group may bind at the site where the 4-oxo
group of the substrate [1] normally binds. Similarly the 3-aminotriazinone [2d] may bind
such that the triazine ring may bind at the pyrimidinone domain, ie the 3-amino group may
bind at the site of the 2-amino group of [1]. All these deficiencies are overcome in 2,3-
dimethyl-6-aminopyrimido[4,5-e][1,2,4]triazin-8-one [2f] which should bind to the binary
complex in the expected manner. This is further supported by the fact that quinonoid 6,7-
dimethyl-7,8(6H)dihydropterin is a very good substrate forDHPR. We have now expressed
human DHPR from cloned eDNA and crystallised the binary complex.13 Crystallisation of
the ternary complex with [2f] is in progress.

REFERENCES

M.Shahbaz, J.A.Hoch, K.A.Trach, J.A.Hural, S.Webber and J.M.Whiteley, (1987) J.Biol.Chem.,

262, 16412-16416
2 K.I.Varughese, M.M.Skinner, J.M.Whiteley, D.A.Matthews and N.H.Xuong, (1992)
Proc.Natl.Acad.Sci.USA, 89, 6080-6084
3 J.E.Craine, E.S.Hall and S.Kaufman (1972) J.Biol.Chem. 247, 6082-6091
4 K.E.Lind (1972) Eur.J.Biochem. 25, 560-562
5 M.M.Chauvin, K.K.Korri, A.Tripak, R.C.Simpson and K.G.Scrimgeour (1978) Can.J.Biochem. 57,
178-187
6 S.Cheema, S.J.Soldin, A.Knapp, T.Hofmann and K.G.Scrimgeour (1973)
Can.J.Biochem. 51, 1229-1239
7 W. L. F. Armarego, (1989) Pteridines, 1, 179
8 P. Waring and W. L. F. Armarego, (1987) Eur. J. Medicinal Chern., 22, 83
9 H. Neunhoffer and P. F. Wiley, Heterocyclic Compounds: Chemistry of 1,2,3-Triazines, 1,2,4-
Triazines, Tetrazines and Pentazines, J.Wiley and Sons, 33, pp.797-842 (1978)
10 T. Sugimoto and S. Matsuura, (1975) Bull. Chern. Soc. Japan, 48, 1679
11 D. Randles, H. Taguchi and W. L. F. Armarego, (1993) Heterocycles, (in press)
12 J.P. Horowitz and C. C. Rila, (1958) J. Am. Chern. Soc., 80, 431; S. J. Angyal and K. Warburton,
(1951) J. Chern. Soc., 2492
13 C. M. Hardy, B. Paal, P. Smooker, R. G. H. Cotton, P. Carr, D. L. Ollis, and W. L. F. Armarego,
(1993 unpublished results)

130
CYS ....,..SER MUTATIONS IN
ch-HUMAN DIHYDROPTERIDINE REDUCTASE

C.M.Hardy,* H.Averdunk,* B.Paal,* R.G.H.Cotton# and

W.L.F.Armarego*

* Division of Biochemistry and Molecular Biology, John Curtin School of Medical

Research, Australian National University, GPOBox 334, Canberra, ACT 2601, Australia
# The Murdoch Institute, Royal Children's Hospital, Flemington Road, Victoria, 3052,
Australia

The eDNA sequence of dihydropteridine reductase (DHPR) showed that the enzyme
has four cysteine residues_l,2 These are cys26, cys85, cys104 and cys161 . Evidence from
titration experiments of human and rat DHPR with thiol reagents and platinum II
complexes suggested that when one of the cysteines was masked by addition of NADH
then it did not react with these reagents and the enzyme was protected from inactivation.3.4
In order to see if one of these cysteine residues was the proton source for the enzymic
reduction of quinonoid dihydropterin substrates, we investigated the effect of replacing the
cysteine residues by serine residues, which are weaker acids, and we examined the kinetics
of the mutant proteins.
For this purpose a new plasmid was constructed from pWLA8 [which expressed
human DHPR in E.coli. K12 (AN1459) when the strain was grown at 300 and then at
45°C],5 and a phagemid vector, pMA200U, which had been derived from pCE30 into
which anjl ori sequence was inserted so as to allow the synthesis of single stranded DNA
for the purpose of sequencing the DNA.6 The original plasmid, pCE30, contained the
tandem A bacteriophage PR and PL promoters, the A ci857ts gene and the AmpR gene.
pWLA8 was digested with the restriction enzyme Bgl I (eleven base cutter) which gave
three fragments (1120 + 1130 + 2760 bps). Two fragments were of the same size so the
products were further digested with Sea I to allow removal of the unwanted fragment ( 1120
....,.. 755 + 365 bps). pMA200U was also digested with Bgl I to give two fragments (1270
+ 2882 bps). The purified (Gene Clean) 1270 bp fragment from pMA200U and the 1130
bp and 2760 bp fragments from pWLA8 were ligated to give the construct pWAM (Figure
1). When pWAM was transformed into E.coli (AN1459) it expressed chDHPR under
conditions similar to those used for pWLA8.
Synthetic 20-mer oligonucleotide primers in which the cysteine coding triplets are
replaced by serine coding triplets were prepared. The plasmid p W AM in the bacterial
strain XLI Blue was used to prepare single stranded DNA. With the Amersham
Mutagenesis System (version 2, code RPN 1523), which uses dCTP(S), we found that the
nicking experiment with the restriction enzyme Nci I was unsuccessful (probably because
pWAM has 13 cleavage sites), but with the enzyme Pvu I (pWAM has two such sites) the
mutated plamids were obtained. The mutageneses were confrrmed by sequencing and
reading only the G tracks because the triplets for cysteine (TGC or TGT) were replaced by
serine triplets (TCC or TCT) with replacement of G by C. Three mutant plasmids
pWAM3, pWAM6 (single mutations) and pWAM8 (double mutation) were prepared
(Figure 2).
The plasmids pWAM, pWAM3, pWAM6 and pWAM8 were transformed into
E.coli (AN1459) and the human proteins were expressed by growing the cells (20-40 L

Chemistry and Biology of Pteridines and Palates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 131
cultures) at 30°C in the presence of ampicillin to a OD595 of 0.3-0.5, and then grown at
45oc overnight. The DHPR was purified by ammonium sulphate precipitation (30%
saturation), affinity chromatography using Procion Brilliant Blue (RS [13324-20-4]
Reactive Blue 2) coupled to Sepharose CL 6B and eluting with a NaCl gradient ( 0 to 0.8

0<1 Q-i

l
-
Bgl I
+
Sea I Bg/1

7 55 bps 365 bps 2882 bps

~
11 30 bps
1270bps
2760 bps
ligate

Figure 1 Insertion of the ch-DHPR gene into vector pWAM for site directed
mutagenesis

ch-DHPR

J
Met
(ATG) I (TAG)
I
Cys26 Cys85
I
Cys104 Cysl61

r.+ +
pW 8
~
pWAM3
+
¥
+
pWAM6
Figure 2 The plasmids pWAM3, pWAM6 and pWAM8 are the same as pWAM
except for the changes in the DHPR genes indicated

132
M), followed by naphthoquinone affinity chromatography (see ref 7). The purity of the
proteins was checked by SDS-PAGE (Pharmacia Phast System). The DHPR protein from
pWAM and pWAM6 gave a single band (27 kDal), whereas pWAM3 (and to a lesser
extent) pWA8 was contaminated with a second minor protein (35 kDal) which could be due
to an oxidised form of DHPR. This was less than 40% in pWAM8 but slightly more in
pWAM3.

Table 1 Kinetic parameters for DHPR (0.1 M Tris/HCl, pH 7.5, 250C)

with quinonoid 6-Methyl-7,8(6H)dihydropterin (NADH at 100 j.!M)

Enzyme app. Km (j.IM) app. Vmax kcat (sec- 1)

(J.Ulloles/min/mg)

WAM 29.4 (± 1.3) 141 61

(Wild-type)

WAM3 23.6 (±1.1) 77* 33*

(cys104 _...ser)

WAM6 38.0 (±1.1) 356 154

(cys161 _... ser)

WAM8 25.5 (±3.7) 137 59

(<;ys26+85 _... ser)
* Approximate, due to contamination by a second protein

Table 2 Kinetic parameters for DHPR (0.1 M Tris/HCl, pH 7.5, 25°C)

with NADH (quinonoid 6-Methyl-7,8(6H)dihydropterin at 90 j.!M)

Enzyme app. Km (j.IM) app. Vmax kcat (sec- 1)

(J.Ulloles/min/mg)

WAM 39.1 (± 9.5) 127 55

(Wild-type)

WAM3 30.1 (± 11.1) 155* 67*

(cys104 _...ser)

WAM6 41.3 (± 1.1) 356 154

(cys161 _... ser)

WAM8 41.0 (± 9.4) 135 58

(cys26+85 _...ser)
* Approximate, due to contamination by a second protein

The absence of E.coli DHPR8 in the preparations was confirmed by the lack of
pterin-independent K3Fe(CN)6 activity in all samples. Amino acid analyses revealed that
the wild-type DHPR contained 2.4 times as much cysteine as the protein from the double

133
mutant pWAM8, and confirmed that indeed a double mutation had been achieved. The
kinetic parameters (Km and Vmax values) for the DHPR produced by WAM, WAM3,
WAM6 and WAM8 were determined for quinonoid 6-methyl-7 ,8(6H)dihydropterin and for
NADH (Tables 1 and 2). The kinetic data show little difference in the parameters (except
perhaps for DHPR from WAM6) when cysteine residues are replaced by serine. These
imply that cysteine residues are either not involved in the chemistry of the reaction and the
binding of substrates, or are not involved in the rate limiting reactions.
Further comparative studies of the mutant DHPR with the wild-type enzyme are
warranted in order to see if the amino acid changes affected enzyme activity in ways other
than in the kinetic parameters. We have examined the thermal stability of the enzymes
under similar conditions and the results are in Table 3. These data were obtained for
measurements in 0.8 M sodium chloride. This was done because in the absence of salt the
rates of inactivation were too fast to measure except for the wild-type enzyme. In the
absence of salt, the activity of this enzyme was unchanged during 4 hours at 40°C, but had
a half life of 3 minutes at sooc. DHPR from WAM6 was the least stable mutant.

Table 3 Thermal stability of DHPR and mutants (50 mM Tris/HCl pH 7.4, 2mM DTT
and 0.8 M NaCl)

Enzyme 50°C 55°C

k (min-1) Half life (min) k (min-1) Half life (min)

WAM Stable> 3 hr Stable> 3 hr 0.136 6.0

(Wild-type)

WAM3 0.065 10.6 0.502 1.4

(cys104 ..,._ser)

WAM6 0.315 2.2 0.768 0.9

(cys161 ..,._ser)

WAM8 0.051 13.6 0.457 1.5

(cvs26+85 ..,._ ser)

Dithiothreitol (DTT) was added to all the enzymes because DHPR loses activity
rapidly in its absence. In the presence of 2 mM DTT human DHPR is stable for over one
year at -2ooc. In the presence of 2 mM DTT the wild-type enzyme is more stable than the
mutant enzymes.
ACKNOWLEDGEMENT- We thank Dr N.E.Dixon for generous supplies of the plasmid
pMA200U.
REFERENCES

1. Dahl, H.-H.M, Hutchinson, W., McAdam, W., Wake, S., Morgan, F.J. and Cotton, R.G.H. (1987)
Nucleic Acids Res., 15, 1921-1936
2. Lockyer, J., Cook, R.G., Milstein, S., Kaufman, S., Woo, S.L.C. and Ledley, F.D. (1987)
Proc.Natl. Acad.Sci.USA, 84, 3329-3333
3. Armarego, W.L.F. and Ohnishi, A., (1987) Eur.J.Biochem. 164, 403-409
4. Webber, S. and Whiteley, J.M. (1981)Arch.Biochem.Biophys. 206, 145-152
5. Armarego, W.L.F., Cotton, R.G.H., Dahl, H.-H.M. and Dixon, N.E. (1989) Biochem.J. 261, 265-268
6. Elvin, C.M., Thompson, P.R., Argall, M.E., Hendry, P., Stamford, P.J., Lilley, P.E. and Dixon, N.E.
(1990) Gene 87, 123-126
7. Cotton, R.G.H. and Jennings, I. (1978) Eur.J.Biochem. 83,319-324
8. Vasudevan. S.. Shaw. D.C. and Armarego, W.L.F. (1988) Biochem.J. 255. 581-588

134
THE SPECTRUM OF MUTATIONS IN DIHYDROPTERIDINE
REDUCTASE DEFICIENCY

Peter M. Smooker, David W. Howells, Irma Dianzani and Richard G.H.

Cotton

Murdoch Institute for Research into Birth Defects

Flemington Road, Parkville
Victoria 3032
Australia

INTRODUCTION
Dihydropteridine reductase (DHPR, EC 1.6.99.7) is the enzyme required for the
recycling of tetrahydrobiopterin, an essential cofactor of the three aromatic amino acid
hydroxylases (1). A rare inherited disorder is due to a deficiency of this enzyme, resulting in
hyperphenylalanemia and disorders of dopamine and serotonin metabolism. DHPR-deficiency
was recognized as an inheritable disorder, distinct from PKU, in the 1960's, and the disease
state was later correlated with an absence of enzyme activity in cultured fibroblasts (2). This
lack of enzymatic activity was shown in some cases to be due to an absence of protein.
We have collected and cultured fibroblasts from a number of DHPR-deficient patients
throughout the world. Using the fibroblasts as a source of genetic material we have embarked
on a programme to determine the molecular defects resulting in DHPR-deficiency. The first
such mutation was found in 1990 (3), which was a three base-pair insertion resulting in the
insertion of a single threonine residue after amino acid 122 (Thr123 ins.). Since this time we
have found a further 9 mutations, the nature of which will be discussed here. Some of the
mutations have been expressed in E. coli and the purified proteins examined by a number of
means. This has enabled the effects of these mutations on the structure and function of the
DHPR enzyme to be characterized.

EXPERIMENTAL PROCEDURES
Identification of mutations

Fibroblasts from patients were cultured in roller bottles and approximately 2 x 107 cells
used for the extraction of RNA. The extraction of whole cell RNA and conversion to eDNA
was as previously described (4). The amplification of first strand eDNA by the polymerase
chain reaction and the subsequent use of the PCR products in the mutation screening CCM
method have also been described (3, 5-6). Briefly, the DHPR coding region was amplified
from fibroblast eDNA by PCR, and this was hybridized to a wild-type PCR product derived
from our reference eDNA clone (7). Mismatched nucleotides were detected by CCM and the
nucleotide change determined by sequencing the relevant portion of amplified patient eDNA.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 135
Expression of mutant proteins

The in vitro mutagenesis ofDHPR and cloning into the expression vector pGEX-2T has
been described in detail in Smooker et al1993 (5). The DHPR enzymes were expressed as a
fusion protein with glutathione-S-transferase, enabling easy purification by absorbtion to
glutathione-agarose beads. The DHPR moiety was released by digestion with thrombin,
which cleaves at the junction between the fused proteins. The mutant DHPR enzymes thus
obtained were examined for enzymatic activity, susceptibility to digestion with chymotrypsin,
and structural integrity (by gel filtration).

RESULTS AND DISCUSSION

The spectrum of mutations

We have found a total of 10 different mutations in the DHPR coding region to date, with
one mutation (Trp36---tArg) reported by Matsubara et al (8). Table 1 shows the mutations we
have found.

Table 1. Mutations causing DHPR deficiency. The list is given in the order in
which mutations appear from the 5'-end of the coding region. All mutations, except
for those in MB and NF, are homozygous. CRM =cross-reacting material, measured
in fibroblasts.

NAME CRM MUTATION AMINO ACID REFERENCES

MB(1),LR,MZ ND G93A Gly23---tAsp 5, 9, 10
DP 38% T346G Trp 108---tGly 9, 11
KA 30% ACT390 ins. Thr123 ins. 3
NF(1) -ve C458T Pro145---tLeu 5, 11
MLJ ND G475A Gly151---tSer 12
PM,MB(2) 100% (PM) C496T His 158---tTyr 5, 9, 13
NP ND G532A Gly170---tThr 5
NF(2) -ve T638G Leu205---tStop 5, 11
AG ND T659G Phe212---tCys 12
AA ND C685T Arg221---tS top 5

The majority of mutations identified are missense mutations, with only two resulting in
premature termination of the peptide chain, resulting in a loss of 40 amino acids (NF) or 24
amino acids (AA). There is an insertion of a single amino acid in KA. Some of the mutations
involve non-conservative substitutions, such as proline to leucine, while others appear to be
conservative, such as glycine to serine (patient MLJ, this may explain the "mild" disease state
in this patient, ref 12). Three of the 9 point mutations occur at the cytosine nucleotide of CpG
doublets, which are susceptible to mutation. The positioning of the mutations in the DHPR
coding region is shown diagrammatically in figure 1.

100 200 300 400 500 600 700

~ G W T
ins.
PG H G LF R WT
D G LS Y T *c * MUT.

Figure 1. Mutations in the DHPR coding region. The coding region is numbered in nucleotides, and the
wild-type amino acid and the substitutions corresponding to each mutation shown below.

136
 The mutations are spread throughout the coding region, albeit with a large gap in which
no mutations are found between nucleotides 100 and 350. As the amino acids corresponding
to the ligand binding sites of DHPR are not definitively known, it is not possible to speculate
that a particular mutation will affect the enzymatic activity of the protein, however after
expression of several mutant proteins some conclusions as to the effects of particular
mutations could be drawn (see below). One mutation, however, did alter a conserved amino
acid in an NADH binding domain. The Gly23-tAsp mutation, found in three individuals,
alters the last of three conserved glycine residues in the dinucleotide binding consensus
sequence Gly-X-Gly-X-X-Gly, which connects the first ~-strand to the a-helix in the ~a~ fold
(14). The glycine residues are required to allow the amino acid backbone to rotate, and
replacement by the larger, charged aspartic acid residue may inhibit this. Thus it would be
expected that NADH binding will be inhibited.

Expression and characterization of mutant proteins

Three mutant proteins were expressed in E. coli and after purification were examined by
a variety of techniques. The mutant enzymes constructed and examined were Gly23-tAsp,
Trp108-tGly and His158-tTyr. The methods used to examine these proteins were (a)
enzymatic activity, (b) susceptibility to protease digestion, and (c) gel filtration. A summary
of the results is given in table 2.

Table 2. Characterization of some mutant DHPR enzymes. Activity is given as a

percentage of the (recombinant) wild-type, and km values are in micromolar.

km km PROTEASE
ENZYME ACTIVITY (DMPH4) (NADH) SUSCEPT- INTEGRITY
IBILITY
Wild-type 100% 40 11 - Dimer
Gly23-tAsp 0% NA NA +I- Dimer
Trp108-tGly 41% 25 24 +++ Some monomer
His158-tTyr Low ND ND ++++ Dimer

The results presented in table 2 give some clues as to the effects of the mutations on the
structure and function of the DHPR enzyme. The Gly23-tAsp mutation renders the enzyme
inactive, but does not grossly perturb protein structure as evidenced by the stability to
protease digestion (the enzyme is only slightly digested by chymotryptic treatment). This
result is expected if, as discussed above, the mutation affects the binding of NADH. The
enzyme harbouring the Trp108-tGly mutation, conversely, has substantial enzymatic activity
but is very susceptible to protease digestion. Examination of the gel filtration profile reveals a
substantial amount of monomeric protein, thus it appears that this mutation affects
dimerization. The level of cross reacting material in fibroblasts from this patient is 38%, hence
the protease sensitivity in vitro is reflected in vivo, and may be due to the monomers being
more susceptible to proteolysis than dimerized protein. The inhibition of dimerization is
probably the result of the mutation being in one of the helices (aE) proposed to be involved in
this process (15). As noted by these authors, the Thr123 insert is in the same region, and the
30% CRM observed in fibroblasts of this patient (KA) may indicate a similar phenotype.
The final mutant examined, His158-tTyr, does not suffer an inhibition of dimerization
but is highly susceptible to protease digestion and has little (and unstable) enzymatic activity.
It appears that this mutation causes a disruption of the overall protein structure such that the
enzymatic activity is severely inhibited. An interesting observation is that although the
mutation results in increased susceptibility to proteolysis in vitro, this is not reflected in vivo,
as there is 100% CRM in fibroblasts of this patient.
For some of the other mutations which were found, but not expressed, the effects can be
inferred. Thus, as patient NF has no CRM in fibroblasts, both mutations must result in
degradation of the protein. Similarly, in patient KA, where there is a homozygous insertion of
one amino acid, the 30% CRM level also indicates that there is some proteolysis of the
enzyme. In summary, the spectrum of mutations in DHPR deficiency reveals a heterogeneous

137
spread of mutations throughout the coding region, with a correspondingly heterogeneous
range of effects which the mutations have on the structure and function of the protein.
REFERENCES
I. J.E. Craine, E.S. Hall, and S.Kaufman, J. Bioi Chem. 47,6082 (1972)
2. F.A. Firgaira, R.G.H. Cotton, and D.M. Danks, Clin. Chim. Acta 95,47 (1979)
3. D.W. Howells, S.M. Forrest, H.H-M. Dahl, and R.G.H.Cotton, Am. J. Hum. Genet. 47,277 (1990)
4. S.J. Ramus, S.M. Forrest, and R.G.H. Cotton, Hum. Mut. 1,154 (1992)
5. P.M. Smooker, D.W. Howells, and R.G.H. Cotton, Biochemistry, in press (1993)
6. R.G.H. Cotton, N.R. Rodrigues, and R.D. Campbell, Proc. Natl. Acad. Sci. USA 85,4397 (1988)
7. H.H-M. Dahl, W. Hutchison, W. McAdam, S. Wake, F.T. Morgan, and R.G.H. Cotton, Nucl. Acids
Res. 15,1921 (1987)
8. Y. Matsubara, I. Hiroyuki, H. Endo, and K. Narisawa, Nucl. Acids Res. 20,1998 (1992)
9. I. Dianzani, D.W. Howells, A. Ponzone, J.A. Saleeba, P.M. Smooker, and R.G.H. Cotton, J. Med.
Genet. in press (1992)
10. F.A. Firgaira, K.H. Choo, R.G.H. Cotton, and D.M. Danks, Biochem. J. 198,677 (1981)
11. F.A. Firgaira, R.G.H. Cotton, and D.M. Danks, In: Chemistry and Biology of Pteridines (Blair J.A.
ed.), de Gruyter, Berlin, pp771 (1983)
12. N. Blau, C.W. Heizmann, W. Sperl, G.C. Korenke, G.F. Hoffmann, P.M. Smooker, and R.G.H.
Cotton, Pediat. Res. 32:726 (1992)
13. R.G.H. Cotton, I.G. Jennings, G. Bracco, A. Ponzone, and 0. Guardamagna, J. Inher. Metab. Dis.
9,239 (1986)
14. R.K. Wirenga, P. Terpstra, and W.G.J. Hoi, J. Mol Bioi. 187,101 (1986)
15. K.I. Varughese, M.M. Skinner, J.M. Whitely, D.A. Matthews, and N.H. Xuong, Proc. Nat[. Acad. Sci.
USA 89,6080 (1992)

138
CLONING AND CHARACTERIZATION OF GENES ENCODING

TETRAHYDROBIOPTERIN BIOSYNTHETIC ENZYMES

Robert A. Levine1' 2' 4 , J. Christopher States3 , Panagiotis Z. Anastasiadis 1 ' 2, and

Donald M. Kuhn 2

1William T. Gossett Neurology Laboratories of Henry Ford Hospital,

Metropolitan Center for High Technology, 2727 Second Avenue, Detroit,
Ml, 48201
2 Cellular and Clinical Neurobiology Program, Department of Psychiatry,

Wayne State University, Detroit, Ml

3 Center for Molecular Biology, Wayne State University
4 Veterans Administration Medical Center, Allen Park, Ml

INTRODUCTION

Tetrahydrobiopterin (BH 4) metabolism is altered at various stages of life. BH 4

deficiencies have been demonstrated in newborns (BH 4 -deficient Phenylketonuria-PKU)
as well as in several neurological diseases including familial dystonia, Parkinson's and
Alzheimer's disease, and normal aging (for review, see 1). BH 4 was originally shown to
be the essential cofactor for phenylalanine hydroxylase2 • BH 4 is perhaps best known as
the essential cofactor for tyrosine and tryptophan hydroxylases, the initial and rate-limiting
enzymes in dopamine and serotonin synthesis, respectively. BH 4 administration in animals
and cultured cells can enhance biogenic amine synthesis, and inhibition of BH 4 synthesis
lowers intracellular BH 4 and reduces biogenic amine synthesis. Thus, BH 4 therapy has
been attempted in several of the diseases mentioned above with some success.
More recently, BH 4 has also been shown to be a necessary cofactor for nitric oxide
synthase activity in the production of nitric oxide3 • BH 4 can also enhance the release of
certain neurotransmitters in brain 4' 5' 6 , and is necessary for DNA synthesis in replicating
murine erythroleukemic cells 7 •

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 139
 In BH 4 biosynthesis, GTP cyclohydrolase catalyzes the conversion of GTP to
dihydroneopterin triphosphate (NH 2TP). NH 2TP is then converted to 6-pyruvoyl
tetrahydropterin (6-PPH 4 ) by 6-PPH 4 synthase. 6-PPH 4 contains 2 keto functions in the
6-position side chain that must be reduced in order to form BH 4 • Sepiapterin reductase
can catalyze sequentially both keto reductions, however 6-PPH 4 reductase may catalyze
the penultimate keto reduction (for review, see 8). A great deal of progress has been
made in the last few years on cloning and characterizing the cDNAs encoding the BH 4
biosynthetic enzymes. The cloning of the cDNAs encoding BH 4 biosynthetic enzymes has
opened the door for our further understanding of BH 4 metabolism, including the
molecular defects in BH 4 -deficient PKU, regulation of the BH 4 biosynthetic pathway, and
other roles that BH 4 and its biosynthetic enzymes may play in mammalian physiology.
This paper will summarize some of the recent work on cloning the cDNAs encoding BH 4
biosynthetic enzymes, our work on cloning of the human brain sepiapterin reductase
eDNA, and some of our initial studies on regulation of expression of the BH 4 biosynthetic
enzymes in brain following exposure of rodents to certain neurotoxins.

METHODS

Animal Experiments

Male Sprague- Dawley rats (250g) were subjected to intrastriatal injections of kainic
acid (2 f.lg in 1 J.ll) under stereotaxic control as previously described 9 • Mice were treated
with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 0.17 mM/kg) once each day
for five days and sacrificed 12 days following the last injection. Substantia nigra and
striatum were dissected and homogenized in ice-cold 50 mM Tris HCI, pH 7.4 made up
in DEPC-treated water to prevent RNA degradation and preserve enzyme activity, which
allowed measurement of all parameters in the same tissue sample. Homogenates were
immediately divided so that appropriate stabilizing solutions (e.g. acid, gel electrophoresis
loading buffer, RNA preparation buffer, etc.) could be added in preparation for assay of
tissue mRNA, enzyme amount or activity, and BH 4, catecholamines, and metabolites.

Northern Blots

For tissue mRNA measurements, homogenates were handled as described by

Biguet and coworkers 10, who published the simultaneous assay of tyrosine hydroxylase
activity and tyrosine hydroxylase mRNA in a single rat substantia nigra. Original extracts
were further homogenized in 10 volumes 0.2 M Tris buffer (pH =8.8) containing 25 mM
EDTA, 0.1 M LiCI, 1% SDS, and 5 J.lg/ml dextran T40, and an aliquot was removed for
total RNA measurements. Following 2 extractions with 2 volumes of
phenol:chloroform:isoamyl alcohol (25:24:1 ), RNA was purified by selective LiCI
precipitation (2.0 N final, overnight at 4°C), followed by centrifugation of RNA. RNA was
reconstituted in 6 f.ll of No Salt buffer containing 1 J.lg/ml proteinase K. Two dilutions of
RNA in the range of 10-20 J.lg/sample from each nigra were analyzed. RNA was
fractionated by electrophoresis on a formaldehyde-containing agarose gel and transferred

140
to modified nylon membranes (Nytran). Tyrosine hydroxylase eDNA and sepiapterin
reductase eDNA probes were labelled by the random primer method. Blots were probed
at 42°( in SO% formam ide (high stringency), SX SSPE, SX Denhardt's, 100 1-1g/ml
denatured salmon sperm DNA, and 0.1% SDS. Filters were then washed successively with
2X SSC containing 0.1% SDS, air dried, and subjected to autoradiography. Tyrosine
hydroxylase sepiapterin reductase mRNAs were assayed by densitometric scanning of
autoradiograms using a Bio Image Laser Scanning Densitometer and calibrated with a
standard curve using dilutions of rat brain mRNA. After removal of the 32 P-Iabelled probe,
these same blots were reprobed with a labelled actin probe to use as an index for
normalizing mRNA changes following treatment conditions. Data are expressed as a
percentage of the density in control lanes.

Enzyme assays

Tyrosine hydroxylase activity was measured by monitoring the release of 3 HOH

from 3,5-3 H-tyrosine as previously described 11 under saturating conditions (1 00 11M
tyrosine, 1 mM BH 4) using the ion exchange method. GTP cyclohydrolase activity was
measured under saturating conditions (1 mM GTP) by monitoring the conversion of GTP
to NH 2 TP as previously described 9 ; neopterin was measured by HPLC with fluorescence
detection after oxidation and dephosphorylation of NH 2 TP9 • SR activity was measured
under saturating conditions (250 11M sepiapterin, 100 11M NADPH) as described
previousli 2 •

RESULTS AND DISCUSSION

Cloning of cDNAs Encoding BH 4 Biosynthetic Enzymes

Much progress has been made in the cloning of cDNAs encoding BH 4 biosynthetic
enzymes. We initially raised antiserum against purified rat erythrocyte sepiapterin
reductase 12 ; the antiserum was then used to screen a eDNA library from rat liver and
isolate a sepiapterin reductase eDNA (SR cDNA) 13 • Oyama and coworkers 14 also isolated
a eDNA from a rat liver eDNA library and confirmed the sequence of the 5' end of the
open reading frame that we originally predicted 13 • We then used the rat liver SR eDNA
to isolate the human brain SR cDNA15 , which will be discussed below.
The second eDNA to be isolated and sequenced encoded rat GTP
cyclohydrolase 16 ; the eDNA was isolated from a rat liver eDNA library using
oligonucleotides derived from the sequence of purified GTP cyclohydrolase.
Subsequently, Togari and coworkers isolated 3 cDNAs from a human liver eDNA library,
which showed divergence at the 3' ends.
Inoue and coworkers purified rat 6-PPH 4 synthase 17 and used synthetic
oligonucleotides to isolate and then sequence a eDNA from a rat liver eDNA library17 •
Based on the rat sequence, Thony and coworkers 18 isolated a eDNA from human liver,
and demonstrated an 82% homology with the rat eDNA.

141
 Following our characterization of rat liver SR eDNA, lchinose and coworkers
isolated and sequenced a human liver SR cDNA19 • We have also isolated and
characterized an SR eDNA by screening a human brain (frontal cortex) eDNA librari 5 with
a 669 bp fragment of the rat SR eDNA according to procedures as previously published 13 •
The human brain SR eDNA contains 1630 bp, as compared to 833 bp for human liver19
and 1157 bp for rat liver13 • The human brain SR eDNA has a larger (260 bp) 5'
untranslated region than the human liver eDNA (22 bp), which contains several potential
regulatory elements; the human brain SR eDNA also has a larger 3' untranslated region
(569 bp compared with 25 bp in human liver). The open reading frame of 783 bp
encodes 261 amino acids comprising a protein of 28,047 M,. There is a 78% nucleotide
and a 68% amino acid homology between the human brain and rat liver SR cDNAs. Two
distinct regions of divergence occur between the two, which may contribute to our
observed poor cross-hybridization of each eDNA with mRNA from the opposite species.

Expression of BH4 Biosynthetic Enzymes

Kainic acid. Kainic acid is a neurotoxin that destroys cell bodies while sparing
nerve terminals of neurons originating in non-lesioned areas; it was originally injected into
rat striatum as a model for Huntington's disease20 • Kainic acid has been shown by several
investigators to activate striatal tyrosine hydroxylase activity21 ' 22 several days after
intrastriatal injection. 7 days after intrastriatal kainic acid injection, GTP cyclohydrolase
activity was increased approximately 70% (p<.05, Student's t-test), and tyrosine
hydroxylase activity was increased 35% (p< .05), while aromatic amino acid decarboxylase
activity was unchanged. Control values (based on supernatant mg protein) were: GTP
cyclohydrolase= .55 ± .04 pmol/min/mg; tyrosine hydroxylase= 1.51 + .05
nmol/min/mg; aromatic amino acid decarboxylase (AAAD)= 1.03 + .08 nmol/min/mg.
Although induction of tyrosine hydroxylase and GTP cyclohydrolase activity has
been shown to occur in the adrenal medulla23 ' 24, these results are the first to demonstrate
induction of any BH 4 biosynthetic enzyme in brain. This paradigm will provide a useful
model for investigating the mechanism(s) underlying coordinate regulation of striatal BH 4
and dopamine synthesis in brain. We are currently investigating the time course and
mechanism by which this elevation in activity occurs.

1-methyl-4-phenyl-1 ,2,3,6-tetrahydropyridine (MPTP). MPTP has been used in

mice to destroy nigrostriatal dopamine neurons and examine the effects on striatal BH 4
and dopamine metabolism. MPTP (0.17 mmol/kg) was administered to mice once each
day for 5 days and mice were sacrificed 12 days after the last injection. Following
sacrifice, substantia nigra! and striatum were dissected and handled as described in
methods. Tyrosine hydroxylase and sepiapterin reductase mRNAs were quantified in
substantia nigra. Striatal tissue was homogenized in ice-cold 50 mM Tris-HCI (pH 7.4)
and immediately divided into aliquots for measuring the following parameters in the same
striatal homogenate: 1) tyrosine hydroxylase content (loading buffer was added in
preparation for SDS Polyacrylamide gel electrophoresis; SDS-PAGE); 2) tyrosine
hydroxylase activity (no further additions necessary); 3) GTP cyclohydrolase, 6-PPH 4
synthase, and sepiapterin reductase activities (no further additions necessary); 4) BH 4

142
content (1.0 N trichloroacetic acid was added in a 1 :10 dilution of sample), and 5)
dopamine and its major metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic
acid (DO PAC) (1.0 N perchloric acid was added in a 1 :10 dilution of sample).
The success of the MPTP lesion was demonstrated by the greater than 70% loss of
dopamine, DOPAC, and HVA. MPTP decreased the activities of striatal tyrosine
hydroxylase and GTP cyclohydrolase, as well as BH 4 content by approximately SO%. The
loss of tyrosine hydroxylase molecules in the striatum MPTP-treated mice was confirmed
by quantification of Western blots probed with anti-tyrosine hydroxylase serum 25 •
However, striatal 6-PPH 4 synthase and sepiapterin reductase activities were not altered,
suggesting that these enzymes are not predominantly localized in dopaminergic terminals
in striatum. Control values expressed as a function of tissue wet weight are: tyrosine
hydroxylase= 15.9 + 0.8 pmol/min/mg; GTP cyclohydrolase= 7.08 .± 0.75 fmol/min/mg;
BH 4 = 0.43 .± 0.03 pmol/mg; DA= 10.35 + 0.44 }Jg/g; DOPAC= 3.31 + 0.14 }Jg/g;
HVA=2.77 + 0.23 }Jg/g. BH 4, dopamine, and metabolites were measured as described
previousl/ 6 •
Northern blots of RNA from substantia nigra of control and MPTP-treated mice
were probed with labelled tyrosine hydroxylase eDNA, and then probed with sepiapterin
reductase eDNA after the blot was stripped of radioactivity from the previous probe.
Quantitative laser-scanning densitometry of the autoradiograms revealed that MPTP
caused a 34% reduction of tyrosine hydroxylase mRNA in the nigra (control=1 00% +
9.4% SEM, MPTP=66o/o + 8.9%, n=8, p < 0.05, Student's t-test). When the same blot
was probed with the 669 bp labelled fragment of rat sepiapterin reductase eDNA, the
content of sepiapterin reductase mRNA rose by 79% (control=100o/o + 9.6% SEM,
MPTP=179o/o + 8.6%, n=8, p < 0.05). It is currently not known what cells in the
substantia nigra contain the elevated level of sepiapterin reductase mRNA or what causes
this increase; however it is possible that there is a compensatory elevation of sepiapterin
reductase synthesis in the surviving nigrostriatal dopamine neurons, which maintains
striatal sepiapterin reductase activity equivalent to control. We are currently exploring the
mechanism and cellular location of this novel elevation of sepiapterin reductase mRNA
in mouse brain. These types of studies will address the extent to which there is coordinate
regulation of expression of genes encoding tyrosine hydroxylase and the BH 4 biosynthetic
enzymes in brain, and how these systems can be manipulated pharmacologically to
produce favorable therapeutic outcomes in neurological and psychiatric diseases where
BH 4 and catecholamine metabolism is altered.

Acknowledgements

Robert A. Levine is supported by NIH grants AG1 0687-01 and NS28800-01. Panagiotis
Z. Anastasiadis is supported by the Bodosaki Foundation, Athens, Greece.

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1. Levine, R.A. Tetrahydrobiopterin and biogenic amine metabolism in neuropsychiatry,

immunology, and aging. "In: Neuroimmunomodulation: Interventions in aging and
cancer - First Stromboli Conference on Aging and Cancer," W. Pierpaoli, N.H.

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 Specter, and B. Jankovic., Annals of New York Academy of Sciences, pp. 129-139
(1987).
2. Kaufman, S.: The structure of the phenylalanine-hydroxylation cofactor. Proc. Nat/.
Acad. Sci., 50: 1085-1 093 (1963 ).
3. Tayeh, M.A. and Marietta, M.A. Macrophage Oxidation of L-Arginine to Nitric
Oxide, Nitrite, and Nitrate: Tetrahydrobiopterin is Required as a Cofactor. }. Bioi.
Chem., 264: 19654-19658 (1989).
4. T.Ohue, K.Koshimura, Y.Akiyama, Y.Watanabe and S.Miwa: Monoamine-mediated
enhancement of acetylcholine release from rat hippocampus by
6R-L-erythro-5,6,7,8-tetrahydrobiopterin. Brain Res. 570, 173-179 (1992).
5. K.Koshimura, S.Miwa, K.Lee, M.Fujiwara, and Y.Watanabe: Enhancement of in vivo
dopamine release from the rat striatum by dialytic perfusion of
6R-L-erythro-5,6,7,8-tetrahydrobiopterin.}. Neurochem. 54, 1391-1397 (1990).
6. Wolf, W.A., Zaija. E., Arthur, R.A. Jr., Anastasiadis, P.Z., Levine, R.A., and Kuhn, D.M.
Effect of tetrahydrobiopterin on serotonin synthesis, release, and metabolism in
superfused hippocampal slices.}. Neurochem., 57: 1191-1197 (1991 ).
7. Tanaka K., Kaufman S. and Milstien S.: Tetrahydrobiopterin, the cofactor for aromatic
amino acid hydroxylases, is synthesized by and regulates proliferation of erythroid
cells. Proc. Nat/. Acad. Sci. USA 86, 5864-5867 (1989).
8. Milstien, S. and Kaufman, S.: The Biosynthesis of tetrahydrobiopterin in rat brain,
"Chemistry and Biology of Pteridines," B.A. Cooper and V.M. Whitehead, (Eds.),
de Gruyter, Berlin, New York, pp. 169-181 (1986).
9. Levine, R.A., Miller, L.P., and Lovenberg, W.: Tetrahydrobiopterin in striatum:
Localization in dopamine nerve terminals and role in catecholamine synthesis.
Science, 214: 919-921, (1981 ).
10. Biguet, N.F., Buda, M., Lamouroux, A., Samolyk, D., and Mallet, J.: Time course of
the changes of tyrosine hydroxylase mRNA in rat brain and adrenal medulla after
a single injection of reserpine. EMBO }. 5(2): 287-291 (1986).
11. Levine, R.A., Pollard, H.B., and Kuhn, D.M.: A rapid and simplified assay method for
tyrosine hydroxylase. Anal. Biochem., 143: 205-208 (1984).
12. Levine, R.A., Kapatos, G., Kaufman, S., and Milstien, S.: Immunological evidence for
the requirement of sepiapterin reductase for tetrahydrobiopterin biosynthesis in
brain. }. Neurochem., 54: 1218-1224 (1990).
13. Citron, B.A., Milstien, S., Gutierrez, J.C., Levine, R.A., Yanak, B.L., and Kaufman, S.:
Isolation and Expression of Rat Liver Sepiapterin Reductase eDNA. Proc. Nat.
Acad. Sci., 87: 6436-6440 (1990).
15. Levine, R.A., Sol us, J.F., Goustin, A.S., Tait, S., Demetriou, S., Citron, B., and Kaufman,
S.: Isolation of a putative eDNA encoding human brain sepiapterin reductase.
"Pterins and Biogenic Amines in Neurology, Pediatrics, and Immunology", N.Biau,
H.-Ch. Curtius, R.A. Levine, and R.G.H. Cotton (Eds.), Lakeshore Publishing, pp.
81-88 (1991 ).
16. Hatakeyama, K., Inoue, Y., Harada, T., and Kagamiyama, H.: Cloning and sequencing
of eDNA encoding rat GTP cyclohydrolase 1: The first enzyme of the
tetrahydrobiopterin biosynthetic pathway. }. Bioi. Chem., 266, 765-769 (1991 ).
17. Inoue, Y., Kawasaki, Y., Harada, T., Hatakeyama, K., and Kagamiyama, H.: Purification

144
 and eDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase.}. Bioi. Chem., 266,
20791-20796 (1991 ).
18. Thony, B., Leimbacher, W., Burgisser, D., and Heizmann, C.W., Human 6-pyruvoyl
-tetrahydropterin synthase: eDNA cloning and heterologous expression of the
recombinant enzyme, Biochem. Biophys. Res. Comm., 189: 1437-1443 (1992).
19. lchinose, H., Katoh, S., Suoka, T., Titani, K., Fujita, K., and Nagatsu, Biochem. Biophys.
Res. Comm., 179: 183-189 (1991)
20. Coyle, J.T. and Schwarcz, R.: Lesion of striatal neurons with kainic acid provides a
model for Huntington's Chorea, Nature, 263: 244-245 (1976).
21. Baring, M.D., Walters, J.R., and Eng, N., Brain Res., 181: 214-218 (1980).
22. Coyle, J.T. and Schwarcz, R., Lesion of striatal neurons with kainic acid provides a
model for Huntington's Chorea, Nature, 263: 244-245 (1976).
23. Abou-Donia, M.M., Daniels, A.J., Wilson, S.P., Nichol, C.A., and Viveros, O.H.,
"Chemistry and Biology of Pteridines," ed. by J.A. Blair, Berlin, Walter de Gruyter,
pp. 777-781 (1983).
24. Viveros, O.H., Lee, C.-L., Abou-Donia, M.M., Nixon, J.C., and Nichol, C.A.: Science,
213: 349 (1981).
25. Kuhn, D.M. and Billingsley, M.L. Tyrosine hydroxylase: Purification from PC12 cells,
characterization, and production of antibodies, Neurochem. Int. 11: 463-475,
(1987).
26. Levine, R.A., Zoephel, G.P., Niederwieser, A., and Curtius, H.C.: Entrance of
tetrahydropterin derivatives in brain after peripheral administration: Effect on
biogenic amine metabolism,}. Pharmacal. Exp. Ther., 242: 514-522 (1987).

145
DROSOPHILA GTP CYCLOHYRODROLASE: MULTIPLE ISOFORM
PRODUCTS OF A SINGLE GENE DERIVE FROM ALTERNATE
TRANSCRIPTS THAT ARE DEVELOPMENTALLY REGULATED AND
FUNCTIONALLY SPECIFIC

J.
M. O'Donnelll, G. Ranganayakulul, X. Chenl, S.
Krishnakumarl, and W. S. Neckameyer2

1Department of Biological Sciences, University of Alabama

Tuscaloosa, AL 35487
2Department of Pharmacological and Physiological Science
St. Louis University School of Medicine
St. Louis, MO 63104

INTRODUCTION

GTP cyclohydrolase I (GTP CH) in Drosophila melanogaster is

strikingly similar to its mammalian cognates by deduced protein homology as
well as by biochemical mechanisms (1, 2; McLean, Krishnakumar and
O'Donnell, submitted). As in mammals, GTP CH is the first and rate-limiting
enzyme in the synthesis of pteridines; beyond the catalytic properties of the
enzyme, the products of the pathway initiated by this enzyme share
functional roles. For example, the aromatic amino acid hydroxylases in
Drosophila require a pteridine cofactor for enzymatic activity (3, 4). The
diversity of pteridine functions in multicellular organisms necessitates a
complexity of regulatory mechanisms. In order to understand this
complexity, an in vivo model is essential. The powerful molecular genetics
of Drosophila make it a cogent system for such studies.

PTERIDINE BIOSYNTHESIS IN DROSOPHILA

GTP CH is encoded by a single gene, the Punch (Pu) locus on

Chemistry and Biology of Pteridines and Folates, Edited by

J .E. Ayling et al., Plenum Press, New York, 1993 147
chromosome 2, named for the mutant eye colors imparted by pteridine
deficiencies (5). More than 60 mutant alleles of this gene have been
generated, and the subsequent genetic and biochemical analysis of the
mutants led to the conclusion that Pu is a complex locus (5-7) because alleles
having functional and developmental specificity, as well as alleles with
apparently general effects, were isolated. Loss of Pu function results in a
multiplicity of phenotypes, summarized below in Table 1.

Table 1. Classes of Punch mutationsl.

Class Genetic characteristics Phenotypes

Null recessive lethal -unpigmented embryonic

cuticle
-poor cuticle integrity
-poor head segment
differentiation
-unfused tracheal
segments
-decreased eye pigment

Eye-specific recessive; homozygous -decreased eye pigment

viable -normal cuticle
pigmentation

Oocyte-specific dominant; most -arrested nuclear

homozygous lethal divisions in early
embryo
-segment pattern defects
-normal cuticle
pigmentation

Proximal homozygous lethal -neural hyperplasia

rearrangements -abnormal segmentation
of nerve cord
-poor larval head
differentiation
-dominant or recessive
eye color defects
-normal cuticle
pigmentation

lPhenotypes are described in detail in (5,6,8 and Ranganayakulu and

O'Donnell, In Prep.

In the cases of developmentally specific mutations, even though pteridine

levels and functions in the mutants appear normal in most tissues and
developmental times, the mutants exhibit loss of pteridines in specific
tissues. Fig. lA illustrates the substantial effects of the allele, Purl, on all

148
pteridines in the adult eye. Even though the homozygous mutant has a
severely abnormal eye color as a result of this pteridine deficit, no other
phenotypic consequences of the mutation are observed. Fig. lB compares the
pteridine profile in wild type and PurWE75 mutant ovaries. This mutant has
wild type eye pigmentation and its ability to perform catecholamine functions
appears normal. In the homozygous state, however, it does not express
normal GTP CH in ovaries (8 and Reynolds, Chen, Ranganayakulu and
O'Donnell, submitted). Consequently, early embryonic nuclear divisions
cannot proceed correctly, and development is aborted at an early stage.
The HPLC profiles of wild type ovaries and eyes also suggest complexity at
another level: that of pathway regulation. In the adult eye, neopterin, the
product of GTP CH activity, is invariably at very low concentrations relative
0.03 . . . , - - - - - - - - - - - - - - - - - - - - - - - ,
A
Heads
·------ Wildtype
rl
-Pu

~ 0.02
c
Q)
(.)
en
Q)
I...
0
::J
u_
0.01

0 2 4 6 8 10 12 14

Ovaries B
0.009
·------ Wildtype
_ PuWE75 1\
l: \~
Q)

I\
(.)
c
Q)
(.)
en
!\
I\
Q)
!.....
0
::J
[;::
0.008

J \ji
---
0 2 4 6 8 10 12 14

Time
Figure 1. HPLC profiles of pteridines in wild t)'Ee and mutant Drosophila eyes and ovaries. A.
Wild type and Purl heads. B. Wild type and Pu WE75 ovaries. Pteridines were extracted from
the tissues in O.lN HCl, oxidized by standard methods, eluted from a Waters 11Bondpak C18
column in 5% methanol, and detected by fluorescence at 365 nm excitation and 465 nm emission
wavelengths. Neopterin elutes at 5 min. and biopterin at 8 min. The remaining peaks are an
unresolved mixture of pterin, 6-hydroxylmethylpterin and isoxanthopterin.
149
 to other pteridines. In contrast, neopterin levels in ovaries and embryos
represent a larger proportion of total pteridine content. This distinction
between pteridine profiles in two different tissues might have several
explanations; the genetic complexity suggests that the basis for the differences
is likely to lie with GTP CH. In general, genetic data of the type observed
here are taken to indicate that the gene encodes a protein with
multifunctional domains or that it expresses multiple products with distinct
functions. Biochemical and molecular genetic analyses support the latter
interpretation. Pu expresses multiple, differentially regulated and
functionally specific GTP CH isoforms.

MOLECULAR CLONING AND CHARACTERIZATION OF PUNCH

After genomic clones of the Pu region were isolated, mutant alleles of

Pu that altered restriction enzyme fragments were mapped by Southern
analysis. These alleles span approximately 25 kb of the region. Subsequent
Northern analysis of transcripts in this region, as well as the characterization
of cDNAs isolated from stage-specific libraries and by PCR amplification of
transcripts, revealed a transcriptionally complex region in which a minimum
of 8-12 developmentally regulated RNAs can be detected (9, 10). A summary
of these studies is presented in Fig. 2. The structural characterization of
eDNA clones established that most of the transcripts arising from this region
result from alternative splicing, and that they overlap significantly in
sequence (McLean, Krishnakumar and O'Donnell, submitted). The common
regions encode a protein that is approximately 70% identical and 80% similar
to those reported for rat and human liver GTP cyclohydrolases (11, 12).
Interestingly, the exons that are unique to each transcript are all 5' to the
common region, and all contain open reading frames which result in GTP CH
proteins with distinct N-terminal domains.
Given the diverse functions of pterins, the complexities in regulation
and expression of this key gene are perhaps to be expected. We speculate that
the distinct N-terminal domains function in the localization of the protein
and in the regulation of GTP CH interactions. In order to explore and test
postulated regulatory mechanisms and relationships, however, it is necessary
first to determine whether these varied products are differentially regulated
and associated with distinct functions, and if so, to identify the product
required for each function. The unique features of the Drosophila model
system permit a dissection of functional relationships and direct tests of
proposed functions and regulatory mechanisms. There are two general ways
to approach this problem. The first is a direct correlation between P u
products and known biochemical functions of pteridines. The second
approach, of value particularly in situations where the precise mechanisms of
pteridine involvement are unclear, involves correlating specific product
alterations in mutants and the resulting mutant phenotypes.

150
 -15 -10 ·5 0 5 10 15

BRHHS S HR H HBS R R S BH RHR R s H R

111•11 ~ I II
L rhd17 t I
t
I~ t~l II! IIU
rH16
r331
r fJ15
• rt rPH30
f
L
rAA4

Figure 2. Molecular Map of the Punch region. Coordinates in the Pu region are indicated in
kilobases on the upper line on the map. Restriction enzyme sites are noted on the second line.
B=Bam Hl; R=Eco Rl; H=Hind III; S=Sal I. The sites of Pu mutations are indicated below the
restriction sites. The small vertical arrows denote sites of chromosomal breaks associated with
rearrangement mutations. Bars indicate deleted segments, and triangles mark transposon
insertion sites. Below the genomic map, exons and introns of alternatively spliced transcripts
are positioned, with filled rectangles indicating shared exons and open, unique exons. Arrows
indicate direction of transcription for these transcripts, which is toward the distal end of the
chromosome. Rectangles containing question marks are exons that have been mapped only by
Northern analysis so that precise positioning is still uncertain. The small arrow in the same
orientation indicates a 1.1 kb transcript for which a eDNA clone is not yet available.
Preliminary evidence suggests that it may also be an alternately spliced product. The two
arrows oriented in the opposite direction indicate two opposite strand transcription units that
are nested within introns.

PUNCH PRODUCTS ARE DIFFERENTIALLY EXPRESSED

The expression patterns of Pu transcripts have been defined by

developmental Northern blot analysis and by in situ hybridization of
genomic and eDNA probes to whole mount embryos and dissected embryonic
and larval tissues (9, 10 and In Prep). In addition, antibodies have been
generated against bacterially expressed Pu proteins that specifically recognize
the isoforms expressed by transcripts A and B. Detailed descriptions of these
results will be reported elsewhere. Here, we summarize the data.
Transcript A corresponds to a 1.7 kb RNA found primarily in the heads
of newly eclosed adult flies (9). It is required for eye pigmentation and is
missing in several Pu mutants that have eye pigment-specific phenotypes.
Similarly, the protein isoform expressed at this time, which is 39 kDA in size,
is also missing in these mutants. Transcript B is 1.75 kb in size and is found in

151
 embryos and adults. Antibodies raised against protein expressed by this
transcript in bacteria detect a Pu protein in the cytoplasm of nurse cells (the 15
sister cells of each developing oocyte which support oocyte growth). The
protein is transported into the cytoplasm of the oocyte in stage 11 of oogenesis
during a bulk flow process that empties the nurse cell cytoplasm into the
growing egg. Once this protein is in the oocyte cytoplasm, it is packaged into
spherical granules (Fig. 3A). These have a morphology of the type described
for a yolk granules. The dynamics and possible functions of the granule
protein are discussed below. The expression of Transcript B itself exactly
parallels that of the protein. Surprisingly, however, it is retained in the nurse
cell cytoplasm during the bulk flow period, and as a result, this transcript is
not found in cleavage stage embryos. After zygotic transcription is initiated,
Transcript B is once again expressed, primarily in the developing larval brain
and ventral nerve cord and in a variety of sensory organ primordia.
Mutations that affect catecholamine functions also alter the level or size of
this transcipt. Transcript C (also 1.75 kb is size) is characterized so far only by
the structure of a eDNA isolated from an embryonic library. It is expressed in
embryos and adults, but tissue localizations have not been completed.
Transcript D, 3.4 kb in length, is ubiquitously expressed in oocytes and
throughout embryogenesis. Transcript E is a 5 kb product that is found in
throughout the cytoplasm of cleavage stage embryos, and to some extent in
mesodermal tissue during gastrulation. However, its most intense
expression is throughout the developing embryonic nervous system. Further
localization experiments are in progress, but it is clear from all of the studies
described here that each product of the locus is expressed in a unique pattern.

CORRELATIONS BETWEEN PUNCH PRODUCTS, MUTANT PHENOTYPES

AND BIOCHEMICAL ROLES OF PTERIDINES

Drosophila tyrosine hydroxylase (DTH) shares striking sequence and

mechanistic conservation with its mammalian counterparts, and so requires
a pteridine cofactor for enzymatic activation (3). The phenotype of mutations
in pale, the genetic locus encoding DTH, are identical to null alleles ofPu ..
This phenotype is characterized by embryonic lethality just prior to hatching,
and unpigmented mouthparts and cuticle (L-DOPA, the product of the
reaction catalyzed by tyrosine hydroylase, is an intermediate in cuticular
sclerotization and melanization; 8,13).
However, there exists in Drosopila two, not three, genes to direct the
hydroxylation of the three aromatic amino acids: DTH, and DTPH, which
hydroxylates both phenylalanine and tryptophan (4). Given that tryptophan
hydroxylase activity should be exclusively confined to serotonin-containing
neurons, and pheynylalanine hydroxylase is expected to have a broader
distribution consistent with its metabolic role, these enzymatic activities must
be differentially regulated both temporally and spatially. In fact, during early
embryogenesis, phenylalanine hydroxylation is the only such enzymatic
activity. Later, during neural differentiation, tyrosine hydroxylase and

152
tryptophan hydroxylase activities are expressed in both the central nervous
system and cuticle.
DTPH is present in two forms: a 45 kilodalton (kDa) subunit found in
ovaries and early embryos, which apparently functions exclusively as a
phenylalanine hydroxylase, and a 50 kDa subunit found in later stage embryos
and larval and adult tissue, which hydroxylates both phenylalanine and
tryptophan (4). The precise structural difference between the 45 kDA and 50
kDA species has not yet been identified; however, it is known that the 45 kDa
protein arises from a post-translational modification (or modifications) of the
50 kDa sybunit, and not from alternative splicing of transcripts, as with Pu.

B c

Fig 3 Locahzatwn of Pu protem Isoforms and DTPH transcnpts Unhke Pu, all DTPH
transcnpts co-locahze with the DTPH gene product (m the embryomc stages shown here, only
the 45kDa DTPH protem IS expressed) A Pu protem m an embryo less than one hour old
recogmzed by antibodies raised agamst protem translated from Transcnpt B This pattern IS
mdistingmshable from that of DTPH Pu Transcnpt E (B) and DTPH (C ) mRNA localization
m early gastrula-stage embryos m-mesoderm D Locahzatwn of Pu protem Isoform ansmg
from zygotic expresswn of Transcnpt B durmg late gastrulation n-neurons E DTPH mRNA
expressiOn m late gastrulation a, p-antenor, postenor gut n-neurons, c-cephahc lobe Scale bar
=50~

153
 The DTPH mRNA and 45 kDa protein co-localizes with one of the Pu
protein isoforms in the a yolk granules, ubiquitously distributed throughout
the early embryo (Fig. 3). As described above, thePu transcript responsible for
this protein is synthesized in egg chambers, but is not itself found in early
embryos. In early gastrulation, the DTPH transcript and protein co-localizes
with the product of the Pu Transcript E (see Fig. 2) in mesodermal tissue. At
this point, the granules have disappeared. However, later in embryogenesis,
but before the 45 kDa species has been replaced by the 50 kDa protein, the
pattern of expression of DTPH and certainPu protein isoforms no longer
completely overlap (Fig. 3). These intriguing differences in regulation
foreshadow the different roles of the Pu gene products required as the
organism develops and gains complexity.

INVESTIGATIONS OF BIOCHEMICALLY UNDEFINED PHENOTYPES

While many of the patterns of expression of Pu and phenotypes of Pu

mutants can be reasonably linked to known functions of pteridines, there are
several phenotypes for which such connections are not apparent. Examples
of these are the neural hyperplasia displayed by some mutants with
alterations in the proximal portion of the locus, and the nuclear division
abnormality in maternal effect mutants. Studies of the neural hyperplasia
phenotype are now just underway. An investigation of the nuclear division
defect has shown a direct link to the expression of a Pu isoform in granules
(Reynolds et al. submitted). Cleavage stage nuclear divisions in Drosophila
embryos proceed in the absence of cell formation and occur in approximately
8 minute cycles. This process is directed almost entirely by maternally
packaged products in the egg cytoplasm. After 14 nuclear division cycles, cell
membranes form around the nuclei, and zygotic transcription begins to direct
development. It is during the precellular stage of embryogenesis that the
nuclear division arrest phenotype in Pu maternal effect mutants is exhibited.
Embryos with the division defects arrest prior to cellularization. Pu WE75 is
one such mutant. It has a reduction in pteridine content in ovaries (Fig. 1),
and it packages little Pu protein into granules. During early embryogenesis,
this mutant has almost none of the protein in the syncytial blastoderm and it
cannot support nuclear division beyond the first one to three cycles. In
normal embryos, the protein granules are abundant and spread throughout
the egg during early division cycles. As nuclear divisions proceed in the
precellular blastoderm, the number and staining intensity of the granules
diminishes. By the time that cellularization occurs, one and a half hours after
fertilization, granules that stain for the protein are gone. Any embryo that
survives to this point, is able to continue development until very late in
embryogenesis. The biochemical basis for the phenotype is unknown, but we
hypothesize that it is related to the roles of biopterin cofactor in erythroid cell
proliferation (14). We are now initiating efforts define the biochemical
processes that are disrupted in these mutants.

154
ACKNOWLEDGEMENTS

This work was supported by NIH grant GM26757 and NSF grant DCB
8608696 to J.M.O. and a grant from the Pharmaceutical Manufacturers'
Association toW. S. N. We wish to gratefully acknowledge the asssistance of
Drs Karen Rose and Harriett Smith-Somerville in the acquisition of the HPLC
data and the preparation of the chromatograms.

REFERENCES

1. Brown, G. M., in "Chemistry and Biology of Pteridines," H.-C. Curtius, S.

Ghisla, and N. Blau, eds., Walter de Gruyter, Berlin (1990).
2. Weisberg, E. P. and J. M. O'Donnell, J. Biol. Chern. 261:1453 (1986).
3. Neckameyer, W. and W. Quinn, Neuron 2:1167 (1989).
4. Neckameyer, W. and K.White, J. Biol. Chern. 267:4199 (1992).
5. Mackay, W. J. and J. M. O'Donnell, Genetics 105:35 (1983).
6. Mackay, W. J., E. R. Reynolds, and J. M. O'Donnell, Genetics 111:885.
7. Reynolds, E. R. and J. M. O'Donnell, Genetics 119:609.
8. Reynolds, E. R. and J. M. O'Donnell, Devel. Biol. 123:430.
9. McLean, J. R., R. Boswell, and J. M. O'Donnell, Genetics126: 1007.
10. O'Donnell, J. M., J. R. McLean, and E. R. Reynolds, Devel. Genet. 10:273.
11. Hatakayama, K., Y. Inoue, T. Harada, and H. Kagamiyama, J. Biol.
Chern.266:765.
12. Togari, A., H. Ichinose, S. Matsumoto, K. Fujita, and T. Nagatsu, Biochern.
Biophys. Res. Cornrn. 187:359.
13. Jurgens, Raux's Arch. Dev. Biol. 193:2283.
14. Tanaka, K., S. Kaufman, and S. Milstein, Proc. Nat. Acad. Sci. USA
86:5867.

155
STUDIES ON GTP CYCLOHYDROLASE I OF ESCHERICHIA COU

Cornelia Schmid 1, Winfried Meining, 1 Sevil Weinkauf, 2 Luis Bachmann,2

Harald Ritz, 1 Sabine Eberhardt, 1 Wolfgang Gimbel,3 Thomas Werner, 4 Hans-
Werner Lahm, 5 Herbert Nar, 6 and Adelbert Bacher1

1Technische Universit:at Mtinchen, Lehrstuhl ftir Organische Chemie und

Biochemie, D-8046 Garching, Federal Republic of Germany
2Technische Universitat Mtinchen, Lehrstuhl ftir Technische Chemie, D-8046

Garching, Federal Republic of Germany

3GSF Forschungszentrum ftir Umwelt und Gesundheit GmbH, Institut ftir

Molekulare Virologie, D-8042 Neuherberg, Federal Republic of Germany

4 GSF Forschungszentrum ftir Umwelt und Gesundheit GmbH, Institut ftir

Siiugetiergenetik, D-8042 Neuherberg, Federal Republic of Germany

5Pharmaceutical Research New Technologies, F. Hoffmann-La Roche AG,

Basel, Switzerland
6Max-Planck-Institut ftir Biochemie, D-8033 Martinsried, Federal Republic
of Germany

INTRODUCTION

GTP cyclohydrolase I (EC 3.5.4.16) has been obtained from Escherichia coli wild type
cells by affinity chromatography. 1•2 The gene coding for the enzyme from E. coli has been
cloned and sequenced3 •4 and has been mapped at 2251 kb of the physical map of the E. coli
chromosome. 5 Strains carrying a plasmid with the gene under the control of its own
promoter expressed about 100-fold increased enzyme levels. The protein has been
crystallized from citrate buffer. 6 GTP cyclohydrolase genes of rat, 7 man, 8•9 and Bacillus
subtilis 10 have also been cloned, sequenced and expressed.

RESULTS

Gene sequence and expression

The gene coding for GTP cyclohydrolase I of E. coli was expressed under control of
the lac repressor in the plasmid pNC0113. E. coli cells (strain XLl) carrying the
recombinant plasmid produced GTP cyclohydrolase in the range of 30 % of total cell
protein. The enzyme could be obtained in pure form from cell extract by chromatography on
DEAE cellulose. The molecular mass was determined by mass spectrometry yielding a value

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 157
of 24700 +1- 2 Da. This molecular mass varied from the mass predicted on the basis of the
published sequence by 70 Da. 4 The entire gene was therefore resequenced using an
fluorescent label DNA sequencer (ABI 373 A). The DNA sequence with minor corrections
is shown in figure 1. The corrected sequence predicts a protein of 24699.5 Da in agreement
with the experimental value. It is also in line with the partial Edman degradation data
reported earlier. 4 It has the EMBL accession number X63910.

AATTTCTTCATCATACCGTTTAATCAATGTGCTGTGAGTAACTTTCACTTCCGTATTTGC 60

ATAACGATGTTTTAACATCTGCTGATGAAAGGCAGCGGCAATTACAATAATTATCGCTGT 120

GAATACTGGATTATGTGCGCCGCCTCACGCACAATAATCAGGCTGTAAATCAGCTTAATA 180

S.D. (M) P S 2
ACTTTGCCCCCACGCAGGGCGGAGGCGTCACACCTGC~AAATCATAAATGCCATCA 240

L S K E A A L V H E A L V A R G L E T P 22
CTCAGTAAAGAAGCGGCCCTGGTTCATGAAGCGTTAGTTGCGCGAGGACTGGAAACACCG 300

L R P P V H E M D N E T R K S L I A G H 42
CTGCGCCCGCCCGTGCATGAAATGGATAACGAAACGCGCAAAAGCCTTATTGCTGGTCAT 360

M T E I M Q L L N L D L A D D S L M E T 62
ATGACCGAAATCATGCAGCTGCTGAATCTCGACCTGGCTGATGACAGTTTGATGGAAACG 420

P H R I A K M Y V D E I F S G L D Y A N 82
CCGCATCGCATCGCTAAAATGTATGTCGATGAAATTTTCTCCGGTCTGGATTACGCCAAT 480

F P K I T L I E N K M K V D E M V T V R 102
TTCCCGAAAATCACCCTCATTGAAAACAAAATGAAGGTCGATGAAATGGTCACCGTGCGC 540

D I T L T S T C E H H F V T I D G K A T 122
GATATCACTCTGACCAGCACCTGTGAACACCATTTTGTTACCATCGATGGCAAAGCGACG 600

V A Y I P K D S V I G L S K I N R I V Q 142
GTGGCCTATATCCCGAAAGATTCGGTGATCGGTCTGTCAAAAATTAACCGCATTGTGCAG 660

F F A Q R P Q V Q E R L T Q Q I L I A L 162
TTCTTTGCCCAGCGTCCGCAGGTGCAGGAACGTCTGACGCAGCAAATTCTTATTGCGCTA 720

Q T L L G T N N V A V S I D A V H Y C V 182
CAAACGCTGCTGGGCACCAATAACGTGGCTGTCTCGATCGACGCGGTGCATTACTGCGTG 780

K A R G I R D A T S A T T T T S L G G L 202
AAGGCGCGTGGCATCCGCGATGCAACCAGTGCCACGACAACGACCTCTCTTGGTGGATTG 840

F K S S Q N T R H E F L R A V R H H N 221
TTCAAATCCAGTCAGAATACGCGCCACGAGTTTCTGCGCGCTGTGCGTCATCACAACTGA 900

TTAAAAGGCAGGAACCATGGAGCGCAACGTCACGCTCGATTTTGTTCGCGGCGTCGCCAT 960

TCTGGGGATCC 971

Figure 1. Nucleotide sequence and deduced amino acid sequence of the gene coding for GTP cyclohydro-
Iase I of Escherichia coli. The putative Shine-Dalgarno region (S.D.) is underlined. The N-terminal
methionine of the deduced amino-acid sequence is posttranslationally removed, and is indicated in
parentheses.

158
 Crystallization

Crystallization experiments were carried out by the vapour diffusion method and by
batch procedures. Crystals were formed in solutions adjusted to 0.42 M sodium citrate pH
7.4, 20 mM phosphate, 5 mM EDTA, 0.02 % sodium azide and 6 mg of protein per ml.
They appeared as rectangular platelets or cubes with maximum length of 0.4 mm within two
weeks (figure 2A). The crystals have space group P2 1, with cell constants a= 204.5 A, b =
210.2 A, c = 72.2 A, ~ = 95.8° (Vm = 3.2 A3/Da, assuming 20 GTP cyclohydrolase I
subunits per asymmetric unit). They diffract x-rays to beyond 3 Aresolution.
Another crystal form was obtained from protein solutions adjusted to 0.2 M sodium
acetate pH 6.0, 0.1 M MES, 20 mM phosphate, 5 mM EDTA and 8 mg protein per ml. Thin
plates with a maximum length of 0.4 mm were formed within 3 days (figure 2B). They
belong to space group C222 1 with cell dimensions a= 313.2 A, b = 227.0 A, c = 131.5 A
and diffract to 3.5 A resolution.
Crystals were also obtained from solutions adjusted to 6% PEG 6000 pH 7.0, 0.1 M
ammonium sulfate, 0.1 M MOPS, 20 mM phosphate, 5 mM EDTA, 0.02% sodium azide
and 8 mg of protein per m1 (figure 2C). They diffract x-rays to 3 A resolution.

Figure 2. Crystals of GTP cyclohydrolase I of Escherichia coli grown at 20 °C. A, 0.42 M sodium citrate
pH 7.4, 20 mM phosphate, 5 mM EDTA and 0.02% sodium azide; B, 0.2 M sodium acetate pH 6.0, 20 mM
phosphate, 5 mM EDTA, 0.02% sodium azide, 0.1 M MES; C, 6% PEG 6000 pH 7.0, 0.1 M ammonium
sulfate, 0.1 M MOPS, 20 mM phosphate, 5 mM EDTA and 0.02 % sodium azide.

Electron microscopy

Crystal suspensions were frozen in their mother liquor by immersion in liquid nitrogen
without chemical prefixation. The crystal surfaces were exposed by deep-etching at -100 oc
and were replicated by shadowing with platinum/carbon. Alternatively, they were decorated
with gold or silver. The experimental procedure has been published. 11
Figure 3 shows micrographs of different planes of crystals belonging to space group
P2 1• On the basis of their 2-D lattice constants, the crystal planes were indexed following the
procedure described by Bacher et al. 12 Whereas it was not possible to label the crystal planes
unequivocally, the identification could usually be narrowed to two possibilities as shown in
the legend to Figure 3.
Micrographs were processed by standard correlation averaging techniques. 13 The
averaged image of the shadowed crystal plane in figure 4A, as well as the decoration pattern
of silver and gold in figures 4B and 4C indicates particles with fivefold symmetry, revealed
by the outline of the metal deposit and by the arrangement of individual metal clusters. Since
GTP cyclohydrolase I consists of identical subunits and the decoration images strongly

159
 Figure 3. Crystal planes of GTP cyclohydrolase I crystals grown m Citrate buffer (space group P21) with
optical d1ffractograms, crystals were freeze-etched and shadowed w1th Pt/C (45°, 1 3 nm) A, ab-plane (0 0
1) or (1 0 -I) plane, B, ac-plane (0 I 0) or be-plane (I 0 0), C, (I I 0) plane or (1-I 0) plane

Figure 4. CorrelatiOn averages of the crystal ab- or (1 0 -I) plane shadowed with Pt/C (A) and decorated
With Ag (B) or with Au (C) (crystals of space group P21 grown from citrate) The decoration pattern of both
metals and the contour of the molecule m shadowed plane strongly md1cate fivefold symmetry

suggest fivefold symmetry, we conclude that the enzyme complex is a decamer, rather than
an octamer as proposed earlier.2 Decoration also reveals a local fourfold rotation axiS
normal to the observed plane and parallel to the fivefold symmetry a:xts of the molecule. The
electron microscopic observatiOns are well m hne with the results of x-ray crystallography as
descnbed below.
Rotational and translational positions of enzyme molecules can also be observed in
decoration images of crystals grown in acetate (space group C222 1). A detailed electron
mtcroscopic analysis of these crystals ts in progress.

Crystallography

Independent information on the particle symmetry was obtained by calculating

selfrotation functions based on x-ray intensity data which were collected from single crystals
of GTP cyclohydrolase I grown in citrate (space group P2 1).
In mitlal searches, Patterson vector lengths of 5-25 A (representing an mtrasuburut
vector set) were used. Hlgh signals for local four- and fivefold axes were found at polar
angles 'I' = 90°, q, = 84°, i.e. parallel to the crystallographic c-axts. The stgnals were
increased when usmg vectors of length 15-50 A (1.e. spannmg the whole enzyme complex
radms range).

160
 Figure 5 shows a plot of the correlation coefficient R1rorr versus rotation angle K in the
rotation axis orientation ('If =90°, cp =84°). Maxima separated by 18° indicate a 20 fold
repeat of the peak pattern, which in tum is indicative of an arrangement with a fourfold
rotation axis parallel to a local fivefold axis. This is in agreement with the above findings
from electron microscopic studies on single crystals of GTP cyclohydrolase I.
Selfrotation functions with K =180° (search for twofold rotation axes) revealed peaks
at ('If, cp = 174°, K = 180°) with 'I'= 9°, 18°, 27°... 90°. This can be interpreted by the
presence of 5 twofold axes in a plane perpendicular to the fivefold axis of one single enzyme
complex (36° repeat) modulated by an additional fourfold axis to give a repeat of 9°
between 20 twofolds. Thus, the enzyme complex is apparently decameric and has 0 5
symmetry.

100

90
...
=
.~
80
u
....
0::
70
C>
u
60
.2=
...
~
. 50

""""C>
u
40

30

20
0 20 40 60 80 100 120 140 160 180

rotation angle K

Figure 5. Crystallographic selfrot:ltion function computed with a Patterson map at 6.0 Aresolution and the
3000 highest Patterson peaks with vector lengths 15 -50 A. Analysis of the dependence of the correlation
coefficient Rm,. from the rotation angle x: with an axis orientation parallel to the crystallographic c-axis
shows a pattern with 18° repeat. This can be explained by the presence of a local fourfold axis parallel to a
fivefold axis (particle symmetry).

DISCUSSION

The gene coding for GTP cyclohydrolase of E. coli could be expressed with high
efficiency under lac promoter control. In the context of this work, it became necessary to
resequence the entire E. coli gene. Minor corrections were introduced.
The protein could be crystallized in several modifications, which appear suitable for x-
ray structure analysis. No satisfactory heavy metal derivatives could be obtained hitherto.
However, the crystal packing of the monoclinic P2 1 crystals obtained from citrate buffer can
be analyzed by freeze-etching electron microscopy. Thus, the translational position of the
protein molecules in the ab crystal plane could be directly observed. Decoration images
indicated that the protein molecules have fivefold symmetry. Moreover, the approximate
orientation of the fivefold molecular axis with respect to the crystal lattice could be
determined. These observations were confirmed by Patterson analysis of x-ray diffraction
data. The combined electron microscopy and x-ray diffraction data suggest that the protein
molecule has D5 symmetry.

161
 An attempt is in progress to calculate an initial electron density based on the reported
crystallographic and non-crystallographic symmetry data. If successful, the electron density
could be improved by cyclic symmetry averaging without the use of multiple isomorphous
replacement.

ACKNOWLEDGEMENTS

This work was supported by grants from the Deutsche Forschungsgemeinschaft and the
Fonds der Chemischen Industrie. We thank Prof. R. Huber for help and support.

REFERENCES

1. A.W. Burg, and G.M. Brown, J. Bioi. Chem., 243:2349 (1968).

2. J.J. Yim, and G.M. Brown, J. Bioi. Chem., 251:5087 (1976).
3. G. Katzenmeier, C. Schmid, and A. Bacher, FEMS Microbial. Letters, 66:231 (1990).
4. G. Katzenmeier, C. Schmid, J. Kellermann, F. Lottspeich, and A. Bacher, Bioi. Chem. Hoppe-Seyler,
372:991 (1991).
5. H. Ritz, G. Keller, G. Richter, G. Katzenmeier, and A. Bacher, J. Bacterial., 175, in press, (1993).
6. C. Schmid, R. Ladenstein, H. Liicke, R. Huber, and A. Bacher, J. Mol. Bioi., 226:1279 (1992).
7. K. Hatakeyama, Y. !none, T. Hamada, and H. Kagamiyama, J. Bioi. Chem., 266:765 (1991).
8. A. Togari, H. Ichinose, S. Matsumoto, K. Fujita, and T. Nagatsu, Biochem. Biophys. Res. Comm.,
187:359 (1992).
9. M. Giitlich, K. Schott, Th. Werner, A. Bacher, and I. Ziegler, Biochim. Biophys. Acta, 1171:133 (1992).
10. P. Gollnick, S. Ishino, S. M.l. Kuroda, D.J. Henner, and C. Yanofsky, Proc. Natl. Acad. Sci., 87:8726
(1990).
11. L. Bachmann, S. Weinkauf, W. Baumeister, I. Wildhaber, and A. Bacher, J. Mol. Bioi., 207:575 (1989).
12. A. Bacher, S. Weinkauf, L. Bachmann, K. Ritsert. W. Baumeister, R. Huber, and R. Ladenstein, J. Mol.
Bioi., 225:1065 (1992).
13. W.O. Saxton, and W. Baumeister, J. Microsc., 127:127 (1982).

162
PARTIAL PURIFICATION AND CHARACTERIZATION OF
GTP CYCLOHYDROLASE I FROM SPINACH LEAVES

Yasuko Sohta, Tomoko Ohta and Masahiro Masada

Department of Bioresources Chemistry,

Faculty of Horticulture,
Chiba University, Matsuda 648, Chiba Japan

INTRODUCTION

GTP cyclohydrolase I catalyzes the formation of dihydro-

neopterin triphosphate and formate from GTP. This reaction is the
first step in the biosynthetic pathways of cofactors such as
tetrahydrofolate and tetrahydrobiopterin. GTP cyclohydrolase I
has been purified and well characterized in several bacterial
species such as Escherichia coLi (1), LactobaciLLus PLantarum (2),
Comamoanus sP. (3), Seratia indica (4), and BaciLLus
stearothermoPhiLus(5). There were also several attempts which
involved purification and characterization of this enzyme from
animals including human(6), chicken(?), rat(8), mouse(9), and
DrosoPhiLa (10).
Dihydroneopterin triphosphate, the enzymatic product, serves
as a key intermediate in the biosynthsis of tetrahydrofolic acid
in bacteria and tetrahydrobiopterin in mammalian species(6). Even
though folic acid has been well analyzed using bacterial species
by many research groups, the properties of GTP cyclohydrolase I in
higher plants have not been reported. For preliminary attempt of
purification of GTP cyclohydrolase I in higher plants, the enzyme
was partially purified from spinach leaves. This is the first
report of GTP cyclohydrolase I from higher plants.

RESULTS

GTP cyclohydrolase I was partially purified from spinach

leaves homogenate by ammonium sulfate fractionation, heat

Chemistry and Biology of Pteridines and Folates, Edited by

J .E. Ayling et al., Plenum Press, New York, 1993 163
 Table 1. Summary of the purification of GTP cyclohydrolase I

Total Total Total Specific Yield

Ftaction volu11e protein activity activity

ml 119 unit unit/mg %

Crude extract 3,400 9,292
Ammon i Ull su 1fate 94 4,541 5,993 1.32 100
Heat treatment 79 2,652 4,740 1. 79 79.1
Ultrogel AcA34 76 182 1,687 9.27 28.1

Table 2a. Effect of heat treatment temperature Table 2b. Effect of heat treatment time on GTP
on GTP cyclohydrolase I activity. Treatment cyclohydrolase I activity at so·c.
time was for 1 min at all temperature.

Relative Relative Specific Relative Relative Specific

Temperature protein activity activity Tille protein activity activity
("C) ( %) ( %) (Ratio) (min) ( %) ( %) (Ratio)

No heat 100 100 1 No heat 100 100

50 56.1 113.0 2.01 0 87.9 314.2 3.60
60 46.0 43.1 0.94 0.5 81.5 144.7 1. 79
70 7.2 8.9 1.24 68.5 122.1 1.79
80 6.9 0 0 2 65.6 209.0 3.08
5 52.4 79.9 1.53
10 47.9 75.9 1.58

Table 3. Effects of various concentrations of

8H., BH2, FAH., and FAH2 on GTP cyclohydrolase I
activity.

BH. BH2 FAH. FAH2

Cone. Relative act. Cone. Relative act. Cone. Relative act. Cone. Relative act.
(mH) ( %) (mM) ( %) (11M) ( %) (111M) (%)

0 100 0 100 0 100 0 100

2.3 100 2.6 100 1.5 100 1.4 100
4.7 100 5.2 100 3.0 100 2.8 100
9.3 100 10.5 100 6.0 100 5.6 100
18.7 100 20.9 100 12.0 100 11.3 100
37.4 100 41.8 100 24.1 100 22.7 100
48.1 100 45.1 100

BH., tetrahydrobiopterin; BH2, dihydrobiopterin; FAH., tetrahydrofo 1ate ; FAH 2, dihydrofolate.

164
 100

.
80
\
M

....>. 60
:~
....u A

<

/~
Ql
>
~ 40
"'
r-
Ql
a:
A
\

?.0
10~
I v ~ 0

10 ll

pH

Fig. 1. Effect of pH on the activity of GTP cyclohydrolase

The reaction mixture contained 50 mM buffers. The activity in Tris-HCl
buffer(pH 8. 0) was taken as 100%. e, Tris-HCl ; Q, Potasium phsphate;
O, Carbonate; b,, Glysine-NaOH; <), Triethanolamine-HCl.

25

20

?
....
0e 2.0
,...-....
Q.
15
",_
"'
ii = 2.2 1.5
>j;
+'
Q.
0
1.0 ...__.,
""'
0

0.5

"',_
+'
I0

"' 0 0. 5
lo9 (S)
"0 -0.5
+'
u
"'
"'"'
~1.0

> -1.5

0. 5 I. 0 I ;

GTP ( mM )

Fig. 2. Effect of GTP cocentration on GTP cyclohydrolase I activity.

165
treatment, and Ultrogel AcA34 column chromatography. The overall
purification is summarized in Table 1. Seventy percent of GTP
cyclohydrolase I activity was found in the fraction precipitated
by 30-50% saturation with ammonium sulfate. Simultaneously about
70% of the protein was precipitated. After fractionation with
ammonium sulfate, to establish the appropriate condition of heat
treatment, several·conditions were applied. Heat treatment for 0
min at 50 ·c resulted in the loss of 12% protein and in 3. 6-fold
specific activity(Table 2). Heat treated fraction was centrifuged
and the supernatant was applied to a column of Ultrogel AcA34. The
activity was eluted as a single peak from the column
chromatography. Active fractions were combined and subjected to
the characterization.
The pH optimum of the enzyme reaction was estimated to be 8.0
under the condition examined(Fig. 1), and the enzyme activity
showed the difference in each buffer.
On the enzyme concentration curve, the pattern of neopterin
produced in the reaction mixture was shown as a sigmoidal curve.
The effect of GTP concentration on the enzyme activity is
shown in Fig 2. A plot of GTP concentration versus velocity
yielded a curve that did not obey the Michaelis-Menten equation.
The Hill coefficient(n) obtained from Hill plot was 2.2.
To study the effect of the end products in the biothynthetic
pathway, various concentrations of tetrahydrobiopterin,
dihydrobiopterin, tetrahydrofolate, and dihydrofolate were
examined. The enzyme activity was not affected in any
concentration and any compound(Table 3).

REFERENCES

1. Yim, J. and Brown, G.M. (1976) J. Biol. Chern. 251, 5087-5094.

2. Jackson,R.J. and Shiota,T. (1975) Biochem. Biophys. Acta 403,
232-244.
3. Cone, J. E., Plowman, J. and Giroff, G. (1974) J. Biol. Chern.
249, 5551-5558.
4. Kohashi, M., Itadani, T. and Iwai, K. (1980) Agric. Biol. 44,
271-278.
5. Suzuki, Y., Yasui, T. and Abe, s. (1979) J. Biochem. 86, 1679-
1685.
6. Blau, N and Niederwieser, A. (1985) J. Clin. Chern. Clin.
Biochem. 23, 169-176.
7. Fukushima, K., Richter, w. E. and Shiota, T. (1977) J. Biol.
Chern. 252, 5750-5755.
8. Hatakeyama, K., Harada. T., Suzuki, s., Watanabe, Y. and
Kagamiyama, H. (1989) J. Biol. Chern. 264, 21660-21664.
9. Cha, K. w., Jacobson, K. B. and Yim, J. (1991) J. Biol. Chern.
266, 12294-12300.
10. Fan, C. L. and Brown, G. M. (1976) Biochem. Genet. 14, 259-
270.

166
DETECTION AND QUANTIFICATION OF GTP CYCLOHYDROLASE I m RNA

Markus Giitlichl, Karin Schott1, Thomas Wemer2, Adelbert Bacher3 and

lrmgard Ziegler1

1 GSF-Institut fiir Klinische Molekularbiologie und Tumorgenetik,

MarchioninistraBe 25, D-8000 Miinchen 70, Germany
2 GSF-Institut fiir Saugetiergenetik, Ingolstiidter Landstr. 1,
D-8042 Neuherberg, Germany
3 Techinsche Universitat Miinchen, Lehrstuhl fiir Organische Chemie
und Biochemie, Lichtenbergstr. 4, D-8046 Garching

INTRODUCTION

There is accumulating evidence that H4biopterin is synthesized in cells which do not

depend on H4biopterin cofactor but undergo cytokine directed proliferation 1. In this case,
H4biopterin has a function which is apparently unrelated to its known cofactor role. It
modulates the clonal expansion of T cells2-4 and the proliferation of erythroid cells5,6.
Closer analysis has shown that it enhances the affinity of the IL-2 receptor complex to its
ligand and subsequently affects various aspects of signal transmission?.
A more detailed understanding of H4biopterin levels in tissues and cells depends on
analysis of the biosynthetic enzymes and the regulatory mechanisms involved. Gradually
increasing activities of GTP cyclohydrolase I and sepiapterin reductase during lectin
stimulation of resting human T lymphocytes satisfactorily explained the accumulation of
H4biopterin during blast transformationS. In primed human T cells, an accelerated synthesis
of H4biopterin results from increased activities of GTP cyclohydrolase I, 6-pyruvoyl-
H4pterin synthase and sepiapterin reductase which are controlled by the synergistic action
of IFN-y and IL-29. In rat thymocytes, H4biopterin synthesis is associated with the cell-
cycle. It culminates at the time of S-phase entry, in parallel with the specific activities of
both GTP cyclohydrolase I and sepiapterin reductase. These increases in enzyme activities
correlate with the steady state mRNA levels for both enzymes, whereas the subsequent
decrease in the activity of the rate limiting enzyme GTP cyclohydrolase I is due to post-
translational modification 10. These preliminary studies make it evident that a closer
understanding of the regulatory steps involved in H4biopterin synthesis of human cells
critically depends on genetic analysis of the rate limiting enzyme GTP cyclohydrolase I.
The genes coding for GTP cyclohydrolase I from man, rat and Escherichia coli have
been cloned and sequencedll-14. This study reports the cloning of a 555 bp fragment of
the human eDNA and of a 578 bp fragment of the murine GTP cyclohydrolase I.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 167
Both Fragments were obtained using a PCR strategy based on the sequence homology
between the bacterial and the rat gene. PCR generated probes were used for
characterization of species and tissue specificity of the GTP cyclohydrolase I mRNA. The
cloned human eDNA segment was also used to isolate eDNA clones that code for the GTP
cyclohydrolase I type 1 and type 2.

MATERIALS AND METHODS

Tissues preparation, cell culture, RNA purification and Northern blot analysis were
performed as described previously14. For reverse transcriptase-PCR the following primers
were used: primer 1 GCAGCGAGGAGGATAACGAG, primer 2 AACTCCTCC
CGAGTCTTNGG (N= A, C, G and T) primer 3 GAGTGTGAGTGATCCTCGACTTGA.
Pairwise combinations of primers 1 and 2 yealded a 555 bp product, wereas primer 1 and 3
lead to a 578 bp product. Radiolabelling of eDNA probes was performed as described
previously14. Pairwise combination of primers 3 ACGAGATGGTGATTGTGAA(G/A)G
and primer 4 GGTAAGGCGTTCTTGAAC(G/f)TG lead to a PCR roduct of 179 bp that
matched to position 480-658 of the rat sequence12 and to position 448-626 and of the
human sequencell.

RESULTS AND DISCUSSION

Sequence analysis

The amino acid sequences of GTP cyclohydrolase I from rat and E. coli show
considerable homology 13. PCR primers were designed to match the rat eDNA sequence in
areas of maximum homology with the E. coli gene. Mixed oligonucleotides were used in
the 3 '-terminal section of the respective primers. Pairwise combinations of primers were

GCAGCGAGGAGGATAACGAGGI'GAACCTCCCCAAAC'IGGCGGCTGCTI'ACTCGTCCATCC'IGCTCTCGC'IGGGC - 7 4
S E E D N E V N L P K L A A A Y S S I L L S L G

GAGGACCCCCAGCGGCAOOGGCTGCTCAAGACGCCC'IGGAGGGCGGCCACCGCCATGCAGTACTI'CACCAAGGGA - 149
E D P Q R Q G L L K T P W S A A T A M Q Y F T K G

TACCAGGAGACCATCTCAGA'IGTCC'IGAATGATGCTATATI'IGA'IGAAGATCA'IGACGAGA'IGG'IGATI'G'IGAAG - 2 24
Y Q E T I S D V L N D A M F D E D H D E M V I V K

GACATAGACA'IGTTTI'CCA'IG'IG'IGAGCATCACCTTGTI'CCATI'IGTAGGAAGSGTCCATATI'GGCTATCTI'CCT - 299
D M D M F S M C E H H L V P F V G R V H I G Y L P

AACAAGCAAGTCCT'IGGTCTCAGTAAACTTGCCAGGATI'GTAGAAATCTACAGTAGACGACTACAAGTI'CAAGAA - 37 4
N K Q V L G L S K L A S I V E I Y S S R L Q V Q E

CGCCTTACCAAACAGATTGCOOTQ3CCATCACAGAAGCCTIGC.AGCCTGC'IGGCGTIU::AGTAGIGAT'IGAAGCG - 449
R L T K Q I A V A I T E A L Q P A G V G V V I E A

ACACACA'IGTGCA'IGGTAATGCGAGG'IGTG:AGAAAA'IGAACAGCAAGAC'IG'ICACTAGCACCATGC'IGGGCG'IG - 524
T H M C M V M R G V Q K M N S K T V T S T M L G V

TTCCGGGAAGACCCAAAGACTCGGGAOOAGTCCC'ICACACTCATCAGGAGC'IGA - 578
F R E D P K T R E E S L T L I R S *

Figure 1. Patrial eDNA sequence of murine GTP cyclohydrolase I. Marked nucleotides are
different from the human eDNA sequence11.14.

168
used for PCR experiments using rat RNA as template. eDNA was prepared from RNA by
reverse transcription and was subsequently amplified. The lengths and the sequences of the
respective rat PCR products were in agreement with the published eDNA sequence12.
The same primer combinations were used in PCR experiments using human and
murine eDNA as templates. PCR products were purified by gel electrophoresis and HPLC
and were sequenced on both strands. The human and murine eDNA segments encompassed
555 bp and 578 bp respectively. Both PCR products were cloned and used as templates for
radiolabelling of eDNA probes.
The partial sequence of the murine GTP cyclohydrolase I eDNA differed from the
human sequence by 56 nucleotides as shown in Fig. 1. It should be noted that most of the
exchanges are located in the wobble positions of the respective codons so that only 10
different amino acids are coded. The deduced amino acid sequence shows 93 % identity
with the human sequence. Among the 10 amino acid residue exchanges, 6 were
conservative.

Tissue speciflty

Total RNA was obtained from liver, kidney, bone marrow, spleen and brain from rats
and from human peripheral blood lymphocytes. Messenger RNA was prepared from several
rat and human cell lines. Northern blot analysis of rat RNA was performed using the 179 bp
rat eDNA probe. This probe hybridized with two rat mRNA species with approximate sizes
of 1.4 and 3.6 kb. The two different species were detected in all rat RNA preparations
analyzed (Fig. 2A). Studies of hybridization stringency indicated that the probes bound
with comparable efficiency to both mRNA species. This indicates that rats produce two

AI 2 3 4 56 7 8 910 2 3 4
c

t-28 s
t- 28 s

t- 18 s f- 18 s

8
1 o.8 1 2.4 1 o.6 1.2 2.4

Figure 2. (A) Northern blot of total RNA from rat organs. Total mRNA (20 or 40 f.lg) of liver (lanes 1 and 2),
kidney (lanes 3 and 4), bone marrow (lanes 5 and 6), spleen (lanes 7 and 8), and bram (lanes 9 and 10) were
hybridized with the radiolabelled rat eDNA probe; (B) Ratio of G1P eyclohydrolase I mRNA species (3.6 kb
mRNA/1.4 kb mRNA) in different rat tissues. (C) Northern blot of human and rat RNA. Hybridization was
performed with the human eDNA probe. Lane 1, 15 f.lg of total RNA of human peripheral blood lymphocytes
stimulated by PHA for 48 h; lane 2, 2 r.tg of mRNA of human T eellline HUT 102; lane 3, 2r.tg of mRNA of
human liver eellline HuH7; lane 4, 2 Jlg of mRNA of rat liver cell line HTC.

169
different messengers for GTP-cyclohydrolase I. The ratio of the two presumed mRNA
species varied between 0.6 and 2.4 in different rat organs (Fig. 2B). The large mRNA
species was relatively more abundant in brain and kidney. It appears likely that the 1.4 kb
species from rat corresponds to the eDNA of 1024 bp reported by Hatakeyama et aL12. This
mRNA would appear to be sufficient for the formation of the GTP cyclohydrolase I subunit
from rat as studied by Hatakeyama. The larger mRNA species has not been reported by
Hatakeyama et a1.12.
For detection of the human GTP cyclohydrolase I mRNA a radiolabelled human
eDNA probe was used that matched all known isoformsll. The human probe detected both
mRNA species of the rat (Fig. 2C). The observed cross-hybridization between the rat and
human sequences is in line with the high degree of DNA sequence homology. In Northern
blots of RNA from human cell lines and tissue, only one mRNA species was found. The
size of this band is comparable to the large mRNA species of rat. Although multiple forms
of eDNA for GTP cycylohydrolase I could be identified 11, only one band could be detected
on Northern blots of human RNA. It remains unclear if all forms of the mRNA share the
same lengths or if two forms are expressed so scarcely that they were below detection limit.
In light of the various metabolic and regulatory functions of H4biopterin, the occurence of
different mRNAs could have regulatory significance. It should be noted that the ratio of the
two mRNA species shows significant differences in the rat organs studied.The cloned
human and murine eDNA probes provide tools for the study of GTP cyclohydrolase I
regulation at the genetic level in mammalian cell systems.
The cloned eDNA Fragment of the human GTP cyclohydrolase I was used to screen a
eDNA library of human liver. Sequence analysis of isolated clones showed two different
coding regions of 250 and 213 arninoacids.

ACKNOWLEDGMENTS

We thank Dr. Elisabeth H. Weiss from the lnstitut fiir Anthropologie und
Humangenetik at the Ludwig Maximillians University Munich for providing us with the
eDNA library.

REFERENCES
1 I. Ziegler, Med. Res. Rev. 10:95 (1990).
2 I. Ziegler, K. Hamm, and I. Berndt, Cancer Res. 43:5356 (1983).
3 S. Webber, and D.J. Jaye. in: Chemistry and Biology of Pteridines, (B.A. Cooper, V.M. Whitehead, eds), pp.
235-238, Walter de Gruyter & Co., Berlin (1986).
4 R.S. Shen, andY. Zhang, Biochemical Archives 7:21 (1991).
5 K. Tanaka, S. Kaufman, and S. Milstien, Proc. Nat/. Acad. Sci. 86:5864 (1989).
6 F. Kerler, L. Htiltner, I. Ziegler, G. Katzenmeier, and A. Bacher, J Cell. Physiol. 142:268 (1990).
7 I. Ziegler, and U. Schwulera,J. Cell. Biochem. 265:17026 (1989).
8 F. Kerler, I. Ziegler, B. Schwarzkopf, and A. Bacher, FEBS Lett. 250:622 (1989).
9 I. Ziegler, K. Schott, M. Liibbert, F. Herrmann, U. Schwulera, and A. Bacher,]. Bioi. Chem.265:11026 (1990).
lO K. Schott, K. Brand, K. Hatakeyama, H. Kagamiyama, J. Maier, T. Werner, and I. Ziegler, Exp Cell Res.
200:105 (1992).
11 A. Togari, H. Ichinose, S. Matsumoto, K. Fujita, and T. Nagatsu, T. Biochem. Biophys. Res. Comm. 187:359
(1992).
12 K. Hatakeyama, Y. Inoue, T. Harada, and H. Kagamiyama,J. Bioi. Chern. 266:765 (1991).
13 G. Katzenmeier, C. Schmid, J. Kellermann, F. Lottspeich, and A. Bacher, Biochem. Hoppe-Seyler 372:991
(1991).
14 M. Giitlich, K. Schott, T. Werner, A. Bacher, and I. Ziegler, Biochim. Biophys. Acta 1171:133 (1992).

170
LOCALIZATION OF GTP CYCLOHYDROLASE I (GTPCH) mRNA IN THE RAT
BRAIN BY IN SITU HYBRIDIZATION

Stephen I. Lentz, Kei Hirayama, and Gregory Kapatos

Cellular and Clinical Neurobiology Program, Department of Psychiatry,

Wayne State University School of Medicine, Detroit, Michigan 48201, USA

INTRODUCTION

GTP cyclohydrolase I (GTPCH) is the first and rate-limiting enzyme in the

tetrahydrobiopterin (BH4) biosynthetic pathway. BH4 is the essential cofactor for th'e
pterin-dependent monooxygenases which include tyrosine and tryptophan hydroxylase,
enzymes that are rate limiting in the biosynthesis of catecholamines and indolamines. BH4
is also required as a cofactor for the family of nitric oxide synthases 1•2• We have obtained
a clone for GTPCH based on the recently published full-length eDNA encoding GTPCH
from rat live.il.4. The in situ hybridization technique was used to study the cellular
localization and the relative levels of expression of GTPCH mRNA in the rat brain.

METHODS

In situ hybridization was performed according to previously published methods5' 6•

Briefly, Sprague-Dawley rats (300-350 g) were decapitated and the brains were removed
and frozen in isopentane on dry ice. Coronal sections of 10 pm were taken every 0.4 mm
between the diencephalon and the myencephalon. Sections were thaw-mounted onto
microscope slides coated twice with porcine gelatin and chromalum, and stored desiccated
at -70°C until use. On the day of the hybridization, mounted sections were quickly air-
dried under a stream of cool air and fixed for 5 min in 3% paraformaldehyde in 0.1 M
sodium phosphate-buffered saline. Sections were rinsed, acetylated in 0.25% acetic
anhydride/0.1 M triethanolamine (pH 8.0), rinsed, dehydrated and air dried. Sections were
prehybridized for 1 h at 52°C in humid chambers with prehybridization buffer containing
40% formamide, 4X saline-sodium citrate (SSC), 10 mM dithiothreitol, 1 mg/ml sheared
salmon sperm DNA, 1 mg/ml yeast tRNA, 1X Denhardt's solution. Prehybridization buffer
was removed and immediately replaced with hybridization buffer (prehybridization buffer
including 10% dextran sulfate) containing 60 fmol of 35 S-CTP-labelled antisense cRNA
probe derived from a clone for GTPCH. The 302 bp coding portion of GTPCH eDNA
(nucleotides 269 through 570), based upon the published sequence for rat liver GTPCH4

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 171
was cloned using the reverse transcription-polymerase chain reaction technique3•
Hybridization was carried out at 52°C in humid chambers for 3.5 h. The slides were then
rinsed, treated with 25 pg/ml RNase A at 37°C for 30 min, cooled to room temperature,
rinsed and dehydrated. For autoradiography, slides were dipped in NTB2 nuclear track
emulsion diluted 1:1 with 600 mM ammonium acetate, exposed at 4°C for 4 to 6 weeks,
developed, counterstained with Harris' hematoxylin/eosin, and coverslipped. Sections were
photographed under darkfield illumination with a Zeiss photomicroscope III.

RESULTS AND DISCUSSION

The nomenclature first described by Dahlstrom and Fuxe7 and Hokfelt et. al. 8 was used
to identify catecholamine and 5-hydroxytryptamine containing cell groups. In the
diencephalon, at the level of the optic chiasm, neurons containing GTPCH mRNA
(GTPCH+) were found scattered along the third ventricle and spread laterally above the
optic chiasm (dopaminergic (DA), Al4). Moving caudally, GTPCH+ neurons spread
laterally at the dorsal (DA, Al2 dorsal) and ventral ends of the third ventricle (DA, A12
ventral). GTPCH+ neurons were also located at the ventral surface of the brain as far
lateral as the supraoptic nucleus (DA, Al5 ventral). A group of GTPCH+ neurons emerged
caudal to the paraventricular nucleus, dorsal to the dorsomedial nucleus and within the zona
incerta (DA, A13). Another appeared caudal to Al3 and medial to the mammillothalarnic
tract (DA, All). As seen in Figure 1, the mesencephalon gave rise to GTPCH+ neurons
in the tegmentum that correspond to the well characterized distribution of the DA-neurons

Figure 1. Localization of G1PCH mRNA in the ventral tegmental area (VTA) and the substantia nigra pars
compacta (SNC). After a 4 week exposure period, GTPCH mRNA was found to be localized to cells
throughout the VTA but relatively few cells were labelled in the SNC. Bar= 100 )lm.

of the ventral tegmental area (VTA; AlO) and substantia nigra pars compacta (SNC; A9).
The GTPCH+ neurons within the SNC, however, appeared to express much lower levels
of GTPCH mRNA than did their counterparts of the VTA. Positive cells were also
observed in the substantia nigra pars lateralis (DA, A9 lateral) and pars reticulata (DA, A9
ventral). Another GTPCH+ group was located in the pineal recess of the third ventricle
that continued caudally to form the pineal gland (melatonin-containing). Figure 2a shows
intensely labelled cells distributed throughout the pineal gland at the level of the inferior

172
 Figure 2. Localization of GTPCH mRNA in the pineal gland and locus coeruleus.
(a) After a 4 week exposure period, cells were intensely labelled throughout the
pineal gland with a low level of background labelling in the inferior colliculus
(IC). (b) After a 4 week exposure period, GTPCH mRNA was found to be
localized to neurons within the locus coeruleus. Other abbreviations: fourth
ventricle, IV. Bars (a and b)= 100 J.lffi.

colliculus (IC). GTPCH+ neurons were distributed around and within the rostral subnucleus
of the interpeduncular nucleus (serotonergic (5-HT) neurons). Near the caudal extent of A9
began another GTPCH+ group designated supralemniscal cells (DA-neurons; A8). At this
level GTPCH+ neurons ran along the midline (5-HT-neurons of the caudal linear nucleus
raphe, B8; and dorsal raphe, B7) and dorsal to the medial lemniscus (5-HT-neurons; B9).
Figure 3 shows heavily labelled GTPCH+ cells in the dorsal raphe. Another midline
GTPCH+ group (5-HT-neurons of the median raphe nucleus) was located caudal to A8.
Several groups of GTPCH+ neurons were located at the rostral end of the metencephalon.
Two were found in the lateral aspects of the pontine reticular formation, one close to the
lateral lemniscus (noradrenergic (NE) neurons, A7) and another in the ventrolateral pons,
medial to the trigeminal and facial nerves (NE-neurons; A5). GTPCH+ neurons were also
located off the midline below the fourth ventricle (Figure 2b) which corresponded toNE-
neurons of the locus coeruleus (A6) and subcoeruleus nucleus (A6 ventral). A fourth group
lined the midline ventral to the dorsal raphe (5-HT-neurons of the raphe pontis nucleus;
B5). GTPCH+ neurons were situated ventrally in the myelencephalon along the pyramidal
tracts comprising the raphe magnus nucleus (5-HT-neurons; B3). Another GTPCH+ group
was found in the lateral part of the roof of the fourth ventricle which forms a dorsolateral
continuation of the NE-neurons of the A6 complex (NE-neurons; A4). Further caudal,

173
 Figure 3. Localization of GTPCH mRNA in the dorsal raphe. After a 4 week
exposure period, cells were heavily labelled throughout the dorsal raphe. Other
abbreviations: cerebral aqueduct, Aq. Bar = 100 pm.

GTPCH+ neurons were located in regions known to correspond to adrenergic neurons along
the midline below the fourth ventricle (C3), lateral to C3 near the nucleus of the solitary
tract (C2), and on the ventral surface lateral to the pyramidal tract (Cl). At this same level,
GTPCH+ neurons were also situated within well defined midline 5-HT neurons of the raphe
pallidus (B2) and raphe obscurus (Bl) nuclei.
GTPCH+ neurons in the rat brain thus correspond to the known monoaminergic cell
groups. The level of GTPCH mRNA expression varied across cell groups with high levels
seen in 5-HT-neurons (Bl-B9) and NE-neurons (A6), moderate levels in DA-neurons (A5,
A7, A8, and AlO), and low levels seen in DA-neurons (A9, A12-A15). Under the
hybridization and autoradiographic conditions used in this study, GTPCH mRNA could not
be unequivocally localized to any cell-type within the olfactory bulb, cerebellum or
hippocampus, brain regions known to contain large numbers of nitric oxide synthase-
positive neurons.

REFERENCES
1. M.A. Tayeh and M.A. Marietta, J. Bioi. Chem. 264:19654-19658 (1989).
2. K. Schmidt, E.R. Werner, B. Mayer, H. Wachter, and W.R. Kukovetz, Biochem. J. 281:297-300 (1992).
3. K. Hirayama, S.I. Lentz, and G. Kapatos, this volume.
4. K. Hatakeyama, Y. Inoue, T. Harada, and H. Kagamiyama, J. Bioi. Chem. 266:765-769 (1991).
5. M.F. Chesselet, L. Weiss, C. Wuenschell, A.J. Tobin, and H.U. Affolter, J. Comp. Neuroi. 262:125-140
(1987).
6. K. Hirayama, S.I. Lentz, and G. Kapatos, J. Neurochem. in press (1993).
7. A. Dahlstrom and K. Fuxe, Acta Physiol. Scand. 62, Suppl. 232:1-55 (1964).
8. T. Hokfelt, K. Fuxe, M. Goldstein, and 0. Johansson, Brain Res. 66:235-251 (1974).

174
EXPRESSION OF GTP CYCLOHYDROLASE I (GTPCH) mRNA IN THE RAT:
TISSUE DISTRffiUTION AND EFFECT OF RESERPINE

Kei Hirayama, Stephen I. Lentz and Gregory Kapatos

Cellular and Clinical Neurobiology Program, Department of Psychiatry,

Wayne State University School of Medicine, Detroit, Michigan 48201, USA

INTRODUCTION

Disruption of vesicular monoamine storage by reserpine results in a marked depletion

of monoamines in the central and peripheral nervous systems and a concomitant increase
in tyrosine hydroxylase (TH) gene expression in response to the resulting acceleration of
nerve impulse flow. This increase in TH mRNA appears to be catecholamine (CA) cell-
type specific in that it is observed within NE-containing neurons of the locus coeruleus
(LC), superior cervical ganglia (SCG) and adrenal gland or adrenal chromaffin cells but not
in DA neurons of the substantia nigra (SN) 1•2• Tetrahydrobiopterin (BH4) is synthesized
by three or four enzymes from GW·4 and is the reduced pteridine cofactor required for CA
and nitric oxide biosynthesis5•6• GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme
in this BH4 biosynthetic pathway3.4. Few studies have examined GTPCH gene expression
in monoamine-containing neurons. A full-length eDNA encoding GTPCH has been isolated
from rat liver7• We have used the reverse transcription-polymerase chain reaction (RT-PCR)
to clone a coding portion of this sequence from rat adrenal gland. With this clone we have
performed an analysis of GTPCH mRNA in rat tissues by Northern blot and nuclease
protection assay and have examined the expression of GTPCH mRNA in CA neurons of
the peripheral and central nervous systems following a single reserpine injection.

METHODS

Male Sprague-Dawley rats (220-250 g) were injected intraperitoneally with a single

dose of reserpine (10 mg/kg). Control animals were injected with drug vehicle. Six to 72
h after injection, rats were sacrificed by decapitation and SCG and brain tissues containing
the LC and SN were dissected and pooled. For Northern blot analysis, RNA was run on
a agarose gel containing 2 M formaldehyde. After transfer, the blot was hybridized in the
solution containing 32P-labelled eRNA probe. GTPCH mRNA was quantitated by nuclease
protection assay with 1 to 100 pg of total RNA from various rat tissues. RNA was
hybridized with 32P-labelled antisense cRNA probe. Non-hybridized probe was digested

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 175
with T1 ribonuclease and then protein digested with proteinase K. Samples were then
electrophoresed in denaturing 6% polyacrylamide gels containing 8 M urea. Gels were
dried and exposed to X-ray film for 16-72 h. Autoradiograms were quantitated by laser
densitometry.

RESULTS AND DISCUSSION

The following oligonucleotides derived from the rat liver GTPCH eDNA sequence
were used: primer A, 5'(dCCACCGCCATGCAGTTCTTCACCA) identical to the coding
sequence 269-292; primer B, 5'(dAGGCTGCAAGGCTTCTGTGATGGC) complementary
to the coding sequence 547-570 for amplification of GTPCH. EcoR1 and BamH1 site
sequences were included in the 5' ends of primer A and B, respectively. This sequence was
chosen for amplification and cloning because it lacks homology with other proteins known
to bind pteridines, including the enzymes, dihydrofolate reductase, sepiapterin reductase and
6-pyruvoyl-tetrahydropterin synthase7•8•9• For amplification of eDNA, total cellular RNA
extracted from rat adrenal gland (1 pg) was transcribed into a single-stranded eDNA, then
further amplification was performed by PCR. Amplification temperatures were as follows,
denaturation at 94°C for 1 min, annealing at 60°C for 1 min, synthesis at 72°C for 2 min
and extension for annealing at 72°C for 10 second per cycle. The total cycle number was
30. Double stranded rat liver eDNA (1 ng) was also used for PCR amplification. The
PCR-amplified products were run on agarose gel. As shown in Figure 1A, PCR performed
following either RT of RNA from rat adrenal gland or with rat liver eDNA generated single
products of approximately 320 bp. To confirm that the amplified DNAs corresponded to
rat GTPCH, restriction endonuclease digestion were performed at an internal BstX1 site
predicted from the published sequence for rat liver GTPCH. Digestion of either the liver-
or adrenal gland-derived PCR product generated two bands of the predicted sizes (94 and
226 bp, Figure. lB). PCR-amplified DNA was extracted from a agarose gel, and DNA was
cloned into a pGEM-3Z vector on BarnH1 and EcoR1 polylinker sites.

A B
2 3 2 3
bp bp

603-
- 603
310 -
-3 10
118-
-1 lfl

Figure 1. A: PCR products run on a 4.0% low melting agarose gel. Lane 1: PCR
product generated with rat liver eDNA. 2: DNA size markers: 1353, 1078, 872,
603,310, 271,234, 194, 118,72 bp. 3: RT-PCR product generated with 1 pg total
cellular RNA from adrenal gland. B: DNAs run on 1.2% agarose gel. 1: undigested
PCR product; 2: digested PCR product with the restriction enzyme BstX1 at 50°C
for 2 h; 3: DNA size markers.

176
Sequence analysis of insert DNA showed it to be identical to the previously reported
sequence for rat liver GTPCH.
The size and relative abundance of GTP':H mRNA in various rat tissues were
determined by Northern blot analysis. As shown in Figure 2A, hybridization at high
stringency to total cellular RNA from tissues known to contain GTPCH enzyme activity
(pineal gland, liver, adrenal gland and brainstem) detected two species of GTPCH mRNA
at 3.8 and 1.2 kb. No hybridization signal was observed on analysis of RNA from the
cerebellum, a brain region that contains extremely low levels of GTPCH enzyme activity
(Figure 2A, lane 1). The ratio of the 3.8 and 1.2 kb forms varied across tissues, with the
larger species predominating in the pineal gland, adrenal gland and brainstem, and the
smaller prominent in the liver. By Northern analysis, GTPCH mRNA in the pineal gland
was estimated to be tenfold more abundant than in the liver and one hundredfold more
abundant than in the brainstem. A preliminary characterization of the relationship of these
two mRNA transcripts to GTPCH protein was performed on whole adrenal gland RNA
following reserpine administration, a treatment known to increase GTPCH enzyme activity
in both the adrenal cortex and medulla. As shown in Figure 2B, 72 h following treatment
with reserpine, the abundance of both large and small forms of GTPCH mRNA were
increased approximately twofold. Heterogeneity of GTPCH mRNA might be the result of
alternative splicing of pre-mRNA, alternative transcription initiation sites or multiple
polyadenylation signals. In order to confirm and quantitate the occurrence of GTPCH
mRNA in rat tissues containing low levels of GTPCH mRNA, a highly sensitive nuclease
protection assay was developed. In agreement with the preliminary data obtained by
Northern analysis, GTPCH mRNA was found to be most abundant in the pineal gland (102
amoVpg RNA), with liver and spleen containing 14.1 and 3.90 amoVpg RNA, respectively.
PC12 cells, whole adrenal gland and lung were found to contain essentially equal levels of
GTPCH mRNA (1.97 to 2.20 amol/pg RNA). Low but quantifiable levels of expression
were observed in the pituitary, brainstem, kidney and thymus(~ 1 amol/ug RNA). No signal
was detected when 100 pg of striated muscle or heart RNA was assayed.

E
.a
a;
.Q
~
iii
CD a;
iii
c
...
CD
.."'
E
G)

c
~
..
CD
c
'Q.
a;
G) c > "CC 'i c0 "'
CD
(J i:i: ::i c( &a (J a:

28S-

18S-

A
Figure 2. Northern blot analysis of GTPCH mRNA. A: Total cellular RNA from
cerebellum (20 pg), liver (20 pg), pineal gland (0.35 pg), adrenal gland (20 pg) and
brainstem (lower midbrain and upper pons, 20 pg) was analyzed. Exposure time
for the lane containing brainstem RNA was approximately tenfold longer that the
other samples. No signal was detectable in the lane containing cerebellar RNA
even at this longer exposure time. 8: Total cellular RNA (20 pg) from control or
reserpinized adrenal gland 72 h after treatment.

177
 300

~ 250 SCG
~
e
...... ----
-
0 200
v
"'<:<$ ~0
0 ()

~ .......

-
;>., 0 150
..= ~
0 ._,
()
;>.,
()
100
A..
G
0 12 24 36 48 60 72
Time (hours)
Figure 3. Time-course of the increase in GTPCH mRNA levels in the rat SCG, LC and SN following
administration of 10 mg/kg reserpine. Total cellular RNA from SCG (12 pg), LC (40 pg) and SN (40 pg)
were analyzed by nuclease protection assay. Data from a single experiment were plotted as a percent of basal
levels. Essentially identical data were obtained from three separate experiments.

In large part, the tissue distribution of GTPCH mRNA determined by this assay appears
to agree with that reported for BH4 and GTPCH enzyme activity. The expression of
relatively high levels of GTPCH mRNA in the spleen, lung, thymus and pituitary, tissues
that do not contain aromatic amino acid hydroxylases, may be related to the role of BH4
in cell proliferation and differentiation or as a cofactor for nitric oxide synthase.
We have also examined by nuclease protection assay the expression of GTPCH mRNA
in CA neurons of the peripheral and central nervous systems following a single reserpine
injection (Figure 3). GTPCH mRNA levels in the SCG were increased over twofold at the
earliest time-point examined (6 hours) and began to decline 72 h following reserpine
treatment. GTPCH mRNA levels in the LC were also increased twofold but this peak was
shifted to 24 h post reserpine. The increase in GTPCH mRNA levels in the SN in response
to reserpine was similar to that found in the LC, with a twofold increase above control
levels at 24 h. The increase of GTPCH mRNA in both LC and SN declined beyond 24 h
but was still elevated 72 h after treatment. In contrast to the reported specificity of the
effect of reserpine on TH mRNA, however, GTPCH mRNA levels were increased in both
NE- and DA-containing neurons in response to this drug. These data suggest that the level
of expression of GTPCH mRNA may be coupled to changes in nerve impulse flow and
accompanying second messenger systems and, that the regulation of GTPCH and TH gene
expression may not be coordinated within DA neurons of the SN.

REFERENCES

1. G.M.Pasinetti, D.G.Morgan, S.A.Johnson, M.A.Myers, and C.E.Finch, J. Neurochem. 55:1793 (1990).

2. S.O.Franklin, Y-S.Zhu, B.C.Yoburn, and C.E.Inturrisi, Mol. Pharmacal. 40:515 (1991).
3. C.A.Nichol, G.K.Smith, and D.S.Duch, Ann. Rev. Biochem. 54:729 (1985).
4. R.A.Levine, G.Kapatos, S.Kaufman, and S.Milstien, 1. Neurochem. 54:1218 (1990).
5. K.Schmidt, E.R.Wemer, B.Mayer H.Wachter, and W.R.Kukovets, Biochem. J. 281:297 (1992).
6. S.S.Gross, E.A.Jaffe, R.Levi, and R.G.Kilboum, Biochem. Biopys. Res. Commun. 178:823 (1991).
7. K.Hatakeyama, T.Harada, S.Suzuki, Y.Watanabe, and H.Kagamiyama, J. Bioi. Chem. 266:20791 (1991).
8. B.A. Citron, S.Milstien, J.C.Guterrez, R.aLevine, B.L.Yanak, and S.Kaufman, Proc. Natl. Acad. Sci. USA.
87:6436 (1990).
9. Y.Inoue, Y.Kawasaki T.Harada, K.Hatakeyama, and H.Kagamiyama, I. Bioi. Chem. 266:20791 (1991).

178
REGULATION OF TETRAHYDROBIOPTERIN BIOSYNTHESIS IN CULTURED
HYPOTHALAMIC AND MESENCEPHALIC NEURONS BY CYCLIC AMP
DEPENDENT GTP CYCLOHYDROLASE I GENE EXPRESSION

Kei Hirayama, Min Zhu and Gregory Kapatos

Cellular and Clinical Neurobiology Program

Department of Psychiatry
Wayne State University School of Medicine
Detroit, Michigan 48201, USA

INTRODUCTION

Tetrahydrobiopterin (BH4) is the essential cofactor for phenylalanine, tyrosine and

tryptophan hydroxylases, enzymes that are rate-limiting in the catabolism of phenylalanine
and the biosynthesis of catecholamines and indoleamines 1• The family of nitric oxide
synthases (NOS), enzymes that produce nitric oxide (NO) from arginine, also require BH4
for activitf· 3•4• GTP cyclohydrolase I (GTPCH) is the first and rate-limiting enzyme in the
BH4 biosynthetic pathway, and catalyzes the formation of D-erythro-7,8-dihydroneopterin
triphosphate and formate from guanosine-5'-triphosphate5•6• The concentration of BH4
within monoaminergic neurons appears to be subsaturating so that changes in BH4 levels
can alter monoamine synthesis. Whether a similar situation exists within neurons that
contain NOS and synthesize NO is currently unknown. In order to investigate the possible
regulation of BH4 biosynthesis within central neurons we have used monolayer cultures of
hypothalamic (HYP) and mesencephalic (MES) neurons derived from day 15 rat embryos.
Our results indicate that BH4 synthesis can be stimulated by a cyclic-adenosine 3'5'-
monophosphate (cAMP)-dependent increase in GTPCH gene expression. If these findings
can be generalized to NOS-containing neurons, their capacity to increase NO production
might also be increased by elevated levels of BH4.

METHODS

HYP and MES neurons were dissociated from day 15 rat embryos, and plated at a
density of 50 k/well in 24- well plates previously coated with a solution of 10 pg/ml of
poly-D-lysine. Cells were cultured in modified N2 medium maintained at 35°C in 10 %
C027• Medium was replaced every other day and contained 10 pM 5-fluoro-2-deoxyuridine
and 100 pM uridine for at least 7 days to suppress the growth of non-neural cells. To

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 179
study regulation of BH4 synthesis, 8-bromo-cAMP and other reagents were added to the
cultures at 14 days after plating. After treatment, cultures were washed twice with
Dulbecco's phosphate buffered saline solution and kept at -80°C until use. Total BH4
content of individual cultures were determined by reverse-phase HPLC with fluorescence
detection8 • GTPCH mRNA was detected by a highly sensitive T1 nuclease protection assay
as described before9 and quantitated by autoradiography and laser densitometry scanning.
RNA was extracted according to the method of Chomczynski and Sacchi10• For protection
assay, 20 pg of total cellular RNA extracted from each culture was used.

RESULTS AND DISCUSSION

HYP and MES neurons were treated with 0 to 5 mM 8-bromo-cAMP for 24 hours.
Total BH4 content was then determined by HPLC with fluorescence detection. As shown
in Figure 1, BH4levels in HYP and MES cultures were significantly increased in a dose-
dependent manner by 8-bromo-cAMP. The maximum increase of BH4 levels was 1.5 fold
greater in MES than HYP cultures. An analogue of cAMP that penetrates through the cell
membrane thus caused an increase in BH4 levels in both types of neuron. Therefore, to
elevate endogenous levels of cAMP, HYP and MES cultures were incubated with forskolin,
an activator of adenylate cyclase or 3-isobutyl-1-methylxanthine, an inhibitor of
phosphodiesterase. Incubation with 100 pM of forskolin for 24 hours produced an increase
in BH4 levels in HYP but not MES cultures. Treatment with 500 pM 3-isobutyl-1-
methylxanthine for 24 hours increased in BH4 levels 2.2 and 1.8 fold in HYP and MES,
respectively. Elevation of endogenous levels of cAMP therefore also increased the level
of BH4. The time course for the increase in BH4 levels produced by 8-bromo-cAMP (5
mM) is shown in Figure 2.

,....... ~ HYP
.....:1 300
0
~
E-< CJ MES
z
0
u 200
~
0
~

'<:t 100
::I:
~

0 0.125 0.50 1.0 2.0 5.0

8-Bromo-cAMP (mM)

Figure 1. Effect of 8-bromo-cAMP on BH4 levels in HYP and MES cultures. HYP and MES neurons were
treated with indicated concentrations of 8-bromo-cAMP for 24 hours. BH4 content was then determined by
HPLC with fluorescence detection. Values expressed as a percentage of the appropriate control and represent
the mean± SE of four separate experiments of six wells each. Control values were; Hyp 941 pg/well, Mes
301 pg/well.

180
BH4 levels were increased in a biphasic manner with a maximum increase at 24 hours of
incubation in both cultures. These increases in BH4 levels could be the result of either an
increase in synthesis, a decrease in degradation, or both. For the analysis of the
degradation rate of BH4, both cultures were treated with 2 mM of 2,4-diamino-6-
hydroxyprimidine, the inhibitor of GTPCH. To increase BH4 levels, cultures were first
incubated with 5 mM 8-bromo-cAMP for 24 hours. Medium was then changed to 2 mM
2,4-diamino-6-hydroxyprimidine with or without 8-bromo-cAMP for 0-8 hours. Then BH4
levels were determined. Turnover time of BH4 was not changed by incubation with 8-
bromo-cAMP. This evidence demonstrated that 8-bromo-cAMP increased BH4 levels by
stimulating BH4 synthesis without altering degradation. Therefore, BH4 biosynthesis in
HYP and MES cultures is regulated by a cAMP-dependent mechanism. In order to study
the role of gene expression in regulating BH4 biosynthesis, HYP and MES neurons were
incubated for 24 hours with 5 mM 8-bromo-cAMP with or without 2 pg/rnl of actinomycin
D, an inhibitor of transcription or 0.5 pg/rnl of cycloheximide, an inhibitor of translation.
The increase in BH4 levels produced by 8-bromo-cAMP was completely prevented by
actinomycin D and cycloheximide. These data suggested that the increase of BH4
biosynthesis by cAMP might be due to an increase in GTPCH gene expression. Therefore,
we measured GTPCH mRNA levels by nuclease protection assay. As shown in Figure 3,
GTPCH mRNA levels were increased approximately 4 and 7 fold in HYP and MES,
respectively after 5 hours treatment with 5 mM 8-bromo-cAMP. These data agreed with
the increase in BH4levels produced by 8-bromo-cAMP.
In conclusion, cAMP increases neuronal BH4 levels by increasing BH4 biosynthesis
and this increase appears to be due to an increase in GTPCH gene expression. In addition,
the regulation of BH4 synthesis by cAMP may be different within HYP and MES neurons.
If BH4 is limiting in the synthesis of NO by NOS, as it is for the synthesis of monoamines,
then these data suggest that NOS activity might be stimulated by an increase in BH4
content brought about by a receptor-mediated increase in cAMP.

400
:l ~ HYP
0
~
E-<
300
c=J MES
z
0
CJ
~
0 200
~
'-'
"<t
::X::
t:rl 100

0 2 6 12 24 36 48
TIME (HOURS)

Figure 2. Tune course for the increase of BH4 levels by 8-bromo-cAMP. HYP and MES neurons were
treated with SmM 8-bromo-cAMP for the indicated times. BH4 content was then determined by HPLC with
fluorescence detection. Values are mean± SE of four separate experiments of six wells each. Control values
were; Hyp 810 pg/well, Mes 251 pg/well.

181
 ;J'
0 1000
~
E-< ~ HYP
z
0
u 800 c=J MES
~
0
~ 600
'-'
<
z
~ 400
E
::t:
u
c.. 200
E-<
Cl
0
Control 5hrs Control 5hrs

Figure 3. Effect of 8-bromo-cAMP on GTPCH mRNA in HYP and MES neurons. HYP and MES neurons
were treated with 5 mM 8-bromo-cAMP for 5 hours. GTPCH mRNA was detected by T1 nuclease protection
assay and quantitated by autoradiography and laser densitometry. Values are mean ± SE of three experiments.
Control Values were; HYP 2.5 amole/pg RNA, MES 2.4 amole/pg RNA.

ACKNOWLEDGEMENT

We thank Mr. Gary M. Bora for his excellent technical assistance. This work was
supported by NIH grant NS 26081.

REFERENCES
1. S. Kaufman, In, Aromatic Amino Acids in the Brain, CIBA Foundation Symposia, Vol. 22, pp. 85-115.
Elsevier, Amsterdam. (1974).
2. N.S. Kwon, C.F. Nathan, and DJ. Stoehr, J. Bioi. Chem. 264, 20496-20501. (1989).
3. M.A. Tayeh, and M.A. Marietta, J. Bioi. Chem. 264, 19654-19658. (1989).
4. K. Schmidt, E.R. Werner, B. Mayer, H. Wachter, and W.R. Kukovetz, Biochem. J. 281, 297-230.
(1992).
5. C.A. Nichol, G.K. Smith and D.S. Duch, Ann. Rev. Biochem. 54, 729-764. (1985).
6. R.A. Levine, G. Kapatos, S. Kaufman, and S.J. Milstein, J. Neurochem. 54, 1218-1224. (1990).
7. G. Kapatos, J. Neurochem. 55, 1995-1201. (1990).
8. T. Fukushima, and J.C. Nixon, Anal. Biochem. 102, 176-188. (1980).
9. K. Hirayama, S.I. Lentz, and G. Kapatos, J. Neurochem. in press. (1993).
10. P. Chomczynski, and N. Sacchi, Anal. Biochem. 162, 156-159. (1987).

182
MYCOPHENOLIC ACID SIMULTANEOUSLY REDUCES INTRACELLULAR
GTP AND TETRAHYDROBIOPTERIN LEVELS IN NEUR0-2A CELLS

Toshie Harada, Kazuyuki Hatakeyama * and Hiroyuki Kagamiyama

Department of Medical Chemistry

Osaka Medical College
Osaka 569, Japan

INTRODUCTION

Tetrahydrobiopterin (BH4) is involved in neural, immune and lipid functions as a

natural cofactor for the aromatic amino acid hydroxylases\ the 0-alkylglycerolipid cleav-
age enzyme 2, and nitric oxide synthases 3- 5• In contrast to other coenzymes, BH4 is thought
to be a regulator of these enzymes, because its intracellular concentration is within the
range capable of affecting enzyme activities and is known to increase in response to the
action of cytokines 6- 9 and in GIS boundary in rat thymocytes 10 , and to decrease with the
differentiation of erythroid cellsh· 12 • BH4 is synthesized from GTP by sequential actions of
GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase.
The biosynthesis of BH4 is mainly regulated at the step of GTP cyclohydrolase I, a rate-
limiting enzyme whose activity is increased by a number of cytokines 13 - 17 • In addition to
the probable regulation of its transcription 10 , we proposed that GTP cyclohydrolase I is
regulated according to the availability of GTP, since we have observed cooperative bind-
ing of GTP to this enzyme 18 • To determine whether or not the level of intracellular GTP is
within a range that can affect GTP cyclohydrolase I activity, we examined the effect of
changes in the GTP level on the level of BH4 19 ; IMP dehydrogenase inhibitors, which
inhibit the rate-limiting and committing step in de novo synthesis of GTP, were used to
reduce the level of intracellular GTP, and guanine or guanosine was used to increase it.
These experiments provided evidence that the intracellular GTP concentration in rat PC-12
pheochromocytoma cells and human IMR-32 neuroblastoma cells is the minimum required
to elicit the maximal activity by GTP cyclohydrolase I. This supports the theory that GTP
might regulate of the reaction catalyzed by GTP cyclohydrolase I19 • In this study, we
attempted to confirm and characterize the GTP-BH4 relationship using mouse Neuro-2a
neuroblastoma cells.

*To whom correpondence should be addressed. Fax: 81-726-81-3723

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 183
MATERIAL AND METHODS

The materials used were the same as those described previously 19 • Neuro-2a cells
were obtained from the American Type Culture Collection (Rockville, MD). The cells
were maintained at 37°C in a atmosphere containing 5% C02 and cultured in Eagle's
minimum essential medium containing 10% fetal calf serum, 0.1 mM nonessential amino
acids, and 2 mM glutamine. The culture medium was changed every 2 days and subcultur-
ing was done every 4 days with the use of a trypsin solution. For experimental use, Neuro-
2a cells were plated on 6-well tissue culture plates (Falcon) at a density of 2.5 X 105 cells
per well (9.6 cm 2) and grown for 2 days. The cells were then washed with Hank's bal-
anced salt solution and incubated for 10 h in serum-free defined medium (Eagle's mini-
mum essential medium containing 0.1 mM nonessential amino acids, 2 mM glutamine, 5
,ug!ml bovine insulin, 5 ,ug!ml human transferrin and 30 nM selenium) containing myco-
phenolic acid and/or guanine compounds as indicated in the text. High performance liquid
chromatographic analysis of nucleotides and pteridines, enzyme assay, and protein assay
were performed as described previously 19 •

RESULTS AND DISCUSSION

As shown in Table I, Neuro-2a cells contained all three BH4 biosynthetic enzymes as
well as the recycling enzyme dihydropteridine reductase. GTP cyclohydrolase I was found
to be the rate-limiting enzyme in Neuro-2a cells and the activity of dihydropteridine reduc-
tase appeared sufficient to regenerate all the BH4 in the cells (Table I). Therefore the reac-
tion rate of GTP cyclohydrolase I in Neuro-2a cells should be reflected in the intracellular
BH4 levels.

Table 1 Activities of BH4 biosynthetic enzymes in Neuro-2a cells.

Enzyme Enzyme activity1

nmol/h·mg ofprotein

GTP cyclohydrolase I 0.079 ± 0.001

6-Pyruvoyltetrahydropterin synthase 1.06 ± 0.02

Sepiapterin reductase 8.11 ± 0.01
Dihydropteridine reductase 1000 ± 50
1Enzymes were extracted and assayed as described under "Experimental Procedures." Values shown repre-
sent means ± S.E. from two identical experiments performed in separate cultures.

Neuro-2a cells were incubated in serum-free medium containing varying concentra-

tions of mycophenolic acid, an inhibitor of IMP dehydrogenase. As shown in Fig. lA,
mycophenolic acid simultaneously reduced the GTP and biopterin levels in Neuro-2a cells,
as had been observed in an earlier study in rat PC-12 cells and human IMR-32 cells 19•
This suggests that the GTP concentration in Neuro-2a cells was adequate to alter the level
of biopterin.
Because GTP cyclohydrolase I was the rate-limiting enzyme in Neuro-2a cells, its
intracellular reaction rate should correlate with the amount of BH4 present in the cells,
assuming that the degradation rate of BH4 was constant. To clarify the relationship be-

184
tween the GTP and biopterin levels, we plotted the GTP values against the corresponding
biopterin values shown in Fig. 1. As shown in Fig. 1B, the shape of the curve was sigmoi-
dal. Mouse GTP cyclohydrolase I extracted from Neuro-2a cells exhibited sigmoidal
saturation kinetics (Fig. 2A), as do rat and human enzymes 19 • The Hill coefficient of the
enzyme was 2.3 (Fig. 2B), which is similar to those of rat and human enzymes (2.9 and 2.5,
respectively). The similarity between the GTP-biopterin curve (Fig. 1B) and the sub-
strate-velocity curve of GTP cyclohydrolase I (Fig. 2A) implies that the enzyme also be-
haved cooperatively in Neuro-2a cells.

0.25-
c 0.3
6 .:: !0 8
c·a; 'Gi
a.

J
i5
i5
0.20
a. OJ
E
0.2
a. 4 E
OJ
~
OJ 0.15 ...... E
E 0 5 0.1
E
~ .=
E 0.10 5
5 2 .= Gi
15.
0.05 !
0.. 0 0
1-
(!) 0
ii'i 0 2 4 6
ii'i GTP (nmol/mg protein)
0 0
0 0.01 0.03 0.1 0.3 1.0
Mycophenolic acid lpMl

Figure 1. A. Effect of mycophenolic acid on intracellular GTP and biopterin levels in Neuro-2a cells.
Neuro-2a cells were treated with various concentrations of mycophenolic acid for 10 h. GTP (•) and biopter-
in (o) were measured by high performance liquid chromatography. The data shown are from two independent
experiments that yielded similar results. Each point represents the mean from the two experiments; the bars
represent the standard error. B. Relationship between intracellular GTP and biopterin levels in Neuro-2a
cells. The GTP values were plotted against biopterin values shown inA.

The level of intracellular GTP in Neuro-2a cells was 6 nmol/mg protein, which is
nearly equal to the values obtained in PC-12 and IMR-32 cells. Since the level of intracel-
lular GTP in PC-12 and IMR-32 cells has been estimated to be 150 .uM19, the level of
intracellular GTP in Neuro-2a cells would also be 150 .uM. On the other hand, the K05
value of mouse GTP cyclohydrolase I (220 .uM, Fig. 2A) was 3-fold than those of the nit
and human enzymes (60 .uM and 70 .uM, respectively) 19• At 150 .uM, GTP cyclohydrolase I
in Neuro-2a cells should exert only 30% of its Vmax value, as estimated from the data in
Fig. 2A. Consistent with this inference, the GTP-biopterin curve for Neuro-2a cells (Fig.
1B) appeared not to be saturated, unlike the curves for PC-12 and IMR-32 cells, which
were saturated19 • Furthermore, if this inference is correct, an increase in the intracellular
GTP level in Neuro-2a cells would be accompanied by an increase in the intracellular
biopterin level. However, since the addition of guanine, guanosine, or GMP did not result
in a higher GTP level in Neuro-2a cells, probably because there was no functioning sal-
vage pathway from these compounds to GTP, we could not verify our inference.
In conclusion, mycophenolic acid simultaneously reduced the GTP and biopterin
levels in mouse Neuro-2a cells. At this lower level, GTP was capable of altering the
intracellular BH4 concentration. Intracellular mouse GTP cyclohydrolase I appeared to
behave in a cooperative manner. The same observation have been made in rat and human
neuronal celllines 19 •

185
 0.4 2 ,---,----,---.
A B
:2
g 0.3 g
E I
)(
>.
"'
~
~ 0.2 0
ti
< ~
OJ
~ 0.1
>.
.3 -1
N
c:
w
0 -2
0.2 0.4 0.6 0.8 1.0 -5 -4 -3 -2

GTP (mM) Log[GTP] (M)

Figure 2. A, Effect of various concentrations of GTP on GTP cyclohydrolase I activity in Neuro-2a cells.
The enzymes were extracted from 108 cells and assayed as described in the text. The data shown are repre-
sentative of two experiments. B, Hill plots. Hill plots were drawn from the data presented inA.

REFERENCES

1. S. Kaufman,Annu. Rev. Biochern. 36:171 (1967).

2. A. Tietz, M. Lindberg, and E.P. Kennedy, J. Bioi. Chern. 239:4081(1964).
3. M.A. Tayeh and M.A. Marietta, J. Bioi. Chern. 264:19654 (1989).
4. N.S. Kwon, C.F. Nathan, and D.J. Stuehr, J. Bioi. Chern. 264:20496 (1989).
5. B. Mayer, M. John, and E. Bohme, FEBS Lett. 277: 215 (1990).
6. R. Kettler, G. Bartholini, and A Pletscher, Nature 249:476 (1974).
7. L.J. Cote, H.H. Benitez, and M.R. Murray, J. Neurobiol. 6:233 (1975).
8. G. Werner-Felmayer, E.R. Werner, D. Fuchs, A Hausen, G. Reibnegger, and H. Wachter, J. Exp. Med.
172:1599 (1990).
9. S.S. Gross, E.A. Jaffe, R. Levi, and R.G. Kilbourn, Biochern. Biophys. Res. Cornrnun. 178:823 (1991).
10. K. Schott, K. Brand, K. Hatakeyama, H. Kagamiyama, J. Maier, T. Werner, and I. Ziegler, Exp. Cell Res.
200:105 (1992).
11. K. Tanaka, S. Kaufman, and S. Milstien, Proc. Nat!. Acad. Sci. U.S.A. 86:5864 (1989).
12. F. Kerler, L. Hiiltner, I. Ziegler, G. Katzenmaier, and A Bacher,.!. Cell Physiol. 142:268 (1990).
13. C. Huber, J.R. Batchelor, D. Fuchs, A Hausen, A Lang, D. Niederwieser, G. Reibnegger, P. Swetly, J.
Troppmair, and H. Wachter,J. Exp. Med. 160:310 (1984).
14. G. Schoedon, J. Troppmair, A Fontana, C. Huber, H.C. Curtius, and A Niederwieser, Eur. J. Biochern.
166:303 (1987).
15. E.R. Werner, G. Werner-Felmayer, D. Fuchs, A Hausen, G. Reibnegger, and H. Wachter, Biochern. J.
262:861 (1989).
16. E.R. Werner, G. Werner-Felmayer, D. Fuchs, A Hausen, G. Reibnegger, J.J. Yim, W. Pfeiderer, and H.
Wachter,J. Bioi. Chern. 265:3189 (1990).
17. I. Ziegler, K. Schott, M. Lubbert, F. Herrmann, U. Schwulera, and A Bacher, J. Bioi. Chern. 265:17026
(1990).
18. K. Hatakeyama, T. Harada, S. Suzuki, Y. Watanabe, and H. Kagamiyama, J. Bioi. Chern. 264:21660
(1989).
19. K. Hatakeyama, T. Harada, and H. Kagamiyama,J. Bioi. Chern. 267:20734 (1992).

186
HUMAN LIVER 6-PYRUVOYL-TETRAHYDROPTERIN SYNTHASE:
EXPRESSION OF THE eDNA, PURIFICATION AND PRELIMINARY
CHARACTERIZATION OF THE RECOMBINANT PROTEIN

Beat Thony, Walter Leimbacher, Nenad Blau, Claus W. Heizmann and

Daniel Btirgisser

Division of Clinical Chemistry

University Children's Hospital
CH-8032 Zurich, Switzerland

INTRODUCTION
Tetrahydrobiopterin (BI4) is the coenzyme for several monooxygenases such as the
aromatic amino acid hydroxylases, the glycerol ether monooxygenase, and the nitric oxide
synthases1,2. A lack of BH4leads to hyperphenylalaninemia and a deficiency of biogenic
amine neurotransmitters, which are responsible for severe mental retardation3. The most
common form, where BH4 biosynthesis is impaired, is a deficiency in 6-pyruvoyl-
tetrahydropterin synthase (PTPS). PTPS catalyzes the second step in the BH4 biosynthetic
pathway, the conversion of 7 ,8-dihydroneopterin triphosphate to 6-pyruvoyl
tetrahydropterin. This triphosphate eliminating reaction requires Mg2+ as a cofactor. As a
means to better characterize biochemically the PTPS, we recently cloned the human liver
cDNA4. Expression of the recombinant enzyme in E. coli allowed us to isolate and purify
the active PTPS in large amounts. This article describes the overproduction and
purification, and gives a prelimi.1ary characterization of some physical properties of the
recombinant human enzyme.

MATERIALS AND METHODS

For construction of a pMAL-c2 vector expressing the PTPS as a fusion with the
maltose-binding protein (MBP; New England Biolabs), the following oligonucleotides
were used for standard PCR reactions: HSYNSAL (5'-AAGATGTCGACGGAAGGTGG
GGCCGTCGC-3'), containing a Sail restriction site and the codons for the first 9 amino
acids; and HSYNTAG2 (5'-CGGGATCCTATTCTCCTTTATAAACC-3'), encoding the
C-terminal 5 amino acids, a stop codon, plus a BamHI restriction site. The resulting Sali-
BamHI restriction fragment was first inserted into pUC18, and subsequently cut with Sail,
blunt-ended with Klenow polymerase, and cut with BamHI. This fragment was inserted
into pMAL-c2 cut with Xmni/BamHI, generating a plasmid with an open reading frame
coding for the fusion protein MBP-PTPS. The affinity of the MBP for maltose could then
be utilized for an efficient purification step. Cleavage of this fusion protein with factor Xa
resulted in MBP and a recombinant PTPS protein of 144 amino acids starting with serine.
One liter of E. coli TBl cells harboring the plasmid pMAL-c2-PTPS were grown and
lysed following the manufacturer's protocol (New England Biolabs). Isoelectric focusing
and SDS-polyacrylamide gel electrophoresis was performed using an immobilized pH

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 187
gradient from pH 3 to 10.5 (Immobiline system, Pharmacia). Isoelectric focusing under
native conditions was performed using the Rotofor cell (BioRad).

RESULTS AND DISCUSSION

The coding region of the eDNA for PTPS from human liver contained 145 amino
acids with a predicted molecular mass of 16,387 Da. Heterologous expression of the
complete coding region of the eDNA in E. coli, using an inducible expression system,
yielded a 16.4 kDa protein subunit on a SDS-polyacrylamide gel, and an active PTPS in
the crude E. coli extract4. After having compared several prokaryotic vector systems (data
not shown), we decided to use the plasmid pMAL-c2 to purify a MBP-PTPS fusion
polypeptide with MBP as a N-terminal fusion partner. The crude lysate from the E. coli
cells was directly applied to an amylose resm column for affinity chromatography. This
first purification step yielded a 16-fold enrichment with a calculated recovery of 98% from
total activity (see Table 1). After cleaving the MBP-PTPS by applying factor Xa, the two
proteins, MBP and PTPS, were separated by size exclusion chromatography (Ultragel
AcA-44), which resulted in a further purification by a factor of 4.7. This yielded 13 mg
recombinant PTPS with a purity of more than 95%, as judged from the 12% SDS-
polyacrylamide gel (Fig. 1). Overall purification was 77-fold with a total yield of 79%.
SDS-polyacrylamide gel electrophoresis of the purified PTPS (Fig. 1, lane 6) showed a
major band with a molecular weight of 16 kDa and a minor band of approximately 32
kDa, corresponding to the monomeric and presumably dimeric forms of PTPS,
respectively. Western blot analysis of this fraction with a polyclonal antibody against the
recombinant PTPS showed an immunoreactive band corresponding also to this larger, 32
kDa band (data not shown). In addition, an aliquot of the protein from the last purification
step was subjected to electrospray ionization-mass spectrometry. The predicted molecular
mass of 16,254.44 Da was confirmed by a smgle maJor peak at a mass of 16,257.33±4.27
Da for the recombinantly expressed PTPS subunit, verifying the presence of only one
major protein. From gel filtration studies using the native, recombinant enzyme we
observed a molecular mass of approximately 83 kDa, suggestmg that the active PTPS
might exist of 5 identical subunits.

6 7 kDa

- 78.0
- 66,3

- 42,7

- 30,0

- 17,0
-12,3

Figure 1. SDS-polyacrylamide gel electrophoresis of fractions from PTPS punfication. Ahquots were
analyzed on a 12% polyacrylamide gel and stamed with Coomassie bnlhant blue. Lane 1, prestamed
molecular mass standards, lane 2, E coil lysate (fraction I, 20 J..tg), lane 3, flow-through followmg loading
onto amylose resm column (20 Jlg); lane 4, eluate from amylose resm column (fraction II, 5 Jlg); lane 5,
fraction II after cleavage by factor Xa (5 Jlg), lane 6, pool of active fractions from gel filtratiOn (fractiOn III,
5 Jlg); lane 7, molecular weight marker (Sigma IV).

188
Table 1. Purification of recombinant human liver PTPS from E. coli TB 1 (pMAL-c2-
PTPS) expressing a MBP-PTPS fusion polypeptide.

Fraction Volume Protein Activity! Specific Yield Purification

activity

ml mg mU mU/mg % -fold
I. Lysate 250 1250 939 0.75 100 1
II. Amylose 6 75 917 12.2 98 16
III. Gel filtration 26 13 746 57.4 79 77

lone unit (U) is defined as the amount of enzyme that catalyzes the production of 1 J..lmo1 Blf4/min6.

10.5 60
'pH 4.8

m
8.5 45 c;·
...
"0
CD
::t ~.
:::J

I• I
Q.
6.5 30 ,....,
:::J
pH • Biopterin
3
0
.:::::::
4.5 15
r
......

2.5,_~~~.-~~~~~-.-.~-r~.-~~~~o

0 2 4 6 8 10 12 14 16 18 20
Fraction number
Figure 2. Determination of the pi value for the active recombinant human PTPS. Isoelectric focusing was
performed using the Rotofor cell (BioRad) and 30 J..lg of purified PTPS (fraction III) in a 40 ml 2%
ampholyte solution (Biolyte, pH gradient from 3 to 10). Subsequently, aliquots from individual chambers
were assayed for activity (Biopterin production). Enzyme activity was detected in a single peak,
corresponding to a pi value of 4.8.

A comparison of the deduced amino acid sequences from the cDNAs between the rat5 and
human liver PTPS revealed an almost identical protein sequence, except for the N-
terminallO amino acids4. TheN-terminal protein sequence of the mature rat liver enzyme
was found to match the residues starting from amino acid 5 when compared to the isolated
rat eDNA Thus, this enzyme appears to be processed and contains 140 residues with a

189
molecular mass of 15,855 Da5. The mature N-terminal end of the human liver enzyme is
not known, however. We expressed and purified also the rat liver eDNA consisting of 140
amino acids, using the same pMAL expression system as described for the human liver
enzyme (unpublished). The calculated isoelectric point of the rat protein was 7.15.
Isoelectric focusing and SDS-polyacrylamide gel revealed two major peaks migrating at pi
values of 7.15 and 7.4, plus several weaker signals migrating at lower pi values (data not
shown). In comparison, the recombinant human liver PTPS had a calculated pi value of
6.67, and upon isoelectric focusing and SDS-polyacrylamide gel showed two major peaks
at 6.7 and 6.9, plus one minor peak at 6.35 (data not shown). Thus, the calculated pi of
both proteins, rat and human, agreed well with the observed values of the unfolded
polypeptides (denatured by urea). In contrast, determination of the pi for the human
recombinant protein under native conditions revealed a value of 4.8 (Fig. 2), resembling a
similar pi value of 4.4 to 4.6 for the native human liver enzyme published earlier?. The
discrepancy for the human PTPS between the unfolded monomeric form (pl=6.7)
compared to the native multimeric protein (pl=4.8) towards a lower pi for the active
enzyme suggests that the native form must be negatively charged, and thus is exposing
more acidic amino acids at the surface.

In summary, this rapid purification procedure allowed the isolation of mg quantities of

highly purified and active PTPS from E. coli needed for further physical and biochemical
studies. We have been able, in a collaboration with the Max-Planck Institute in
Martinsried (Drs. R. Huber and H. Nar), to obtain preliminary X-ray diffraction data from
PTPS crystals. Hopefully, this will allow us to analyze the 3-dimensional structure of the
human PTPS in the near future. Besides characterizing the wild-type PTPS, it became now
possible to examine mutant enzymes from patients exhibiting an altered enzyme activity.

ACKNOWLEDGMENTS
We thank F. Neuheiser for skillful technical assistance. This work was supported by
a grant from the Swiss National Science Foundation (No. 31-33897.92), and in part by the
Helmut Horten Stiftung, the Stiftung fiir wissenschaftliche Forschung an der Universitat
ZUrich, and the Ciba-Geigy-Jubilaums-Stiftung.

REFERENCES

1. D.S. Ouch and G.K. Smith GK, J. Nutr. Biochem. 2:411 (1991).
2. R.C. Prince and D.E. Gunson, Trends Biochem. Sciences 18:35 (1993).
3. C.R. Scriver, S. Kaufman and S.L.C. Woo, The hyperphenylalaninemias, in: "The Metabolic Basis of
Inherited Disease," C.R. Scriver, A.L. Beaudet, W.S. Sly and D. Valle, eds., McGraw-Hill, New York
(1989).
4. B. ThOny, W. Leimbacher, D. Biirgisser and C.W. Heizmann, Biochem. Biophys. Res. Comm. 189:1437
(1992).
5. Y. Inoue, Y. Kawasaki, T. Harada, K. Hatakeyama and H. Kagamiyama, J. Bioi. Chern. 266:20791
(1991).
6. S.-I. Takikawa, H.-Ch. Curtius, U. Redweik, W. Leimbacher and S. Ghisla, Eur. J. Biochem. 161:295-302
(1986).
7. D. Heintel, W. Leimbacher, U. Redweik, B. Zagalak and H.-Ch. Curtius, Biochem. Biophys. Res. Comm.
127:213 (1985).

190
ENZYMATIC PROPERTIES OF 6-PYRUVOYL TETRAHYDROPTERIN
SYNTHASE PURIFIED FROM FAT BODIES OF SILKWORM LARVAE

Masahiro Masada

Department of Bioresources Chemistry

Faculty of Horticulture, Chiba University
Matsudo 648, Chiba 271, Japan

INTRODUCTION

Tetrahydrobiopterin is a cofactor of the aromatic amino acid hydroxylases 1. 6-Pyruv-

oyl tetrahydropterin(PTP) synthase is essential in the biosynthesis of tetrahydrobiopterin.
This enzyme catalyzes the conversion of dihydroneopterin triphosphate to PTP in the
presence of Mg2+•2 • The decrease of the amount of this enzymatic activity is the most fre-
quent cause of atypical phenylketonuria3•4 • PTP synthases were highly purified from human
liverS, salmon liver6, and Drosophila head7• Recently, eDNA sequence of PTP synthase
from rat liver was reported 8• However, although PTP synthases were highly purified from
several sources, the information on the enzymatic properties has been reported hardly any-
thing.
In this report, the author describes in detail the properties of PTP synthase purified
from fat bodies of silkworm larvae.

RESULTS

PTP synthase activity was measured based on the observation that pterin was quantita-
tively produced from the enzymatic product, PTP, by the chemical degradation under the
acidic condition when the enzyme reaction was terminated9• This method had made
possible to investigate directly the enzymatic properties.
PTP synthase was purified from fat bodies of silkworm larvae by procedures including
heat treatment, ammonium sulfate fractionation and column chromatographies on hydroxy-
lapatite, DEAE-Toyopearl, and Ultrogel AcA44. Finally, the preparation was purified to
homogeneity by a mean of preparative disc electrophoresis. ·
The molecular weight was estimated by three different methods. The molecular weight
of enzyme was calculated to be about 87,000 and 85,000 by the gel filtration using Ultrogel

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 191
 ,_
CD 0.5 • •
x
~

.E
c
0
u
(lj
~
~
c
~ 0.1

10 20
Mg 2 + concentration (mM)
Figure 1. Effect of Mg2+ concentration on the activity

Table 1. Effects of some divalent cations on the activity

with and without of a low concentration of Mg2+

Relative activity (%)

Cations
(5 mM) +1 mM Mg2+
None 0 33.5
Mg2+ 100
Ca 2+ 38.4 75.3
Mn2 + 15.8 29.6
Cu2+ 0 0
Zn 2+ 0 0
Ni2+ 0 0
Sr 2+ 3.0 85.2
sit+ 15.9 95.4

192
AcA44 and TSKgel G3000SW, respectively. By the method with native PAGE, the molecu-
lar weight was estimated to be about 72,000. The purified enzyme gave a single band on
SDS-PAGE and the molecular weight of the band was estimated to be 18,500. These re-
sults indicate that PTP synthase from fat bodies of silkworm larvae is composed of four
identical subunits.
The absorption spectrum of PTP synthase showed a single peak at 280 nm.
The pH optimum of the enzyme activity was around 8.5, and the activity showed
significant difference in the buffers used (data not shown).
The enzyme activity was inhibited by sulfhydryl reagents such as pCMB, monoiodoac-
etate and N-ethylmaleimide. The enzyme activity had not been affected by other inhibitors
examined.
Effect of Mg2+ concentration on the activity showed a biphasic pattern as shown in
figure. The Km values for Mg2+were calculated to be 2.0 x w- 3 and 2.1 x w-4 M, respec-
tively. On the other hand, the Km values for dihydroneopterin triphosphate in the presence
of 1 or 5 mM of Mg2+ were calculated to be almost the same value, 1.28 x 10-5 and
1.69 x 10-5 M, from each Lineweaver-Burk plot.
Effects of some divalent cations were demonstrated as shown in table. Some divalent
cations such as Ca2+ , Mn 2+ and Ba2+ also stimulated the enzyme activity. However, the
additional effect of Mn2+in the presence of Mg2+ at various concentrations was competitive
and/or inhibitory on the activity. The addition of Ca2+ to the reaction mixture containing of
a low concentration of Mg2+ (1 mM) was revealed to be an additive effect. On the other
hand, when each 5 mM of Ba2+ and Sr2+ was added to the reaction mixture containing the
same low concentration of Mg2+ (1 mM), the activity was greatly accelerated. Those addi-
tions of both Ba2+ and Sr2+ had revealed to be the synergistic effects on the activity in the
presence of Mg2+ .
When each 5 mM of Ca2+ and Ba2+ was, respectively, added to the reaction mixture,
the effect of Mg2+ concentration on the activity showed typical Michaelis-Menten kinet-
ics on the both cases. However, the activity in the presence of Mn 2+ (5 mM) had de-
creased in proportion to the increase of Mg2+ concentration. Under the condition of the
reaction mixture containing a low concentration of Mg2+ (1 mM), effect of Ca2+ concen-
tration showed typical Michaelis-Menten kinetics, while that of Ba2+ concentration on the
activity showed a biphasic pattern.
From these results, the physicochemical properties of this enzyme was almost similar to
those of other enzymes reported. Concerning to the enzymatic properties, the result ob-
tained in this experiment could not be compared with those of other enzymes because the
assay method was completely different. On the properties of silkworm enzyme, the posibili-
ty may be speculated that this enzyme has two binding site for Mg2+ and each site may
independently contribute to the expression of enzymatic activities. One site of them has the
replaceable ability to other divalent cations. However, another binding site for Mg2+ may be
essential for the expression of enzymatic activities.

REFERENCES

1. S.Kaufrnan, Metabolism of the phenylalanine hydroxylation cofactor, J.Biol.Chem.

242:3934(1967)
2. M.Masada,M.Akino,T.Sueoka,and S.Katoh, Dyspropterin, an intermediate formed from
dihydroneopterin triphosphate in the biosynthetic pathway of tetrahydrobiopterin,
Biochim.Biophys. Acta. 840:235(1985)
3. S.Yoshioka,M.Masada, T.Yoshida,T.Mizokami,M.Akino,N.Matsuo,T.Tsuchiya,T.Seki,

193
 S.Arashima,and M.Kawaguchi, A defective enzyme in hyperphenylalaninaemia due
to biopterin deficiency, J.Inher.Metab.Dis. 6:127(1983)
4. H.Shintaku,A.Niederwieser,W.Leimbacher,and H.-Ch.Curtius, Tetrahydrobopterin
deficiency:assay for 6-pyruvoyl tetrahydropterin synthase activity in erythrocytes, and
detection of patients and heterozygous carriers, Eur.J.Pediatr. 147:15(1988)
5. S.Takikawa,H.-Ch.Curtius,U.Redweik,W.Leimbacher, and S.Ghisla, Biosynthesis of
tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl tetrahydropterin
synthase from human liver, Eur.J.Biochem. 161:295(1986)
6. T.Hasler,and H.-Ch.Curtius, Purification and characterization of 6-pyruvoyl
tetrahydropterin synthase from salmon liver, Eur.J.Biochem. 180:205(1989)
7. Y.S.Park,J.H.Yim,K.B.Jacobson,and J.J.Yim, Purification and characterization of
6-pyruvoyl tetrahydropterin synthase from Drosophila melanogaster,
Biochim.Biophys.Acta. 1038:186(1990)
8. Y.lnoue,Y.Kawasaki,T.Harada,K.Hatakeyama,and H.Kagamiyama, Purification and
eDNA cloning of rat 6-pyruvoyl tetrahydropterin synthase,
J.Biol.Chem. 266:20791(1991)
9. M.Masada,J .Matsumoto,and M.Akino, Biosynthetic pathway of pteridines and their
association with phenotypic expression in vitro in normal and neoplastic pigment cells
from goldfish, Pigment Cell Research. 3:61(1990)

194
NORTHERN BLOT ANALYSIS OF SEPIAPTERIN
REDUCTASE mRNA IN MAMMALIAN CELL LINES
AND TISSUES

JosefMaier,1,2 Karin Schott, I Thomas Werner, 3 Adelbert Bacher,4

and lrrngard Zieglerl
1GSF-Gesellschaft fiir Umwelt- und Gesundheitsforschung
lnstitut fur Klinische Molekularbiologie und Tumorgenetik
Marchioninistr. 25, D-8000 Mtinchen
2present address: Universitat Ttibingen, Institut ftir Chemische
Pflanzenphysiologie, Corrensstr. 41, D-7 400 Ttibingen
3GSF-Gesellschaft fiir Umwelt- und Gesundheitsforschung
lnstitut fiir Siiugetiergenetik, Ingolstiidter Landstr. 1, D-8042 Neuherberg
4Technische Universitlit Miinchen, Lehrstuhl fiir Organische Chemie und
Biochemie, Lichtenbergstr. 4, D-8046 Garching, Germany

INTRODUCTION

Recent evidence has shown that H4biopterin is synthesized in cells which undergo cy-
tokine-directed proliferation and do not use H4biopterin as a hydroxylation cofactor.
H4biopterin, in turn, enhances the proliferation of erythroleukemic cells and modulates vari-
ous aspects of the interleukin-2-induced clonal expansion ofT cells. 1 In these cells the ac-
tivity of sepiapterin reductase (SR) is in the range of 100 pmol min·l mg-l.Lectin stimulation
of resting T cells causes a slowly progressing increase, starting from levels below detection
and a transient enhancement of SR activity is found after treatment of activated T cells by
interferon-'Y plus interleukin-2.1 The NK-like human cell line YT and the murine erythroleu-
kemic cell line B8/3 lack any SR activity. The molecular basis of SR regulation has not been
explained in any of these cases.
Based on the published sequence of rat SR cDNA2 we constructed eDNA probes
which specifically hybridize with rat, murine, or human SR mRNA, respectively. They were
used for Northern blot analysis of mRNA expression in different cell lines and tissues of
these mammalian species.

EXPERIMENTAL PROCEDURES AND RESULTS

PCR Amplifications and Sequence Analysis

PCR amplification of eDNA derived from mRNA, purification, cloning and sequencing
of PCR products were performed as described previously.3A Fragments of eDNA specifi-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 195
cally coding for rat, mouse and human SR were amplified by PCR via primers derived from
the rat sequence.2 Primer positioning close to the 3'-end of the coding region yielded eDNA
fragments of all three species. The products were reamplified and purified by HPLC. The
human fragment was cloned in a T-tailed vector. The identity of the amplificates was con-
firmed by direct sequencing (Figure 1).
Rat and mouse eDNA fragments are 92 % identical. This value drops to 77 % and
74% when human eDNA-fragment is compared with rat and mouse, respectively. At the
amino acid level the identity is 85 % among both rodents, but it decreases to 68 and 66 %
respectively, when the human sequence is compared with rat or mouse SR. SR shows a
markedly lower degree of evolutionary conservation than does GTP cyclohydrolase I. Rat
and human GTP-cyclohydrolase I demonstrate a 98 % similarity at the eDNA leveP The
sequence of the human fragment was identical to that published previously.6

rat 614 AG TTG GCC CGG GAA ACC TCC ATG GAC CCA GAG TTG AGG AGC AGA CTG
mouse .. T .A. .. . . . A . . . . . . . A.
man . . . . . . . . . . . . . G . . . . . . G. . . . . . . . . . C A .. C.A .AA G.G .. .

rat 661 CAG AAG TTG AAT TCT GAG GGG GAG CTG GTG GAC TGT GGG ACT TCA GCC
mouse •• G •• G •• T .C.
man ... G.. C . . . . G G.A A . . . . . A . . . . . . . . . . T .. C AA. GTG . . . . . .

rat 715 CAG AAA CTG CTG AGC TTG CTG CAA AGG GAC ACC TTC CAA TCT GGA GC 755
mouse G.. .A. ..G
man . . . . . . . . . . . . . . . . . A ... G . . . A . . . . GAG ... A.G . . . . . . . .

Figure 1. Sequence alignment of the eDNA fragments obtained by PCR primers GGACACCAACATGCA-
GC and TCATAGAAGTCCACGTGG. Identical primary structure is indicated by points. The positions are
indicated according to the published eDNA sequence of rat SR. 2

Northern Blot Analysis

The low steady state mRNA levels in cells need isolation of poly-A+-mRNA prior to
Northern blot analysis. Probes labeling of PCR products with [a- 32P]dCTP and hybridiza-
tion was performed as described.3,4 Two mRNA species for SR were detectable (Figure 2).
Their sizes were 1.3 and 2.1 kb for rat, 1.3 and 2.3 kb for mouse and 1.6 and 2.1 kb for hu-
man cell lines. Analysis of the rat system showed that the 2.1 kb mRNA species, which was
found in the liver cell line HTC and activated rat thymocytes was absent in kidney, liver,
brain and spleen (Figure 2). The lengths of the 1.3 kb rat mRNA and of the 1.6 kb human
mRNA both agree with the published eDNA clone lengths, respectively.2. 6,7

For further characterization of both murine mRNA species the signal intensity of hy-
bridization obtained under washing conditions of varying stringency was compared. This
shows a more stable interaction of the 1.3 kb mRNA species with the eDNA probe as com-
pared to the 2.3 kb species. This may argue against the hypothesis that both mRNA species
are alternatively spliced forms with identical protein coding regions. The detection of two
SR mRNA could support the existence of SR isozymes. The similarity of eDNA fragments
was higher among both rodents than between the rodents and man. Due to these species-
specific differences cross-hybridization of a rat probe with murine mRNA yielded a mark-
edly stronger signal than equal amounts of mRNA from human sources (Figure 3).
No SR activity had been detected in both the human NK-like cell line YT and in the
murine erythroleukemia cell line B8/3. Northern blot analysis of both cell lines demonstrated
that the absence of enzyme activity correlates with a lack of mRNA expression (Figure 4).
The specific activities of SR vary among different cell types and cell lines of a species. A

196
 28 s
28 s
18 s
18 s

man rat
u ..., .... c:
·ac:
>-
.r::
N N N u >-
f- I
I I 2 c.:> f- Q) Q) Q)
f- c:
.,
I ....J ~
~
Q)
I
....J t-
a_ -o 0..
1..
..0
f- rn :y
u I
:J
I
"'
Figure 2. Northern blot analysis of mRNA (5 jlg) specific for SR in rat, murine, and human cell lines and
tissues. The positions of 18 Sand 28 S rRNA are indicated.

A
•
B

rat mouse
- man
>-U N' N N
..r:: I- I I Q<..:l
I- I _J~

_J 3
I - a.
1-[IJ :J Q)
u :r:I

Figure 3. A, Northern blot analysis of mammalian SR mRNA with a rat-specific

probe; B, rehybridization with a probe specific for human ~-tubulin.

comparison among the cell lines of a species demonstrates that a decrease in specific activity
correlates with a decrease in the steady state mRNA levels.
The data demonstrate a major difference between the regulation of GTP-cyclohydro-
lase I and of SR activity. Previous studies have suggested that a post-translational modifica-
tion essentially contributes to the regulation of GTP cyclohydrolase I. 3,8 In contrast, protein
modification does not appear to contribute to the regulation of SR activity. SR mRNA ex-
pression is absent in human NK-like cell line YT and in the murine erythroleukemia subclone
B8/3 which both lack SR activity. Moreover, the relative mRNA expression correlates with
the enzymatic activities of different cell lines within the same species (Figure 4). This indi-
cates that SR activity is regulated by its steady state mRNA levels.

Acknowledgment
Support by Grant ZI 153/5-2 of the Deutsche Forschungsgemeinschaft is acknowl-
edged.

197
 "E>- lO A
N >- 0.8
~~ 0.6
1):;::
> u
:;: 0 0.4
0
...
"ii 0.2
0
< lO B
z
a:: c 0.8
E ·~ (II 0.6
CD
-~ ...Q.
CD
0.4
0 Ill
...
"ii 0.2
0
c
a::
VI
(II

0
:0
.....,c:
=
... .s
z
0
3
D

.&J
~

i
<0..

m en ret mouse
1- N N >- (.) N ..., z ,.,.,
>- ~ <-' ~
1-
1- I I -.t .........
.....
:I:
..,Q.
..J
1- ..J ~ 00
m
~
:I:
1- m
:I: (.)

Figure 4. A: Relative enzymatic activities of SR; B: relative SR mRNA expression; C: Northern blot analy-
sis of SR mRNA with species-specific probes; D: rehybridization with a eDNA probe for ~-tubulin ; the rela-
tive values of the liver cell lines were set to 1.0. 5 11g of poly-A+-enriched mRNA was applied to each lane;
b.d., below detection limit; Thy, thymocytes 48 h after activation.

REFERENCES

1. I. Ziegler, K. Schott, M. Lubbert, F. Herrmann, U. Schwulera, and A. Bacher, J. Bwl. Chem.265:17026

(1991).
2. B.A. Citron, S.S. Milstien, I.C. Gutierres, R.A. Levine, B.L. Yanak, and S. Kaufman, Proc. Nat/. Acad.
Sc1. USA 87:6436 (1990).
3. K. Schott, K. Brand, K. Hatakeyama, J. Maier, T. Werner, and I. Ziegler, Exp. Cell Res. 200:105 (1992).
4. J. Maier, K. Schott, T. Werner, A. Bacher, and I. Ziegler, Exp. Cell Res. 204, in press (1993).
5. M. Gutlich, K. Schott, T. Werner, A. Bacher, and I. Ziegler, Bwch1m Bwphys. Acta 1171:133 (1992).
6. H. Ichinose, S. Katoh, T. Sueoka, K. Titani, K. Fujita, and T. Nagatsu, Bwchem Bwphys. Res. Commun.
179:183 (1991).
7. R.A. Levine, J. Solus, S. Goustin, S. Tait, S. Demetriou, B. Citron, and S. Kaufman, m: "Ptenns and
Neurogenic Ammes in Neurology, Pediatncs and Immunology," N. Blau, H.-Ch. Curtms, R.A.
Levme, and Cotton R.G.H., eds., Lakeshore, Grosse Pointe, (1991).
8. K. Schott, M. Gutlich, and I. Ziegler, J. Cellul. Physwl., in press (1993).

198
PURIFICATION AND PROPERTIES OF HUMAN
SEPIAPTERIN REDUCTASE FROM PLACENTA

Josef Maier, 1,2 and Irmgard Ziegler!

1GSF-Gesellschaft fur Umwelt- und Gesundheitsforschung
Institut fur Klinische Molekularbiologie und Tumorgenetik
Marchioninistr. 25, D-8000 Miinchen
2present address: Universitat Tiibingen, Institut fur Chemische
Pflanzenphysiologie, Corrensstr. 41, D-7400 Tiibingen, Germany

INTRODUCTION

Recently H4biopterin was found to be synthesized in cells which do not use it as a hy-
droxylation cofactor. This is the case during cytokine-directed proliferation and differentia-
tion of cells which participate in hematopoiesis or immune response.!
Sepiapterin reductase (EC 1.1.1.153) (SR) catalyzes the last step in the biosynthesis of
H4biopterin. The enzyme reduces 6-pyruvoyl-H4pterin in two steps to H4biopterin using
NADPH as source of the hydride equivalents. In the above mentioned cells the activity of
sepiapterin reductase was in the range of 100 pmol min-I mg-1. A similar range was found in
human placenta. This tissue also does not use H4biopterin as a hydroxylation cofactor,
which, in contrast, is the case for liver or brain.
This study describes a method for purification of sepiapterin reductase from human
placenta to a homogeneity of 90 % with a final recovery of 4.7 %. A purification scheme
based on ammonium sulfate fractionation, hydroxyapatite chromatography, QAE-Sephadex
chromatography, isoelectric focusing in granulated gels and gel filtration by HPLC was ap-
plied. A polyclonal rabbit-antiserum was produced. Physicochemical and catalytical proper-
ties of the enzyme were investigated and were compared with those of the rat erythrocyte
enzyme and the human liver enzyme previously reported.2,3

EXPERIMENTAL PROCEDURES AND RESULTS

Enzyme Purification

Preparation of Crude Extract. Frozen, human placenta (500 g) was homogenized

with four volumes of 5 mM sodium phosphate buffer pH 5.5. This and all following buffers
contained 0,25 mM PMSF, 5 mM EDTA, and 1 mM DTE. The homogenate was centri-
fuged at 20,000 x g for 30 min.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 199
 Ammonium Sulfate Fractionation. The pH of the crude exact was adjusted to 5.5
while stirring on ice. Solid ammonium sulfate was added to 38 % saturation and the mixture
stirred for 30 min. After centrifugation additional ammonium sulfate was added to the su-
pernatant to 60 % saturation. After further stirring for 30 min and centrifugation the precipi-
tate was dissolved in buffer A of 20 mM sodium phosphate pH 6.5. The solution was fil-
trated and desalted by gel filtration with Sephadex G-25.

Hydroxyapatite Chromatography. Enzyme solution containing 300 mg of protein

was applied to a column of hydroxyapatite (type fast flow, Fluka, Ni.irnberg, Germany;
2.6x30 em) equilibrated with buffer A. After washing the column with buffer A until the
280 nm absorbance reading of the eluates had decreased to the starting value, the enzyme
was eluted with 0,5 1 linear gradient of 20-300 mM sodium phosphate buffer of pH 6.0
(Figure 1). The activity was eluted at a molarity of about 220 mM. The active fractions of
10 chromatographies were pooled, concentrated by ammonium sulfate precipitation and de-
salted by gel filtration with Sephadex G-25 using a column equilibrated with buffer B
(20 mM Tris-Cl pH 7.3).

........,
::E
..........
-Protein SR activity ~ ... Gradient f""'
0.6 '[' 3.0 3.0
CJ)
E
L.
....
Q) E
.... I
I
:J 0.5
·ec
/I 2.5
.0
12.5 II
II
Q)
+-
0
0.4 s:: 2.0
0 II 2.0 0
..s:: +: I I E
c
c. 0 I I ..............
Ill
0
0.3 L.
+- 1.5 I I 1.5 ..........
..s:: s:: >-
c. Q) 1.·1" +-
·s;:
....0 0.2 (.)
c 1.0 .·r I 1.0 +:
0 I (.)
(.)
I 0
+-
s:: 0.1 c 0.5 0.5 Q)
Q) ·o; E
'6 +-
>-
0 0 0
L. 0 0 N
c
L.
a..
~
0 25 50 75 100 125 150 175 200 w
Fraction number
Figure 1. Chromatography on hydroxyapatite; elution with a 20-300 mM sodium phosphate buffer (pH 6.0)
gradient; flow rate 0.8 ml min·1; 4-ml fractions.

QAE-Sephadex Chromatography. Enzyme solution containing 200 mg of protein

was applied to a column of QAE-Sephadex (Q-25-120, Sigma, Deisenhofen, Germany; 25 x
1.9 em) equilibrated with buffer B (Figure 2). All of the enzyme was eluted by washing the
column with 100 ml ofbuffer B; elution ofthe absorbed proteins was carried out with a lin-
ear gradient of0-1 M NaCl in buffer B (Figure 2).

Isoelectric Focusing in a Layer of Granulated gel. Preparative isoelectric focusing

was performed by the method ofB.J. Radola4 on glass plates (26.5 x 12.5 em) which were
coated with 122 ml of a thick suspension of Sephadex G-200 superfine containing 4% car-
rier ampholytes (Servalyt 4-9 T, Serva, Heidelberg, Germany). The gel was dried in air until
a layer was obtained which did not move when the plate was inclined at an angle of 45°. Af-
ter pre-focusing over night at a voltage gradient of 30 V cm· 1 (Multiphore II electrophoresis
unit, LKB, Sweden) the enzyme solution containing 100 mg of protein was mixed with dry
gel (30 mg per ml) and poured into a slot 8 em towards the anode. Focusing started with a
voltage gradient of30 V cm- 1 and after stepwise amplification ofthe voltage gradient, ter-
minated 5 h later at 68 V cm·1. After the coloured standard proteins had been spread over
the whole gel width (22.5 em) the gel layer was fractionated by a metal grid and removed

200
 -- Protein
f""'
Gradient SR activity Ol
f""' 0.8 E
1.0 E 'T
4 c
Ol
.E
..sc: 0.6
~

~
(3 0.8 0

-- -
0 0 3 E
z :;:::

-
c
.......
0
0.6 ...0 0.4
c: 2 ·;;:>-
cQ) 0.4 Q)
0 :;:::
c:
'5 0
0.2
0
... 0

-
0 0
(.!) 0.2 c Q)
·a; E
>-
0 ...0
a...
0 0 w
N
c
0 5 10 15 20 25 30 35 40 45 50
Fraction number
Figure 2. Chromatography on QAE-Sephadex; elution with 0-1 M NaCl in buffer B; flow rate 0.9 ml min-I;
5-ml fractions.

with a spatula. The pH of the gel fractions was measured and the proteins were eluted with
buffer C (100 mM sodium phosphate buffer pH 6.8, 100 mM KCl). The pi of the enzyme
was 8.4 (Figure 3).

Gel Filtration. Enzyme solution containing 0.4 mg of protein was applied to a TSK-
3000 HPLC column equilibrated with buffer C. The enzyme showed a retention time as if it
had a molecular weight of 45.3 kDa compared with standard proteins (Figure 4). The results
of the purification procedures are summarized in Table 1.

Properties of the Enzyme

SDS gel electrophoresis indicated an apparent molecular weight of 28 kDa. From pro-
tein band quantification data the activity of the pure SR of human placenta was estimated to
be about 1 !lmol min-I mg-1. The pi was 8.4. The~ values were 20 11M for sepiapterin and
6 11M for NADPH. A polyclonal rabbit-antiserum was produced. It precipitated the enzy-
matic activity of SR and stained two protein bands of molecular weights 28,000 and 56,000
in a Western blot analysis. The published eDNA of human SR encodes also a 28 kDa pro-

f""' __.. SR activity o-o Protein .._.pH f""'

Ol
E 35 10 1.4 E
Ol
'T 30 1.2 .......
E
c 9
.E c:
25 1.0 0

-
0 1? 8 :;:::

- -
s
E 20 Q 0.8 ...0
7 c:
0 Q)
>- 15 0.6 0
c:
·;;: =a.6 0
:;::: 10 0.4 0

-...
0
0 c
Q) 5 5 0.2 ·a;
E 0
>-
N 0 4 0 a...
: :
c
w 0 :s: !10! !15! 20: Fraction No.
Protein print 111 II I 111111111111111 1111111111111111 II II II II II I I I
Figure 3. Isoelectric focusing in a granulated gel of Sephadex G-200. Enzyme solution was applied between
fraction 1 and 5. The protein print was obtained by rolling down a cellulose acetate strip on the gel layer and
staining it with Ponceau S (Serva, Heidelberg, Germany).

201
tein. 5 The data indicated that human SR from placenta resembles the rat enzyme which is an
homodimer of 56 kDa. 2 A second protein band, as described for human liver3 was not found
to be associated with enzyme activity in placenta.

~
-Protein Enzyme activity ~
I
I 0>
E 150 E
:l
0> 1000 'ic:
........ .E

--
c:
.Q 100 0
0 E

-
I.. c:
........
c: 500
Gl
(J >-
c:
0 50 '>
+=

-
(J
(J
c: 0
'i Gl
0 0 0 E
I..
a.. >-
N
20 25 30 35 40 45 50 55 60 c:
L&J
Fraction number
Figure 4. HPLC gel filtration on TSK-3000; flow rate 100 J!l min-1; 0.5 ml fractions.

Table 1. Purification of sepiapterin reductase from human placenta.

Procedure Volume Total Spec. act. Recovery Purifi-

[ml] protein [nmol % cation
[mg] min-1 mg-1] (-fold)

Crude extract 1200 35000 0.02 1

Ammonium sulfate 100 9800 0.1 140 5
Hydroxyapatite 20 218 2.7 84 135
QAE-Sephadex 3.5 95.5 4.0 55 200
IEF 0.2 3.43 30 14.7 1500
Gel filtration 0.05 0.035 930 4.7 46500

ACKNOWLEDGEMENTS

This work was supported by grant ZI 153/5-2 of the Deutsche Forschungsgemeinschaft.

REFERENCES

1. I. Ziegler, K. Schott, M. Liibbert, F. Herrmann, U. Schwulera, and A. Bacher, J. Bioi. Chem.265:17026

(1991).
2. T. Sueoka, and S. Katoh, Biochim. Biophys. Acta 717:265 (1982).
3. B. Zagalak, F. Neuheiser, and U. Redweik, in: "Chemistry and Biology ofPteridines 1989," H.-Ch. Cur-
tins, S. Ghisla, and N. Blau, eds., Walter de Gruyter & Co., Berlin (1992).
4. B.J. Rado1a, Biochim. Biophys. Acta 386:181 (1975).
5. H. Ichinose, S. Katoh, T. Sueoka, K. Titani, K. Fujita, and T. Nagatsu, Biochem. Biophys. Res. Commun.
179:183 (1991).

202
STIMULATION OF TETRAHYDROBIOPTERIN SYNTHESIS BY CYTOKINES IN
HUMAN AND IN MURINE CELLS

Ernst R. Werner, Gabriele Werner-Felmayer, Giinter Weiss and Helmut

Wachter

Institute for Medical Chemistry and Biochemistry, University of Innsbruck

A-6020 Innsbruck, Austria

INTRODUCTION

Initiated by the finding of increased concentrations of neopterin in urines of patients

suffering from malignant tumors and viral infections 1, a variety of studies have
investigated neopterin concentrations in body fluids of patients suffering from various
diseases and compared these to clinical findings and other laboratory tests. Several reviews
of this work have been published2· 3.4· 5•6 and the clincal picture was found compatible with
the hypothesis that neopterin concentrations are increased in disease states with activated
cell mediated immunity. For example, neopterin concentrations are increased during the
course of allograft rejections7 • In mammals other than humans, however, similar
observations have not been possible to make, since neopterin does not occur in amounts
comparable to humans in body fluids 8 • Nevertheless, biochemical work using cultured cells
has demonstrated that in human as well as in murine cells cytokines lead to increased
intracellular tetrahydrobiopterin concentrations due to stimulation of GTP cyclohydrolase
I. Subject of the present article will be to summarize the present knowledge on this topic.
A more detailed compilation will be published elsewhere9• The significance of the rise in
tetrahydrobiopterin concentrations for nitric oxide formation is dealt with in another
chapter of this volume10 •

STIMULATION OF PTERIDINE BIOSYNTHESIS IN HUMAN CELLS BY

CYTOKINES

Impact of Cytokine Treatment on Pteridine Levels

In early studies, induction of pteridine synthesis was studied by analyzing supernatants

of cultured cells for neopterin. Peripheral blood mononuclear cells released neopterin when
activated by allogeneic stimulation or by lectins 11 •12 • A more detailed analysis revealed that
neopterin originates from macrophages, which are stimulated by interferon-gamma, which
in turn derives from activated T-cells 13 • Interferon-gamma was found to lead to an increase

Chemistry and Biology of Pteridines and Folates, Edited by

J .E. Ayling et al., Plenum Press, New York, 1993 203
of GTP-cyclohydrolase I in the cells14 • From the lack of 6-pyruvoyl tetrahydropterin
synthase activity, the absence of biopterin derivatives and the increase in GTP pools it was
subsequently speculated that lack of feedback inhibition by tetrahydrobiopterin might be
important for the accumulation of neopterin derivatives by human macrophages 15 • On the
other hand, work by Ziegler showed that both neopterin and biopterin derivatives are
formed during activation of human peripheral blood mononuclear cells16 •
Using a sensitive analytical technique17 , we then found that stimulation of pteridine
synthesis by interferon-gamma is a more general phenomenon than previously assumed: It
does not only occur in immune cells, but is seen in all investigated cells or cell lines
responsive to interferon-gamma, what was tested by increased expression of major
histocompatibility complex antigens. In these human cells, both 7,8-dihydroneopterin and
5,6,7,8-tetrahydrobiopterin derivatives accumulate. In particular, tetrahydrobiopterin was
unambiguously detected in stimulated, but not in unstimulated human macrophages 18 • In
addition to biopterin and neopterin derivatives, stimulated macrophages contain 6-threo-
1'2'3' trihydroxypropylpterin. The achiral column cannot distinguish between the L-and
D-enantiomers, which are called monapterin and umanopterin. Umanopterin has recently
been isolated from urine of cancer patients 19 • It remains to be seen whether the isomer
found in activated human macrophages is identical to umanopterin. Treatment of THP-1
myelomonocytoma cells leads to exactly the same pteridine pattern as in macrophages20 •
This demonstrates that biopterin found in human macrophage preparations originates from
cells of the monocytic lineage itself rather than from impurites in the macrophage
preparation.

Impact of Cytokine Treatment on Tetrahydrobiopterin Biosynthetic Activities

As a next step, we determined the activities of the three enzymes catalyzing the
pathway from GTP to tetrahydrobiopterin in the cells 21 • We found that only GTP-
cyclohydrolase I is stimulated by interferon-gamma. The long time required for reaching
the maximal activity (about 24 hours) as well as the unchanged Km value suggest that the
mechanism of this stimulation is a de novo synthesis of GTP-cyclohydrolase I protein.
Upon interferon treatment, the activity is stimulated up to 100 fold, as measured at
saturating concentrations of GTP. Compared to this pronounced increase in biosynthetic
activity, the observed up to two-fold increases in intracellular GTP15 might be only of
minor importance for the regulation of pteridine synthesis in interferon treated cells. As
can be deduced from work with neuronal cell lines 22 , a twofold change in intracellular
GTP corresponds to an about 25% change in intracellular pteridine concentrations.
Following treatment with interferon-gamma, however, intracellular pteridine concentrations
increase 10 to 30-fold18 •
Enzyme activities subsequent to GTP-cyclohydrolase I, i.e. 6-pyruvoyl tetrahydropterin
synthase and sepiapterin reductase are constitively present in the cells and remain
unchanged by the interferon-treatment21 • The activity of the induced GTP-cyclohydrolase
I relative to the activity of the constitutive 6-pyruvoyl tetrahydropterin synthase was found
in good agreement with the observed pteridine patterns. Human macrophages, which have
the lowest 6-pyruvoyl tetrahydropterin synthase activity, form 50 times more neopterin
than biopterin derivatives. Nevertheless, biopterin concentrations in macrophages are still
controlled by GTP-<:yclohydrolase I activites. The reason for this is the comparatively high
Km of 6-pyruvoyl tetrahydropterin synthase, which in human liver 3 as well as in cultured
human cells24 was found to be 10 .uM. In human fibroblasts both enzyme activities are of
the same order of magnitude, resulting in about equal concentrations of neopterin and
biopterin in these cells. In T-24 cells, 6-pyruvoyl tetrahydropterin synthase activity is
considerably higher than the induced GTP-cyclohydrolase I, so that 50 times more

204
biopterin than neopterin derivatives are formed21 • The reasons for the varying activities of
6-pyruvoyl tetrahydropterin synthase in human cells are unclear. It will be interesting to
learn whether these cells contain different amounts of 6-pyurvoyl tetrahydropterin synthase
protein. Another possibility is that the human enzyme has become more vulnerable to
damage by the environment than enzymes from other mammalian species. This
environment may be more aggressive in macrophages than in other cells, causing the
extraodinarily low activity of 6-pyruvoyl tetrahydropterin synthase activities of human
macrophages.
Comparing intracellular and extracellular pteridine concentrations 18 , it is evident that
tetrahydrobiopterin is held more efficiently in the cells compared to neopterin derivatives.
7, 8-Dihydroneopterin triphosphate accumulating in the cells is cleaved by phosphatases 21 ,
and the resulting dihydroneopterin and neopterin leak from the cells and give rise to the
increased neopterin concentrations in body fluids of patients with diseases, in which
cytokines are produced due to activation of cell mediated immunity. The retention of
tetrahydrobiopterin in the cells may explain why biopterin levels are only slightly increased
in humans with immunology-associated increase of neopterin concentrations.
Our in vitro investigations clearly demonstrate that the increase of neopterin in humans
due to cytokine action originates from increased tetrahydrobiopterin synthesis due to
stimulation of GTP-cyclohydrolase I. In children with 6-pyruvoyl tetrahydropterin synthase
deficiency, in contrast, the block of the enzyme metabolizing 7 ,8-dihydroneopterin
triphosphate to 6-pyruvoyl tetrahydropterin causes a drop in biopterin concentrations along
with the increase of neopterin25 •

What Kind of Cytokines Stimulate GTP-cyclohydrolase I in Human Cells?

Due to the rapid discovery of new cytokines and the limited availability of many of
these new mediators, no complete survey of activating cytokines is available. In human
cells, interferon-gamma is the most active single agent13 •20•26-29 • Some of the cells like
macrophages 26-28 , human THP-1 myelomoncytoma cells 20 , human umbilical vein endothelial
cells30 also respond to lipopolysaccharide by increase of GTP cyclohydrolase I activities.
In presence of T-lymphocytes, however, lipopolysaccharide acts in an indirect way by
triggering formation of cytokines such as interferon-gamma from T-cells, which then cause
the activation of GTP-cyclohydrolase I in other cells. Tumour necrosis factor-alpha as a
single agent stimulates the activity in fibroblasts 9 and endothelial cells30 , but not in
macrophages27 •28 and not in THP-1 cells 20 • Like many other agents, however, tumour
necrosis factor alpha acts costimulatory to interferon-gamma in these cells. In human
peripheral blood mononuclear cells, interferon-alpha and -beta can stimulate the activity in
an indirect way by triggering the formation of a 15 KDa protein, which then causes
formation of interferon-gamma31 • In T-lymphocytes, the stimulatory action of interleukin-2
was found to be supported by interferon-gamma formed upon the interleukin-2 treatment32 •

STIMULATION OF TETRAHYDROBIOPTERIN BIOSYNTHESIS IN MURINE

CELLS BY CYTOKINES

In murine fibroblasts 33 , tumour necrosis factor alpha can induce GTP-cyclohydrolase I

activity in a manner comparable to human fibroblasts 9 • A pronouced difference in the
activities of the biosynthetic enzymes leading from GTP to tetrahydrobiopterin is the two
orders of magnitude higher activity of 6-pyruvoyl tetrahydropterin synthase. This causes

205
 Guanosine 5'-triphosphate induced by cytokines

C?
(GTP) in human and in

I
murine cells
GTP cyclohydrolase I
0 OH OH

~ 2 3 9
7,8-dihydro
neopterin
phosphatases
N H
H H
7,8-d ihydroneopterin increased in humans
triphosphate with immunological challenge

0
H H

Nt~~CHg
OHOH

~ H H ~.------------------------,
LJ Increased by cytokines in
human and in murine cells
5,6, 7 ,8-tet rahyd robiopteri n
Figure 1. Schematic presentation of impact of cytokines on tetrahydrobiopterin synthesis in human and in
murine cells. Among the cytokines tested so far, interferon-gamma and tumour necrosis factor alpha were
the most active agents in stimulating GTP-cyclohydrolase I activity.

206
a triggering of tetrahydrobiopterin synthesis in murine cells without the additional
accumulation of neopterin derivatives as in the case of the human cells. Other differences
of human versus murine cells relate to the action of stimulatory agents and inhibitors.
Whereas interferon-gamma is the most active agent in the human, it is only marginally
active as single agent in stimulating pteridine synthesis in murine cells. Nevertheless small
doses of interferon-gamma led to a pronouced increase in the expression of major
histocompatibility complex antigens, indicating that the used interferon-gamma preparation
is highly active on the murine cells 33 • The effect of cycloheximide on cytokine induced
pteridine synthesis is also different in human and murine cells, indicating that the cytokine
signal may be processed differently in the two species33 • Dispite all these points, the
overall effect of cytokine treatment is the same for both human and murine cells. These
agents lead to increased intracellular tetrahydrobiopterin due to activation of GTP-
cyclohydrolase I activity.

CONCLUSION

A schematic summary of the findings is presented in Figure 1. In human and in murine

cells, cytokines like interferon-gamma or tumour necrosis factor alpha stimulate the
activity of GTP-cyclohydrolase I. Due to the constitutive presence of the two subsequent
enzymes, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase, this triggers an
increased formation of tetrahydrobiopterin in human and in murine cells. Caused by the
comparatively low activity of 6-pyruvoyl tetrahydropterin synthase in human cells, the
increased biosynthesis of tetrahydrobiopterin is accompanied by the accumulation of
neopterin derivatives, which after dephosphorylation leak from the cells and give rise to
the increased concentraions of neopterin in body fluids of patients suffering from diseases
challenging the cell-mediated immunity.

ACKNOWLEDGEMENT

Support by the Austrian Research Funds "Zur Forderung der Wissenschaftlichen

Forschung", project 9685, is gratefully acknowledged.

REFERENCES

1. H. Wachter, A. Hausen, and K. Grassmair, Erhohte Ausscheidung von Neopterin im Harn von
Patienten mit malignen Tumoren und mit Viruserkrankungen, Hoppe Seyler's Z. Physiol. Chem.
360:1957 (1979).
2. D. Fuchs, A. Hausen, G. Reibnegger, E.R. Werner, M.P. Dierich, and H. Wachter, Neopterin as
marker for activated cell mediated immunity: Application for HIV infection, lmmunol. Today 9:150
(1988).
3. H. Wachter, D. Fuchs, A. Hausen, G. Reibnegger, and E.R. Werner, Neopterin as marker for the
activation of cellular immunity: Immunological basis and clinical application, Adv. Clin. Chem. 27:81
(1989).
4. M.M. Miiller, H. C. Curtius, M. Herold, and C. Huber, Neopterin in clincal praxis, Clin. Chim.
Acta 201:1 (1991)

207
5. A. Hausen, D. Fuchs, G. Reibnegger, E.R. Werner, and H. Wachter, Neopterin in clinical
medicine, Pteridines 1:3 (1989).
6. H. Wachter, D. Fuchs, A. Hausen, G. Reibnegger, G. Weiss, E.R. Werner, G. Werner-Felmayer
and H. Wachter. Neopterin, Biochemistry, Methods, Clinical Application. Walter de Gruyter,
Berlin-New York (1992).
7. R. Margreiter, D. Fuchs, A. Hausen, C. Huber, G. Reibnegger, M. Spielberger, and H. Wachter,
Neopterin as a new biochemical marker for the diagnosis of allograft rejection, Transplantation
36:650 (1983).
8. D.S. Duch, S.W. Bowers, J.H. Woolf, and C.A. Nichol, GTP-cyclohydrolase, neopterin and
biopterin in tissues and body fluids of mammalian species, Life Sci. 35:1895 (1984).
9. E.R. Werner, G. Werner-Felmayer, and H. Wachter, Tetrahydrobiopterin and cytokines, Proc. Soc.
Exp. Bioi. Med. 203: May, in press (1993).
10. G. Werner-Felmayer, E.R. Werner, G. Weiss, and H. Wachter, Modulationofnitricoxidesynthase
activity in intact cells by tetrahydrobiopterin levels, this volume.
11. D. Fuchs, A. Hausen, C. Huber, R. Margreiter, G. Reibnegger, M. Spielberger, and H. Wachter,
Pteridinausscheidung als Marker fiir Alloantigen-induzierte Lymphocytenproliferation, Hoppe
Seyler's Z. Physiol. Chem. 363:661 (1982).
12. C. Huber, D. Fuchs, A. Hausen, R. Margreiter, G. Reibnegger, M. Spielberger, and H. Wachter,
Pteridines as a new marker to detect human T-cells activated by allogeneic modified self major
histocompatibility complex (MHC) antigens, J. Immunol. 130:1047 (1983).
13. C. Huber, J.R. Batchelor, D. Fuchs, A. Hausen, D. Lang, D. Niederwieser, G. Reibnegger, P.
Swetly, J. Troppmair and H. Wachter, Immune Resonse associated production of neopterin-Release
from macrophages primarily under control of interferon-gamma, J. Exp. Med. 160:310 (1984).
14. G. Schoedon, J. Troppmair, G. Adolf, C. Huber, and A. Niederwieser, Interferon-gamma enhances
biosynthesis in peripheral blood mononuclear cells by induction of GTP-cyclohydrolase I activity,
J. Interferon Res. 6: 697 (1986).
15. G. Schoedon, J. Troppmair, A. Fontana, C. Huber, H.C. Curtius, and A. Niederwieser,
Biosynthesis and metabolism of pteridines in peripheral blood mononuclear cells and leukemia lines
of man and mouse, Eur. J. Biochem.166:303 (1987).
16. I. Ziegler, Synthesis and interferon-gamma controlled release of pteridines during activation of
human peripheral blood mononuclear cells. Biochem. Biophys. Res. Commun. 132:404 (1985).
17. E.R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, and H. Wachter, Simultaneous determination
of neopterin and creatinine in serum with solid phase extraction and on-line elution liquid
chromatography. Clin. Chem. 33:2028 (1987).
18. E.R. Werner, G. Werner-Felmayer, D. Fuchs, A. Hausen, G. Reibnegger, and H. Wachter,
Parallel induction of tetrahydrobiopterin synthesis and indoleamine 2,3-dioxygenase activity in
human cells and cell lines by interferon-gamma, Biochem. J. 262:861 (1989).
19. S. Ogiwara, T. Nagatsu, R. Teradiera, K. Fujita, and T. Sugimoto, Occurence of umanopterin, a
new diastereomer of neopterin, in the urine of cancer patients, Tetrahedron Lett. 33:1341 (1992).
20. G. Werner-Felmayer, E.R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, and H. Wachter.
Neopterin formation and tryptophan degradation by a human myelomonocytic cell line, Cancer Res.
50:2863 (1990).
21. E.R. Werner, G. Werner-Felmayer, D. Fuchs, A. Hausen, G. Reibnegger, J.J. Yim, W. Pfleiderer,
and H. Wachter. Tetrahydrobiopterin biosynthetic activites in human macrophages, fibroblasts,
THP-1 and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, 6-pyruvoyl
tetrahydropterin synthase and sepiapterin reductase are constitutively present. J. Bioi. Chem.
265:3189 (1990).
22. K. Hatekeyama, T. Aharada, and H. Kagamiyama, IMP dehydrogenase inhibitors reduce
intracellular tetrahydrobiopterin levels through reduction of intracellular GTP levels, J. Bioi. Chem.
267:20734 (1992).
23. S.l. Takikawa, H.C. Curtius, U. Redweik, W. Leimbacher, and S. Ghisla, Biosynthesis of
tetrahydrobiopterin. Purification and characterization of 6-pyruvoyl tetrahydropterin synthase from
human liver. Eur. J. Biochem.161:295 (1986).
24. E.R. Werner, G. Werner-Felmayer, D. Fuchs, A. Hausen, G. Reibnegger, G. Wels, J.J. Yim, W.
Pfleiderer and H. Wachter, 6-Pyruvoyl tetrahydropterin synthase assay in extracts of cultured human
cells using high performance liquid chromatography with fluorescence detection of biopterin, J.
Chromatogr. 570:43 (1991).
25. J.C. Nixon, C.L. Lee, S. Milstien, S. Kaufman, and K. Bartholome, Neopterin and biopterin levels
in patients with atypical forms of phenylketonuria, J. Neurochem. 35:898 (1980).
26. C.F.Nathan, Peroxide and pteridine: A hypothesis on the regulation of macrophage antimicrobial

208
 activity by interferon-gamma. in: "Interferon 7", I. Gresser, J. Vilcek, eds, Academic Press,
London (1986)
27. J. Troppmair, K. Nachbaur, M. Herold, W. Aulitzky, H. Tilg, G. Gastl, P. Bieling, B. Kotlan, R.
Flener, B. Mull, W.O Aulitzky, H. Rokos and C. Huber, In vitro and in vivo studies on the
induction of neopterin biosynthesis by cytokines, alloantigens and lipopolysaccharide (LPS), Clin.
Exp. Irnrnunol. 74: 392 (1988).
28. G. Werner-Felmayer, E.R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, and H. Wachter,
Tumour necrosis factor alpha and lipopolysaccharide enhance interferon-induced tryptophan
degradation and pteridine synthesis in human cells, Bioi. Chern. Hoppe Seyler 370:1063 (1989).
29. B. Hofmann, H. Bass, P. Nishanian, M. Faisal, R.A. Figlin, G.P. Sarna and J.L. Fahey, Different
lymphoid cell population produce varied levels of neopterin, beta-2 microglobulin and interleukin 2
receptor when stimulated with interleukin-2, interferon-gamma or tumour necrosis factor alpha,
Clin. Exp. Irnrnunol. 89:548 (1992)
30. G. Werner-Felmayer, E.R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, G. Weiss, and H.
Wachter, Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide mediated
formation of cyclic GMP, J. Biol. Chern. 268:1842 (1993).
31. M. Recht, E. C. Borden, and E. Knight, A human 15 KDa interferon-induced protein induces the
secretion of interferon-gamma, J. Irnrnunol. 147:2617 (1992).
32. I. Ziegler, K. Schott, M. Lubbert, F. Herrmann, U. Schwulera, and A. Bacher, Control of
tetrahydrobiopterin synthesis by synergistic action of interferon-gamma and interleukin-2, J. Bioi.
Chern. 265:17026 (1990).
33. E.R. Werner, G. Werner-Felmayer, D. Fuchs, A. Hausen, G. Reibnegger, and H. Wachter, Impact
of tumour necrosis factor-alpha and interferon-gamm on tetrahydrobiopterin synthesis in murine
fibroblasts and macrophages, Biochern. J. 280:709 (1991).

209
INTERFERON-y AND KIT LIGAND ARE PRIMARY REGULATORS OF
GTP CYCLOHYDROLASE ACTIVITY: MECHANISMS AND IMPLICATIONS

hmgard Zieglerl, Karin Schottl, and

Lothar Hilltner2

lGSF-Institut ftir Klinische Molekularbiologie

2GSF-Institut ftir Experimentelle Hiimatologie
Marchioninistr.25
D-8000 Miinchen 70

INTRODUCTION

(6R)-f4biopterin (Bf4) is synthesized from GTP in different types of tissues and in cells of
different lineages. It is the natural and immediate electron donor for aromatic amino acid
monooxygenases. Thus, it serves as a cofactor in tissues committed to degradation of
phenylalanine and the synthesis of the neurotransmitters dopamine, epinephrine, and serotonin
(for review, see ref.l). The activity of GTP cyclohydrolase I (GTP-CH), the first and rate
limiting enzyme of BH4 synthesis, appears to be constitutively expressed in all competent
tissues such as liver, adrenal medulla, and brain2. Changes in its activity may occur in response
to physiological conditions and pharmacological treatrnent3. No specific regulatory factors have
been identified yet.
Recent evidence has shown that BH4 is also synthesized in cells that do not require
involvement of BH4 in its role as a cofactor. Instead, they are rather characterized by cytokine-
directed proliferation and differentiation such as occurs during hematopoiesis4-6 or during the
clonal expansion ofT cells7 (for review, see ref.&). The final product, BH4, functions as a
feedback modulator ofT cell expansion (for review, see ref.&) and positively affects the
multiplication of erythroid cells6.9. In these proliferating cells BH4 synthesis is subject to
stringent regulation. GTP-CH activity in monocytes/macrophages is induced by interferon-y
(IFN-y) and results in the release of neopterin 10. In activated T cells, its effect is further
enhanced by synergistic action with interleukin 2 (IL-2)7.
Bone marrow-derived mast cells (BMMC) also undergo cytokine-directed proliferation and
differentiation dependent on both IL-311 and kit ligand (KL)12. Moreover, rodent BMMC
produce serotonin 13, a function which depends on the presence of BH4 as cofactor. In the
mouse mutant genotypes W/WY and Sl!Sld the expression of functional kit receptorl4 and of

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 211
KL15 is severely impaired. Consequently, both genotypes are virtually devoid of mast cells 16.
Moreover, KL is able to promote in vitro proliferation of BMMC derived from normal mice, but
not those from the mutant mice of genotypes WfWY and Sl!Sld12.
In the present study we report the identification of KL as a major and specific regulator of
GTP-CH activity in mast cells and compare it with the IFN-y- induced upregulation of this
enzyme in cells associated with the immune system. The unaffected GTP-CH activity in the
brain of both murine mutant genotypes W rwv and Sl/Sld serves to contrast both cytokine-
directed regulatory mechanisms with the constitutive expression of activity in the brain and other
tissues.

MATERIALS AND METHODS

The culture of the HTLV-1-infected human T cell line MT-2 was described in7. The
macrophage-like cell line U937 was maintained in a similar manner. The culture of murine
BMMC was described in17. Recombinant murine KL (hisKL) was expressed in£. coli and
purified by affinity chromatography as described in 18 . Due to the lower specific activities of
hisKL preparations, about 400 ng rnl-1 were required to equal a saturating dose of 100 ng rn1-1
yeast-derived KL in the mast cell cultures. The HPLC determination of biopterin and the
determination of GTP-CH activity were described previously?. Serotonin was determined
according to19. The expression of human and rodent GTP-CH mRNA was determined using a
555 bp eDNA segment generated by polymerase chain reaction after reverse transcription (RT-
PCR)20. The specificity of the eDNA probe was confirmed by direct sequencing. Radiolabeling
was performed by PCR strategy as described21 . Isolation and separation of total RNA, vacuum
blotting onto Nylon membranes and hybridization were also previously described21 . The
signals produced by GTP-CH mRNAs were not only calibrated against the amounts of 28S and
18S ribosomal RNA loaded on the gel, but also against the B-tubulin signal. Both parameters
agreed well. The signals on the autoradiograms were quantitated with the 2D program of the
Ultro Scan XL Laser Densitometer (Pharmacia, Freiburg) .

RESULTS

Regulation of GTP-CH Activity by IFN-y in Cells Associated with the Immune

System

The cell line U937 synthesizes neopterin upon activation with IFN-y identically to
monocytes/macrophages from peripheral blood22 . The total (cellular plus released) neopterin
production averaged 2.22 pmol/1 06 cells. Treatment with IFN-y increased it approximately 2.8-
fold (Fig. I A). Northern blot analysis with a eDNA probe specific for human GTP-CH detected
a mRNA species consisting of 3.6 kb20. Quantitative analysis of the signals showed that the
induction of neopterin synthesis by IFN-y correlated with a 4-fold increase in GTP-CH mRNA
expression (Fig.lB).
The CD4+ T cell line MT-2 lacks expression of IFN-y and was therefore used as a model
system to study the progress of GTP-CH activation upon IFN-y treatment. This induction was
further enhanced by IL-2 which is ineffective per se. It culminated after 44 h7 (Fig.2A).
Northern blot analysis demonstrated that the steady state levels of GTP-CH mRNA correlate
with the increase in apparent enzyme activity (Fig.2B). Subsequently, the activity declined
rapidly even though the mRNA expression remained nearly constant.

212
 A 5 B

-=a
E
5
E: 4
t::
·;::: 3
£
0.
0
~ 2

0
controls IFN-y controls IFN-y
Fig.l Induction of neopterin synthesis in the macrophage-like cell line U937 by IFN-y (500 U ml-1) after
30h. A: neopterin synthesis • released neopterin, !.Cl cellular neopterin; B: relative GTP-CH mRNA
expression.

0 400
:~ A
I
B
u ~
CJ
"'
u .....
0
<C
~::.....,
·~= 300 .9 0
o.o
"'t:
-~::
"'"'
"'"'
o -
'-< 0
0 0 0.'-<
"'u x-o>..
~ .....0 0
0 <:-§]
.a ~ 200
>..~
.c ~~
0 E u
u>.. 0
(.) .:::
d. ~
t-< 100 ~
CJ
0 20 40 60 0 27 44 65
time [h] time [h]
Fig.2 Induction of BH4 synthesis in the CD4+ T cell line MT-2 by IFN-y (500 Uml-1) plus IL-2 (20 U mt1 ).
A: activity of GTP-CH; B: relative GTP-CH mRNA expression. • control; ~ IFN-y plus IL-2

Regulation of GTP-CH Activity by Kit Ligand in Primary Murine Mast Cells

Murine BMMC grown with IL-3 produce 6.1 pmol biopterin/106 cells. Culturing the cells
with KL instead of IL-3 increased the production about 4-fold, but was not further enhanced by
combination with IL-3 (Fig.3A). A panel of cytokines, such as IL-4, IL-9 or NGF, which
synergistically increase the proliferation of IL-3 induced BMMC, could not substitute for KL
with respect to promotion of biopterin synthesis (data not shown). Fig.3B demonstrates that the
KL-mediated increase in Bf4 production is caused by upregulation of GTP-CH activity. The
other enzymes involved in Bf4 synthesis remained unaffected.
Northern blot analysis of rat RNA with a eDNA probe for GTP-CH previously had detected
two mRNA species of approximately 1.4 and 3.6 kb size. Both were present in all rat tissues
investigated. The ratio of the 3.6 kb species to the 1.4 kb species varied between 0.8 in liver and

213
 @'"""
u ~ I (<!

~B 1
25 A 0!=:""'
'-' ~0::: B
..... "<tiZl
......., I:: ::r: '-'
"' 20
~
·...-1~"7
S ~-S
u '"7,....; s
s ...
'Cl
OJ) ~­

s bn
0

0
15 BS S 8
s ro "7
..c: Oll·c:
;::::
~ ~S B 6
!=: 10 O;...o.,
..c: <!.) 0
·c: .8< ~ :.0 4
~ !:l ~ ('l

:.0 5
"§ 0
O..·p s
*
i=:"Oi:I:
2
~n::
!=: -
o.. 0 _,_,_<..LL..__
IL-3 KL IL-3+KL - 0 SR
~ §,
0..

Fig.3 Production of biopterin in murine BMMC cultured in the presence of IL-3 (20 ng mtl) or KL
(100 ng ml-1) A: biopterin levels, f"&l 6-biopterin, • 7-biopterin; B: activities of Bl4 synthesizing
enzymes, • IL-3, @. KL

Exp.#1 Exp.#2
a b a b

- 288
3.6 kb -

- 188
1.4kb - .

Fig.4 Northern blot analysis of GTP-CH mRNA expression in murine BMMC. a: cells cultured with KL
(100 ng mi-l; b: cells cultured with IL-3 (20 ng mJ-1). The signal intensities of the 1.4 plus 3.6 kb species were
calibrated against the methylene blue staining intensities of blotted RNA (18S plus 28S). The mean values for
the mRNA signals obtained were 1.9 for KL cultured cells and 0.99 (arbitrary units) for IL-3 cultured cells.

2.4 in kidney20. Similarly, both mRNA species were present in murine BMMC at a ratio of0.18
(±0.04 S.D.). No difference in this ratio was found between cells grown in KL or IL-3 (Fig.4).
The level of both GTP-CH mRNA species increased significantly upon KL treatment. Quantitive
estimates are given in the legend of fig.4. They revealed an approximately 2-fold increase in
GTP-CH mRNA whereas the catalytic activity increased approximately 6-fold. Given the
limitations of quantitative Northern blot evaluation a potential contribution by post-translational
modification requires further investigation. On basis of the amino acid sequence deduced from
the cloned rat GTP-CH eDNA, phosphorylation by growth-associated histone Hl kinase or
casein kinase II appears to be possibly involved23.
Assuming a homogenous distribution of BH4 and a cell volume of 8 x 10- l. 0 ml, the
concentration of the BR4 cofactor is approximately 8 j.lM in IL-3 grown cells. KL triggers an
increase to approximately 30 j.lM. In care of non-neuronal tryptophan 5-monooxygenase the
in vitro Km value for BR4 is 22 j.lM24. Thus, the KL-induced increase occurs at the Km value
of tryptophan 5-monooxygenase and results in maximum changes of activity.

214
 Furthennore, analysis of the effect of the growth factors on the activity of tryptophan 5-
monooxygenase in murine BMMC demonstrated that the activity of 47.6 (± 7.4) and 50.8
(±13.8) pmol mg-1 rnin-1 in IL-3 or KL grown cells increased about 5-fold when both growth
factors were combined (Fig.5A). No other factors could substitute for KL25. Thus, the
concerted action of IL-3 and KL on the hydroxylation of tryptophan enhances the production of
serotonin approximately 20-fold (Fig.5B). KL selectively causes the release of about half of
total serotonin produced (Fig.5B)25.
We previously found that 7-biopterin in mast cells is only fonned during ongoing
hydroxylation of tryptophan!?. Fig.3A demonstrates that the combination of KL with IL-3 has
no effect on the total amount of BI4 but increases the portion of7-biopterin from 8.0 to 35.6%.
Thus, the upregulation of tryptophan 5-monooxygenase activity by the concerted action ofiL-3
and KL25 fully explains the pattern of 6-biopterin and 7-biopterin.

350
A
e 100
I:: ;::!
E
'6 300 '><<'<l
Co E
E 250 .....
0
0 ~
5.
-
200 c 60
c<'<l .9
<'<l
..c !:l
9' c 40
II)
(.)

s
0.

I
...4
c
0
(.)
c 20
:I:
·a
90
0
,;., ...
II) 0
IL-3 KL IL-3+KL "' ll..,-3 KL ll..,-3+KL
Fig.S Synthesis of serotonin in murine BMMC cultured in the presence of IL-3 (20 ng mt·l), KL (100 ng mt· l)
or both growth factors. A: activity of tryptophan 5-monooxygenase; B: serotonin production,
• cellular serotonin , ~ released serotonin

BH4 Synthesis and Serotonin Production in the Brain of the Mutant Genotypes
W/Wv and Sl/Sld

In tl{e brain and both other tissues investigated (liver, spleen) in the wild type and both the
WJWV and SVSld mutant genotypes only the 6-isomer of biopterin was found. The following
Bl4 concentrations were found in the brain (pmol g·l): 276 (±28) in wild type, 277 (±15) in the
genotype w;wv and 285 (±24) in the genotype SVSld. The values for serotonin (nmol g-1) were
as follows: 3.75 (±0.09), 3.67 (±0.32) and 3.77 (±0.35), respectively. These data demonstrate
that the lack of a functional kit receptor or of KL neither affects the synthesis of the BH4
cofactor, nor the activity of tryptophan 5-monooxygenase. In addition, the Bl4 concentration in
the liver and the capacity for phenylalanine degradation were not significantly different in both
mutant genotypes as compared to the wild type (data not shown). Thus, the GTP-CH activity
in these tissues is independent of the KL.
Together with the IFN-y- induced increase of GTP-CH mRNA expression in the cells of the
immune system, the data obtained with murine BMMC suggest that GTP-CH is subject to a cell
lineage and tissue specific regulation. This is further supported by the existence of multiple
mRNA species and their tissue specific expression20,26. A similar situation is found in the case
of tryptophan 5-monooxygenase which is discussed in more detail in ref.25. The question
whether IFN-y and KL induce the expression of specific isoforms of GTP-CH in cells of the

215
 immune system, in BMMC and in tissues such as brain or liver or whether KL post-
translationally activates GTP-CH, awaits further investigation.

ACKNOWLEDGEMENTS

The authors thank Dr. Wolf Endres (Kinderklinik der Universitiit Innsbruck) for
determination of urinary phenylalanine levels, Wolfgang R&ll as well as Hannelore Broszeit for
qualified technical assistance.

REFERENCES

S.Kaufman and D.B.Fisher, in Oxygenases (O.Hayaishi, ed.) pp 285-370, Academic Press New York
(1972)
2. T.Fukushima and I.C.Nixon, Analyt.Biochem.l02: 1767 (1980)
3. O.H.Viveros, M.M.Aboud-Donia, C.L.Lee, S.P.Wi1son, and C.A.Nicho1, in Function and Regulation of
monoamine enzymes: Basic and Clinical Aspects (E.Usdin, N. Weiner, and M.B. Youdim, eds.) pp 241-
259, Mac Millan Publ.Ltd., London (1981)
4. !.Ziegler and St.Thierfelder, Z.fNaturf.42c:461 (1987)
5. F.Kerler, L.Hiiltner, !.Ziegler, G.Katzenmeier, and A.Bacher, J.Cell.Physiol.l42:268 (1990)
6. K.Tanaka, S.Kaufman, and Sh.Milstien, Proc.Natl.Acad.Sci.(USA) 86:5846 (1989)
7. !.Ziegler, K.Schott, M.Liibbert, F.Herrmann, U.Schwulera, and A.Bacher, J.Biol.Chem 265: 17026 ( 19907)
8. I.Ziegle.-, Med.Res.Rev.l0:95 (1990)
9. F.Kerler, !.Ziegler, C. Schmid, and A.Bacher, Exp.Cell Res.l89: 151 (1990)
10. C.Huber, J.R.Batchelor, D.Fuchs, A.Hausen, A.Lang, D.Niederwieser, G.Reibnegger, P.Swetley,
J.Troppmair, and H.Wachter, J.Exp.Med.l60:310
11. J.N.Ihle, J.Keller, S.Oroszlan, L.E.Henderson, T.D.Copeland, F.Fitch, M.B.Prystowsky, E.Goldwasser,
J.W.Schrader, E.Palaszynski, M.Dy, and B.Lebel, J.Immunol.l31:282 (1983)
12. J.G.Flanagan, D.C.Chan, and P.Lever, Cell64: 1025 (1991)
13. L.B.Schwartz and K.F.Asuten, Prog.Allergy 34:271 (1984)
14. K.Nocka, J.C.Tan, E.Chin, T.J.Chu, P.Ray, P.Traktman, and P.Besmer, EMBO 1.9:1805 (1990)
15. K.M.Zsebo, D.A.Williams, E.N.Geissler, V.C.Broudy, F.H.Martin, H.L.Atkins, R.Y.Hsu, N.S.Birkett,
K.H.Okino, D.C.-Murdock, F.W .Jacobsen, K.E.Langley, K.A.Smith, T.Takeishi, B.M.Cattanach,
S.J.Galli, and S.V.Suggs, Cell63:213 (1990)
16. Y.Kitamura, H.Nakayama, J.Fujita in Mast Cell and Basophil Differentiation and Function in Health and
Disease (S.J.Galli and K.F.Austen, eds) pp 15-25, Raven Press New York (1989)
17. !.Ziegler and L.Hiiltner, FEBS Letters 307:147 (1992)
18. G.Reisbach, I.Bartke, B.Kempkes, J.Ellwart, A.Bimer, K.Thalmeier, R.Mailhammer, G.W.Bomkamm,
A. Ullrich, and P.DOrmer, Exp.Hematol. in press (1993)
19. E.Traiffort, P.Hubert, N.Tayeb, and N.Aymard, J.Chromat.571:231 (1991)
20. M.Giitlich, K.Schott, Th.Wemer, A.Bacher, and !.Ziegler, Biochim.Biophys.Acta 1171:133 (1992)
21. K.Schott, M.Giitlich, !.Ziegler, J.Cell.Physiol, in press (1993)
22. K.Rokos, R.O.F.Kunze, M.A.Koch, H.Rokos, and K.Nilsson in Unconjugated Pterins and Related
Biogenic Amines (H.Ch.Curtius, N.Blau, and R.A.Levine eds.) Walter de Gruyter Berlin (1987)
23. K.Hatakeyama, Y.lnoue, T.Harada, and H.Kagamiyama, J.Biol.Chem.266:765 (1991)
24. D.M.Kuhn, M.A.Meyer, and W.Lovenberg, Arch.Biochem.Biophys.l99:355 (1980)
25. !.Ziegler, H.Hiiltner, D.Egger, B.Kempkes, R.Mailhammer, St.Gillis, and W.Rbdl, J.Biol.Chem. (in
press) (1993)
26. A.Togari, H.Jchinose, Sh.Matsumoto, K.Fujita, and T.Nagatsu, Biochem.Biophys.Res.Comm.l87:359
(1992)

216
DIFFERENTIAL METABOLISM OF TETRAHYDROBIOPTERIN IN MONOAMINE
NEURONS: A HYPOTHESIS BASED UPON CLINICAL AND BASIC RESEARCH

Gregory Kapatos, Kei Hirayama, Stephen I. Lentz, Min Zhu and Susan Stegenga

Cellular and Clinical Neurobiology Program, Department of Psychiatry, Wayne State

University School of Medicine, Detroit, Michigan, 48201, U.S.A.

INTRODUCTION

Neurons that synthesize and secrete monoamines have been extensively studied
because of their hypothetical involvement in the symptomology associated with certain
neurological and psychiatric disorders, and, by inference, their possible involvement in the
disease process itself. 5,6,7,8-tetrahydrobiopterin (BH4) is the essential cofactor for the
family of pterin-dependent enzymes that includes tyrosine and tryptophan hydroxylase, the
rate-limiting enzymes in the biosynthesis of catechol and indoleamines1• The family of nitric
oxide synthases, enzymes that convert arginine to nitric oxide, also require BH4 for activity,
although at extremely low concentrations2• While the concentration of BH4 within
monoaminergic neurons is unknown, it appears to be subsaturating with respect to these
enzymes3.4. Increases or decreases in BH4levels might therefore be expected to alter basal
enzyme activity and, in some cases, also modify allosteric regulatory mechanisms. The
capacity of monoaminergic and possibly nitric oxide-producing neurons to synthesize
neurotransmitter and thereby perform their physiological functions is thus highly dependent
upon the intracellular concentration of BH4.
In man, unequivoval evidence exists of deficiences in monoamine neurotransmission
that are the direct result of genetic defects in BH4 biosynthetic or regenerative processes5 •
Individuals afflicted with these genetic diseases are typically detected at birth by the
diagnosis of hyperphenylalanemia. With the exception of the "peripheral" form of BH4
deficiency 6, severe neurological and cognitive dysfunctions typically occur in these patients
later in development despite dietary control of phenylalanine intake. To date, genetic
diseases that increase rather than decrease levels of BH4 have not been observed in the
clinic. Although rarely if ever referred to in the neurology and mental health literature, the
existence of genetic mutations of BH4 metabolism that interfere with normal monoamine
biosynthesis would seem to support theories that hypothesize abnormal monoamine
synthesis as the cause of numerous neuropsychiatric diseases. Because these diseases are
believed to involve specific populations of neurons, however, for any such theory to include
a role for aberrant BH4 metabolism would require that alterations in BH4 metabolism also
be restricted to specific populations of neurons. This requirement would seemingly go
against the typical characterization of BH4 as simply an "interested bystander" in
monoamine neurotransmitter biosynthesis.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 217
 This chapter will attempt to describe and integrate clinical and basic research
findings in support of our hypothesis that BH4 metabolism is normally heterogeneous
across different populations of monoamine-containing neurons.

CLINICAL EVIDENCE

Numerous studies have demonstrated abnormally low BH4levels in neuropsychiatric

diseases such as Alzheimer Disease7 , Parkinson Disease8, dystonia9 and depression10 • The
role of BH4 in the etiology of these diseases might seem questionable inasmuch as
decreased levels of BH4 would presumably effect the capacity of all monoaminergic
neurons to synthesize neurotransmitter equally. This lack of specificity would be in conflict
with the specific behavioral deficits characteristic of these diseases. Levels of the various
monoamines within the brain and periphery of BH4 synthesis deficient patients have,
however, actually been shown to be differentially effected by the loss of BH411 • For
example, marked declines in the concentrations of serotonin, dopamine and their metabolites
in CSF, plasma and urine are typically observed in these patients while levels of
norepinephrine and epinephrine and their metabolites appear to be quite normal. These
differences in monoamine levels in response to BH4 deficiency were initially attributed to
differential rates of turnover i.e., monoamines that turnover more rapidly would be most
susceptible to the resulting partial blockade of their synthesis in response to decreased BH4
levels11 • We propose here the alternative hypothesis that BH4 levels are normally
heterogeneous across monoamine neurons; i.e., that monoamine neurons most susceptible
to BH4 deficiency would be those that normally express lower levels of BH4 biosynthetic
enzymes and, therefore, lower steady-state levels of BH4.
Additional evidence for heterogeneity in BH4 metabolism across monoamine-
containing cell types can be found in the physiological processes that are known to be
regulated by monoamine secretion. For example, we have previously reported that a "leaky"
BH4 synthesis diffident patient with approximately 10% of normal levels of BH4 exhibited
movement disorders characteristic of patients with Parkinson Disease12• From this it can be
inferred that low levels of neurotransmitter exist within the dopaminergic nerve terminals
of the caudate and putamen that arise from cell bodies in the midbrain. In contrast, this
same patient exhibited normal prolactin secretion when challenged with a dopamine
receptor antagonist, demonstrating that hypothalamic dopamine neuron function remained
intact. A second analysis of hypothalmic control of endocrine function in this same patient,
a challenge with combined insulin and arginine administration, also indicated normal
monoaminergic regulation of growth hormone secretion. That hypothalamic dopamine
function is dependent upon BH4 and can be compromised by the loss of BH4 is evidenced
by the hyperprolactemia observed in a patient with a more profound blockage of BH4
biosynthesis 13 • This "leaky" patient also displayed a normal diurnal rhythm in the pineal
gland hormone melatonin, whose synthesis is dependent upon BH4 within pinealocytes 14
and whose regulation by light is dependent upon an intact and functional preganglionic
norepinephrine inpue5• These physiological measures suggest that dopamine synthesis in
hypothalamic neurons, melatonin synthesis by the pineal gland, and norepinephrine
synthesis by sympathetic neurons may all be less suceptible to the lowering of BH4 levels
than is dopamine synthesis within midbrain nigrostriatal neurons. Finally, based upon
clinical observations there is also reason to believe that there are differences in the
biosynthesis of BH4 in brain and peripheral tissues6•

LABORATORY OBSERVATIONS

In our initial studies to test the hypothesis of heterogeneity in BH4 metabolism

across populations of monoaminergic neurons we examined hypothalamic and midbrain

218
neurons maintained in primary tissue culture16 • These brain regions were chosen because
they include identified populations of dopamine-containing neurons that are relatively
devoid of other monoamine-containing cell types. In addition, as described above, clinical
studies indicated that there was reason to believe that BH4 metabolism would be different
within hypothalamic and midbrain dopamine neurons.
Kinetic parameters describing BH4 metabolism in hypothalamic and midbrain
cultures were determined by following the decline in steady-state levels of BH4 after
inhibition of BH4 synthesis. In all experiments, we observed that levels of BH4 were
significantly greater in hypothalamic than midbrain cultures. The average fractional rate
constants for the loss of BH4 after synthesis inhibition in these cultures were virtually
identical and demonstrated that approximately 15% of the intracellular pool of BH4 turns
over per hour. The half-life for BH4 determined in either culture system (4.5 hours) was
therefore equal. When combined with the observed difference in steady-state levels, these
fractional rate constants yield a rate of BH4 synthesis in hypothalamic neurons significantly
greater than that observed in midbrain dopamine neurons. Hypothalamic dopamine neurons
maintained in culture thus appear to express higher steady-state levels of BH4 and
synthesize BH4 at a more rapid rate than do their counterparts isolated from the
mesencephalon. In contrast to this difference, BH4 biosynthesis by either population of
neurons was equally sensitive to the inhibitors N-acetyl serotonin (NAS) and 2,4-diamino-6-
hydroxypyrimidine (DAHP) 16•
We have recently reported that there are several notable differences between central
and peripheral nervous system monoaminergic neurons regarding BH4 metabolism and their
response to inhibitors of BH4 biosynthesis. Owing to the heterogeneity of neuronal cell
types present in cultures of CNS, no absolute comparison between central and peripheral
neurons can be made regarding the rate of BH4 biosynthesis. Comparisons can be made,
however, between fractional rate constants for the loss of BH4 following BH4 synthesis
inhibition, because these values are independent of whether BH4 content is expressed on
a per well, protein or cell basis. BH4 metabolism within CNS neurons maintained in culture
exhibited an average half-life of 4.5 hours. The half-life for BH4 with PNS neurons was
almost twice as rapid17• Although the basis for this difference has not been established, it
again illustrates that BH4 metabolism can differ between populations of BH4-containing
cell types and between neurons derived from the central and peripheral nervous systems.
Cellular heterogeneity of BH4 metabolism is also suggested by differences we have
detected in the ability of the compounds N-acetyl-serotonin (NAS) and 2,4-diamino-6-
hydroxypyrimidine (DAHP) to inhibit BH4 biosynthesis. Concentrations of 100 uM NAS
and 5 mM DAHP have previously been shown to produce maximum levels of inhibition
in central neurons 16• In contrast, maximum levels of inhibition of BH4 biosynthesis in
peripheral neurons were obtained at concentrations of 2.5 mM NAS and 1 mM DAHP17 •
BH4 synthesis by peripheral neurons of the superior cervical ganglia was therefore twenty-
five times less sensitive to NAS and 5 times more sensitive to DAHP than in central
neurons. While these differences could reflect an artifact of tissue culture or the differential
metabolism of these compounds, they are also consistant with a possible difference in the
BH4 biosynthetic pathway within central and peripheral neurons. This premise is supported
by the clinical reports mentioned above of a novel form of hyperphenylalanemia,
characterized by a defect in hepatic BH4 biosynthesis that does not involve the central
nervous system6 •
The differential sensitivity to NAS exhibited by central and peripheral
monoaminergic neurons that we have observed may find its biochemical basis in the
seemingly redundant use of the enzymes sepiapterin reductase and 6-pyruvoyl reductase in
the reduction of precursors of BH4 during the last two steps of the BH4 biosynthetic
pathway18 • Based on this hypothesis, cells of the periphery would express equal levels of
both of these reductases and, as a result, BH4 synthesis would be less sensitive to inhibition

219
of sepiapterin reductase by NAS. In contrast, neurons of the CNS would primarily utilize
sepiapterin reductase in the terminal reductions of BH4 intermediates and would thus be
more sensitive than peripheral neurons to NAS. This hypothesis is supported by our
observation reported at the last International Pteridine Symposium that a concentration of
NAS (200 uM) that was completely ineffective in inhibiting endogenous BH4 biosynthesis
by peripheral neurons was at the same time capable of blocking the conversion of
exogenously supplied sepiapterin to BH419• It would therefore appear that in these neurons
BH4 synthesis continues despite inhibition of sepiapterin reductase, perhaps because 6-
pyruvoyl reductase can function in BH4 biosynthesis.
We have also used these culture systems to examine the regulation of BH4
biosynthesis and here too we find differences between hypothalamic and midbrain dopamine
neurons and between central and peripheral neurons. Initial studies have focused on the role
of 3 '5 '-cyclic-adenosine monophosphate (cAMP)-dependent regulatory mechanisms because
previous studies have shown that elevations in cAMP levels are capable of inhibiting BH4
biosynthesis in the pineal gland14 while stimulating BH4 synthesis in the adrenal medulla20 •
In hypothalamic and midbrain cultures cAMP has been found to increase BH4 levels by
stimulating biosynthesis21 • This increase in biosynthesis in response to elevated cAMP is
significantly greater in midbrain than in hypothalamic dopamine neurons and follows an
extended time-course that is indicative of altered gene expression. This possibility is
supported by the observations that levels of mRNA encoding for GTP cyclohydrolase I
were increased by cAMP and inhibition of gene transcription or translation completely
blocked the ability of cAMP to stimulate BH4 biosynthesis. BH4 biosynthesis within central
neurons appears, therefore, to be positively regulated by a cAMP-dependent mechanism in
a manner similar to that observed previously in the adrenal medulla but certainly different
from the pineal gland. The knowledge that the adrenal medulla and sympathetic ganglia are
both derived embryologically from the neural crest led us to investigate whether a cAMP-
dependent mechanism is also involved in the regulation of BH4 biosynthesis in these
peripheral neurons. Elevation of cAMP levels within peripheral neurons maintained in
culture, however, did not significantly alter BH4 levels. In contrast, BH4 levels in these
sympathetic neurons were sensitive to the neurotrophin nerve growth factor and to the
cytokine leukemia inhibitory factor. These observations demonstrate that even cells that are
derived from the same embryonic tissues generate diverse mechanisms for regulating BH4
levels.
The introduction of molecular biology to the field of unconjugated pteridines22 has
also allowed us to investigate the expression of GTP cyclohydrolase I in intact brain and
sympathetic ganglia at the molecular levee3•24 •25 • The results from these studies have done
nothing but reinforce our earlier observations regarding heterogeneity of BH4 metabolism.
In contrast to the original report of a single form of GTP cyclohydrolase I mRNA in rat
liver2, we have detected two mRNA species of 1.2 and 3.8 kilobases in rat liver and other
tissues23 ' 25 • The relative abundance of these mRNAs varied, with the short form predominant
in the liver and the long form the major species in the pineal gland, adrenal gland,
brainstem and hypothalamic neurons maintained in culture. This heterogeneity in GTP
cyclohydrolase I mRNA may reflect alternative splicing of pre-mRNA, alternative
transcription initiation sites or multiple polyadenylation signals and may help to explain the
disparate range of sizes reported for the subunit form of mammalian GTP cyclohydrolase
f 6•27•28•29 • Although these apparent structural differences might suggest otherwise, the
possibility of two distinct GTP cyclohydrolase I genes is unlikely because mutations that
eliminate GTP cyclohydrolase I enzyme activity and BH4 biosynthesis in man30 and
mouse31 would have to involve simultaneous loss of both genes. It remains to be determined
whether these apparent differences in GTP cyclohydrolase I protein are related to the two
forms of mRNA which may have the potential to code for separate translation products with
different catalytic and regulatory capabilities. As demonstrated by in situ hybridization to

220
localize mRNA at the cellular level and quantitative RNA protection experiments23 •24 •25 there
are dramatic differences in the level of GTP cyclohydrolase I mRNA expression within the
dopamine neurons of the midbrain inthat neurons localized to the substantia nigra contain
far less GTP cyclohydrolase I mRNA than do their neighbors in the ventral tegmentum. If
mRNA content is a reflection of the amount of GTP cyclohydrolase I protein, then it is
highly probable that levels of BH4 are also substantially lower in these neurons. Dopamine
synthesis within substantia nigra neurons might therefore be highly sensitive to the further
decline in levels of BH4 that is characteristic of BH4 deficient patients. Perhaps this is the
answer to the mystery of why these patients exhibit movement disorders reminiscent of
Parkinson Disease yet display normal hypothalamic, sympathetic and pineal gland function.

SUMMARY

This chapter has attempted to describe and integrate some of the clinical and basic
research that support our hypothesis that the metabolism of BH4 is normally heterogeneous
across different populations of monoamine-containing neurons. Based upon this hypothesis,
there may now be reason to support the idea that certain neuropsychiatric illnesses, which
are though to be the result (at least in part) of altered monoamine metabolism, might find
their roots in an abnormal metabolism of BH4 within specific monoaminergic cell groups.
Such a specific dysfunction might not be apparent in the rest of the brain or peripheral
nervous system, thereby being difficult to detect. Perhaps the application of molecular
biological techniques to studies of BH4 metabolism in man will shed new light on these
problems.

ACKNOWLEDGEMENT

We thank Mr. Gary M. Bora for his expert technical assistance. We also thank our
colleagues in the Cellular and Clinical Neurobiology Program for their intellectual support.
This work was supported by a grant from the National Institute of Neurological Diseases
and Stroke (NS 26081).

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10. J.C. Garbutt, D.S. Duch, C.A. Nichol and H. Woolf, Psychiatry Res. 16:181-187 (1985).
11. I.J. Butler, S.H. Koslow, A. Krumholz, N.A. Holtzman, and S. Kaufman, Ann. Neurol. 3:224-230
(1978).

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12. S. Kaufman, G. Kapatos, W.B. Rizzo, J.D. Schulman, L. Tamarkin and G.R. van Loon, Ann.
Neural. 14:318-325 (1983).
13. R.R. Mcinnes, S. Kaufman, JJ. Warsh, G.R. van Loon, S. Milstien, G. Kapatos, S. Soldin, P.
Walsh, D. MacGregor and W.B. Hanley, J. Clin. Invest. 73:458-469 (1984).
14. G. Kapatos, S. Kaufman, JL. Weller and D.C. Klein, Science 213:1129-1131 (1981).
15. J. Axelrod, Science 184:1341-1348 (1974).
16. G. Kapatos, J. Neurochem. 55:129-136 (1990).
17. G. Kapatos, K. Hirayama and H. Hasegawa, J. Neurochem. 59:2048-2055 (1992).
18. R.A. Levine, G. Kapatos, S. Kaufman, and S. Milstien, J. Neurochem 54:1218-1224 (1990).
19. G. Kapatos, H. Hasegawa, K. Hirayama and V. Kemski, in Chemistry and Biology of Pteridines,
H.Ch. Curtius, S. Ghis1a, N. Blau, eds. Walter deGruyter & Co., Berlin (1990).
20. M.M. Abou-Donia S.P. Wilson, T.P. Zimmerman, C.A. Nichol and O.H. Viveros, J. Neurochem.
46:1190-1199 (1986).
21. K. Hirayama, M. Zhu and G. Kapatos, this volume.
22. K. Hatak.eyama, Y. Inoue, T. Harada and H. Kagamiyama, J. Bioi. Chern 266:765-769 (1991).
23. K. Hirayama, S. Lentz and G. Kapatos, this volume.
24. S. Lentz, K. Hirayama and G. Kapatos, this volume.
25. K. Hirayama, S. Lentz and G. Kapatos, J. Neurochem. in press.
26. K.W. Cha, K.B. Jacobson, J.J. Kim, J. Bioi. Chern. 266:12294-12300 (1991).
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Neurochem. 50:655-657 (1988).

222
TISSUE DISTRIBUTION OF TETRAHYDROBIOPTERIN GENERATING
ENZYMES

Masaaki Hoshiga, Kazuyuki Hatakeyama* and Hiroyuki Kagamiyama

Department of Medical Chemistry

Osaka Medical College, Osaka 569, Japan

INTRODUCTION
Tetrahydrobiopterin (BH 4) is the natural cofactor of phenylalanine, tyrosine and
tryptophan hydroxy lases as well as nitric oxide synthaset-s. BH4 is synthesized from
GTP by successive actions of at least three enzymes2,6, namely, GTP cyclohydrolase I, 6-
pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR)2,6. BH4 is
regenerated by dihydropteridine reductase (DHPR) from quinonoid dihydrobiopterin 2.
The activities of these BH4-generating enzymes have been measured in various tissues7- 10 ,
but the expression of their genes has not been fully elucidated. Herein, we examined the
transcriptional regulation of BH4-generating enzymes in various tissues. Northern blot
analysis was performed with eDNA probes of these enzymes to determine the mRNA
levels of the BH4-generating enzymes. Our purposes are: to examine the spatial spectra
of the mRNA of the BH4-generating enzymes and compare them with their enzyme
activities; 2) to determine whether the mRNA expression of these enzymes differ with the
tissue.

MATERIAL AND METHODS

Adult male Wistar rats were purchased from Japan Clea. Total cellular RNA was
prepared from various tissues by the guanidine isothiocyanate/cesium chloride gradient
methodll. The 260nm/280nm ratios of isolated RNA were more than 1.8. Five p.g of
total cellular RNA of each organ was electrophoresed by 1.2% agarose/formaldehyde gel
and subsequently transferred to Hybond N + membranes (Amersham, Inc.) as described
by the manufacturer. Filters were prehybridized for 4 hand hybridized for 18 hat
65°C. The eDNA probes we used were: a 748-bp Hind III-Nco I fragment including
full coding sequences of rat liver GTP cyclohydrolase I cDNA12; a 420-bp Pst I-Pst I
fragment including from + 1 to +349 bases of rat PTPS cDNA13; SR and DHPR eDNA
probes were prepared using polymerase chain reaction (PCR) as follows. First strand
eDNA was synthesized with M-MLV reverse transcriptase (BRL) using a eDNA
synthesis kit (BRL). To prepare the SR eDNA probe, the following oligonucleotide
primers were synthesized: a 43-mer (5'-GGGCTGCAGGAATGCCGGCAGGCA-
GGTTGCGCTGTCTGCGTG-3') containing the 5' end of the rat SR coding regiont4 and
5' overhangs of a Nae I site; a 32-mer (GAAGCTTGGCGCTGTGACAG-
GACATGGGCTTA-3') containing antisense nucleotides of the 3' end of

*To whom correpondence should be addressed. FAX: 81-726-81-3723

Chemistry and Biology of Pteridines and Palates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 223
its coding region 14 and 5' overhangs of a Hind III site. PCR was performed with the
following thermocycle parameter: 93°C for 3 min, 60°C for 1.5 min and noc for 3 min
for 25 cycle. PCR products were cloned into the pCR™ Vector (Invitrogen Corp.) and
the plasmids were digested with Nae I and Hind III. The resulting 803 bp fragments
were used as an SR eDNA probe. To prepare the DHPR eDNA probe, oligonucleotide
primers were synthesized as follows: a 37-mer (5'-CCGGATCCCGGGGCGGC-
TTCGGGCGAGGCGCGCCGAG-3') containing the 5' end of the rat DHPR coding
regionts and 5' overhangs of BamH I and Sma I sites; a 32-mer(5'-
GAAGCTTACTGAGGTGCAACTTAGAAATAGGC-3') containing the 3' end of its
coding region ts and 5' overhangs of a Hind III site. The thermocycle conditions were
94°C for 1 min, 55°C for 1 min and 72°C for 1 min for 40 cycles. PCR products were
digested with Rsa I and Sau3A I and the resulting fragment identical to +70 to +636 bp
of the DHPR coding regionts was cloned into pBluescript digested at the BamH I and
Hinc II sites. The plasmids were digested with Xba I and Xho I and the 584 bp
fragment was obtained as a DHPR eDNA probe. These eDNA probes were labeled with
[cx-32P]dCTP (specific activity, 3000 Ci/mmol; Amersham) using a random prime
synthesis kit (Amersham). After washing in 0.1x SSPE-0.1% SDS as the final
condition, the filters were exposed on phosphor imaging plates (Fujix BAS 2000 system ;
Fuji Film Corp.) for 4 days at room temperature. The signal intensity was determined
by computer-assisted densitometry (Fujix BAS 2000 system).

RESULTS AND DISCUSSION

Fig.1 shows the results of Northern blot analysis with each BH4-generating enzyme
eDNA probe. There were two species (1.3 and 3.2 kb) of mRNA that hybridized to the
GTP cyclohydrolase I eDNA probe (Fig. 1a). These two species of GTP
cyclohydrolase I mRNA are probably due to the use of alternative polyadenylation sites,
because no evidence suggesting the existence of the GTP cyclohydrolase I isozyme was
obtained in the purification 16 and because only the 3.2 kb mRNA hybridized to the DNA
probe which contains only the 3' noncoding region from 100 to 660 bp downstream of
the first polyadenylation signal (unpublished observation). Every tissue in which GTP
cyclohydrolase I mRNA was detected contained both mRNA species of large and small
sizes. The ratio of signal intensity (3.2kb/1.3kb) varied in each organ; e.g., 0.6 for
liver, 1.6 for adrenal gland, 1.2 for spleen, 0.6 for bone marrow, 0.8 for small intestine
and 2.0 for cerebellum. Recently, Gutlich et al. reported similar results 17. The
physiological implications of different mRNA species is not clear.
In contrast, Northern blot with PTPS (Fig. lb), SR (Fig. lc) and DHPR (Fig. ld)
revealed that each mRNA of these enzymes consists of a single species in all the tissues
examined. The length of PTPS and SR mRNA, 1.4 and 1,6 kb, respectively, is
similar to that described previouslyt3,t 8 • The length of rat DHPR mRNA, 1.4 kb, is
similar to that of human DHPR mRNA, 1.5 kb19.
The tissue distribution of mRNA expression corresponds with that of each enzyme
activity previously reported 7-to. This finding suggests that steady state levels of
activities of these BH4-generating enzymes are regulated mainly at their transcriptional
level. All four enzymes have already been purified in rat tissuest3,t6,20,2t. The hepatic
contents of these enzymes can be estimated by the use of specific activities of these
enzymes in liver crude extracts and purified preparations; the levels of GTP
cyclohydrolase I, PTPS, SR and DHPR were 0.1, 0.6, 0.3 and 38 pmol/mg of total
protein, respectively. The relative levels of the estimated contents of these enzymes
were consistent with those of the mRNA levels observed in the present study.
There are some differences in tissue distribution of mRNA expression for BH 4-
generating enzymes. In the kidney and thymus, the GTP cyclohydrolase I mRNA level
was much lower than the levels of mRNAs for other three enzymes. GTP
cyclohydrolase I is the committing enzyme from GTP to BH 4 and is induced by
interferon-r22. The mRNA expression of this enzyme in the kidney and thymus is
limited at steady state and may be induced under some conditions_ In comparison of SR
and DHPR mRNAs in tissue ditribution, the hematopoietic and cardiopulmonary tissues
and the testis expressed mainly SR, whereas the brain did mainly DHPR. The reason
why these enzymes are highly expressed in those tissues is not clear.
In conclusion, this study revealed that the spatial spectra of mRNA levels at steady

224
 a
~28S rRNA
3.2 kb ~
~18S rRNA
1.3 kb ~

b
~28S rRNA

~18S rRNA
1.4 kb ~

c
~28S rRNA

1.6 kb ~ ~18S rRNA

d
.....C:28S rRNA,

1.4 kb ......

~28SrRNA

-.<18S rRNA

Figure 1. mRNA expression of BH4-generating enzymes in various tissues. Total RNA ( 5 p.g) from rat
16 tissues were hybridized to each eDNA probe of GTP cyclohydrolase I (a), PTPS (b), SR (c) and DHPR
(d) as described in "MATERIAL AND METHODS ". Equal amounts of RNA application in each lane
were estimated by ethidium bromide-stained gel (e).

225
state correlate with those of the enzymatic activity of each BH4-generating enzyme.
Therefore, the steady state levels of these BH4-generating enzymes are suggested to be
regulated mainly at the transcriptional level.

REFERENCES
1. S. Kaufman (1967) Annu. Rev. Biochem. 36, 171-184.
2. C.A. Nichol, G.K. Smith and D.S. Duch (1985) Annu. Rev.Biochem. 54, 729-764.
3. N.S. Kwon, C.F. Nathan and D.J. Stuehr (1989) J. Bioi. Chern. 264, 20496-20501.
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5. B. Mayeh, M. John and E. Bohme (1990) FEBS Lett. 277, 215-219.
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Blau, ed., Walter de Gruyter, Berlin.
7. D.S. Duch, S.W. Bowers, J.H. Woolf and C.A. Nichol (1984) Life Sci. 35, 1895-1901.
8. S. Yoshioka, M. Masada, T. Yoshida, K. Inoue, T. Mizokami and M. Akino (1983) Biochim. Biophys.
Acta 756, 279-285.
9. G.K. Smith (1987) Arch. Biochem. Biophys. 255, 254-266.
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11. J.M. Chirgwin, A.E. Przybyla, R.J. MacDonald and W.J. Rutter (1979) Biochemistry 18, 5294-5299.
12. K. Hatakeyama, Y. Inoue, T. Harada and H. Kagamiyarna (1991) J. Bioi. Chern. 266, 765-769.
13. Y. Inoue, Y. Kawasaki, T. Harada, K. Hatakeyama and H. Kagamiyarna (1991) J. Bioi. Chern. 266,
20791-20796.
14. B.A. Citron, S. Milstien, J.C. Gutierrez, R.A. Levine, B.L. Yanak and S. Kaufman (1990) Proc. Nat!.
Acad. Sci. USA 87, 6436-6440.
15. M. Shahbaz, J.A. Hoch, K.A. Trach, J.A. Hural, S. Webber and J.M. Whitley (1987) J. Bioi. Chern.
262, 16412-16416.
16. K. Hatakeyama, T. Harada, S. Suzuki, Y. Watanabe and H. Kagamiyarna (1989) J. Bioi. Chern. 264,
21660-21664.
17. M. Gutlich, K. Schott, T. Werner, A. Bacher and I. Ziegler (1992) Biochim. Biophys. Acta 1171,
133-140.
18. J. Maier, K. Schott, T. Werner, A. Bacher and I. Ziegler (1993) Exp. Cell Res. 204, in press.
19. H.H.M. Dahl, W. Hutchinson, W. McAdam, S. Wake, F.J. Morgan and R.G.H. Cotton (1987) Nucl.
Acids Res. 15, 1921-1932.
20. T. Sueoka and S. Katoh (1982) Biochim. Biophys. Acta 717, 265-271.
21. S. Webber, T.L. Deits, W.R. Snyder and J.M. Whitley (1978) Anal. Biochem. 84, 491-503.
22. E.R. Werner, G. Werner-Felmayer, D. Fuchs, A. Hausen, G. Reibnegger, J.J. Yim, W. Pfleiderer
and H. Wachter (1990) J. Bioi. Chern. 265, 3189-3192.

226
CO-INDUCTION OF TETRAHYDROBIOPTERIN (BH 4) LEVELS AND

TYROSINE HYDROXYLASE ACTIVITY IN CULTURED PC12 CELLS

Panagiotis Z. Anastasiadis1' 2, J. Christopher States3, Donald M. Kuhn 2, and

Robert A. Levine 1·2.4

1William T. Gossett Neurology Labs, Henry Ford Hospital, Detroit, Ml

2 Cellularand Clinical Neurobiology Program, Department of Psychiatry,
Wayne State University, Detroit, Ml
3 Center for Molecular Biology, Wayne State University

4Veterans Administration Medical Center, Allen Park, Ml

INTRODUCTION

BH 4 is the cofactor for phenylalanine hydroxylase, as well as tyrosine and

tryptophan hydroxylases, which are the rate-limiting enzymes in catecholamine (CA) and
serotonin synthesis, respectively. GTP cyclohydrolase (GTP-CH) and sepiapterin reductase
(SR) catalyze the initial and final steps in BH 4 biosynthesis, respectively. Because no
intermediates are detected, GTP-CH is thought to be the rate-limiting enzyme in non-
primate BH 4 biosynthesis. In humans, the existence of neopterin in fluids suggests that
other enzymes following GTP-CH may also play a regulatory role in BH 4 biosynthesis.
CA depletion elevates tyrosine hydroxylase (TH) and GTP-CH activities, as well as
BH 4 1evels in rat adrenal medulla1' 2 , suggesting that "coordinate regulation" ofTH and BH 4
biosynthetic enzymes may occur. Coordinate regulation refers here to a coordinated
response in the long-term induction of TH, GTP-CH and SR activities when BH 4 and/or
CA metabolism is altered. It is possible that a co-induction of TH and BH 4 -related enzyme
activities is crucial for maintaining increased tyrosine hydroxylation in vivo.
Cultured rat pheochromocytoma (PC12) cells3 were used to test the co-induction
of GTP-CH, SR, and TH following drug treatments influencing BH 4 and CA metabolism.
Diamino-hydroxypyrimidine (DAHP) and N-acetyl-serotonin (NAS) were used to inhibit
GTP-CH and SR, respectively, and decrease PC12 cell BH 4 1evels4 • BH 4 was added to test
for saturation of TH in situ. Vasoactive intestinal peptide (VIP) was used to induce TH
gene expression and increase TH activity5 • Nerve growth factor (NGF) was used to induce
BH 4 metabolism and increase BH 4 levels6 •

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et at., Plenum Press, New York, 1993 227
MATERIALS AND METHODS

Cell culture

PC12 cells were maintained in tissue culture flasks in a humidified (90o/o)

atmosphere of 95o/o air and So/o C02 at 37°C. The medium consisted of Dulbecco's
Modified Eagle's Medium (DMEM; Gibco, 4500 mg!L glucose with L-glutamine), 44 mM
sodium bicarbonate and 24 mM HEPES, 7o/o heat-inactivated horse serum, 7o/o fetal bovine
serum, and Penicillin-streptomycin (1 o/o v/v of 10,000 U/ml penicillin solution and 10,000
J.lg/ml streptomycin).
Cells were harvested by mechanical dislodgment and collected by centrifugation
at R.T. (7 min, 900 x g). The supernatant was removed by aspiration and the cell pellet
was washed once with prewarmed (37°C) HEPES-buffered Krebs-Ringer solution (KRH):
125 mM NaCI, 4.8 mM KCI, 2.6 mM CaCI 2 , 1.2 mM MgS04 , 10 mM glucose, 25 mM
HEPES adjusted to pH 7.4 with 1 M Tris base), 1.0 mM ascorbic acid and 1.0 mM
KH 2 P04 • Cell viability was determined by dye exclusion (0.01 o/o Trypan blue, viability
always >90o/o) and cells were resuspended to the desired final concentration of cells in
appropriate buffer solutions.

Tissue Homogenization

PC12 cells were cultured in 25 cm 2 flasks (12 X 104 cells/cm 2 ), dislodged with
Versene (Gibco), centrifuged at 900 x g for 15 min, and then the pelleted cells were
homogenized in 50 mM Tris-HCI pH 7.4. The homogenate was split immediately for
preparation of each assay. For determination of BH 4 and CA levels, cells were cultured
in 24-well plates at the same plating density, washed with Dulbecco's Phosphate-Buffered
Saline and homogenized as previously.

Enzyme activity assays

TH activity was measured by monitoring the release oflHOH from 3,5-3 H-tyrosine
as previously described 7• Saturating conditions for measuring TH Vmax were 100 JiM
tyrosine and 1 mM BH 4 • GTP-CH activity was measured by monitoring the conversion of
GTP to NH 2 TP 8 • 1 mM GTP was used to saturate GTP-CH. Neopterin was measured by
H PLC with fluorescence detection 9 after oxidation and dephosphorylation of N H2 TP. SR
activity was measured by monitoring the conversion of sepiapterin to dihydrobiopterin 10,
using HPLC with fluorescence detection to measure biopterin, following manganese
dioxide oxidation of dihydrobiopterin. Saturating conditions for measuring SR Vmax were
250 JiM sepiapterin and 100 JiM NADPH.

Measurement of pterins, catecholamines, and metabolites

BH 4 1evels were measured as biopterin by HPLC with fluorescence detection after

acid oxidation 9 • BH 4 levels were also determined directly by HPLC with electrochemical
detection 9 • 3,4-dihydroxyphenylalanine (DOPA), dopamine (DA), homovanilic acid,
dihydroxyphenylacetic acid, norepinephrine and epinephrine were measured in a single
chromatographic run by HPLC with electrochemical detection8 • Protein was measured by
the method of Bradford 11 •

228
 BH 4 and biopterin were purchased from Schirks Labs (Switzerland). NGF (2.5S)
was purchased from Collaborative Res. Inc.. VIP (porcine) was obtained from Bachem
(California). All other chemicals and reagents were of the highest grade available from
commercial suppliers.
Statistical analysis was carried out by one-way analysis of variance. Post hoc
comparisons were made with the Newman-Keuls test.

RESULTS AND DISCUSSION

The inhibitory effect of NAS, and DAHP on BH 4 biosynthesis in PC12 cells was
tested following a 24 hour incubation with varied concentrations of either NAS or DAHP.
Both NAS and DAHP maximally depleted PC12 cells of BH 4 at a concentration of 2 mM.
At this concentration, both compounds caused significant reduction of the cell number
(approximately 60% of control), which could be due either to cellular toxicity or inhibition
of cellular proliferation. NGF caused a 2.5-fold increase in intracellular BH 4 Ievels. DAHP
blocked the NGF-induced increase and depleted endogenous BH 4 • The NGF-induced
BH 4 levels were reduced by NAS but depletion of intracellular BH 4 levels was not
achieved even at high NAS concentrations (2 mM). 94 o/o of total intracellular biopterin
was present as BH 4 • Control biopterin levels were 2.57 ± 0.17 pmoles/1 05 cells.
The effect of BH 4 on PC12 cell CA synthesis was then studied. PC12 cells were
incubated in media containing 300 J.lM BH 4, 1 mM NAS (suboptimal concentration for
inhibition of BH 4 synthesis), or both for 2 hours, and then intracellular and extracellular
CA levels were measured. Significant changes (p< 0.01) were seen only in the
intracellular levels of DOPA and DA (table 1). BH 4 increased both DOPA (not detectable
in control cells) and DA levels. NAS (1 mM) inhibited control CA synthesis in PC12 cells,
and this inhibition was reversed by the addition of BH 4 in the incubation media.

Table 1. Effect of exogenous BH 4 on intracellular catecholamine levels+ of control or

NAS-treated cultured PC12 cells after 2 hours.

DOPA (pmoles/1 06 cells) DA (pmoles/1 06 cells)

Control below det. limits 2913.63 ± 149.13

300J.1M BH 4 250.64 ± 18.32 4659.36 ± 4.39

1mM NAS below det. limits 2429.44 ± 82.54

NAS + BH 4 427.07 ± 12.43 3695.86 ± 103.41

+values represent the mean ± SEM (n = 6)

The effects of NGF (50 nglml), or VIP (10 J.lM) on intracellular BH 4 Ievels were also
tested in PC12 cells. As mentioned previously, NGF caused a 2 .5-fold increase in
intracellular BH 4 levels. Treatment of PC12 cells with VIP increased intracellular BH 4
levels 4-fold.
SR, GTP-CH and TH activities were determined in the supernatant of PC12 cell
homogenates following a 24 hour incubation with tested compounds at 37°C. Incubation
of cells with NGF increased both GTP-CH and THin vitro activities 2-fold. VIP increased

229
GTP-CH activity 2-fold and TH activity 3-fold. SR activity was not altered by any
treatment. Control values for SR, GTP-CH, and TH, activities were 12.57 ± 3.3, 1.60 ±
0.26, and 22.1 ± 3.6 pmoles/mg protein/min, respectively.
According to the hypothesis of "coordinate regulation", conditions causing the long-
term activation of TH will also elevate the activity of BH 4 biosynthetic enzymes to provide
more BH 4 when elevated CA synthesis is required. Insufficient evidence exists supporting
the hypothesis of coordinate regulation. The co-induction of TH and BH 4 -related enzyme
activities was tested as a first step in the elucidation of the regulatory mechanisms involved
in the long-term induction of BH 4 - and CA-related enzymes. Our results (table 1) suggest
that TH is subsaturated with BH 4 in PC12 cells and confirmed a previous report by
Brautigam et al. 4 • Regarding the co-induction of TH and BH 4 -related enzymes, our results
suggest that the long-term activation of TH and GTP-CH may be coupled in vivo. Because
GTP-CH is believed to be the rate-limiting enzyme in BH 4 biosynthesis in non-primates,
an induction of GTP-CH should result in increased intracellular BH 4 levels without
requiring the induction of the other BH 4 biosynthetic enzymes. It is possible that a
transient increase in SR activity occurs before or after the time tested (24 hours). Future
studies are needed to determine whether this co-induction is coordinately regulated. It
is also necessary to test whether this co-induction occurs at the level of TH and GTP-CH
gene transcription, or at the post-transcriptional or post-translational levels.

Acknowledgements

P.Z. Anastasiadis is supported by the Bodosaki Foundation, Athens, Greece. R.A.

Levine is supported by NIH grants AG10687-01 and NS28800-01.

REFERENCES

1) Abou-Donia, M.M., Wilson, S.P., Zimmerman, T.P., Nichol, C.A., and Viveros,
O.H.: J. Neurochem., 46: 1190-1199, 1986.
2) Baruchin, A., Weisberg, E.P., Miner, L.L., Ennis, D, Nisenbaum, L.K., Naylor, E.,
Stricker, E.M., Zigmond, M.J., and Kaplan, 8.8.: J. Neurochem., 54: 1769-1775,
1990.
3) Greene L.A. and Tischler A.S.: Proc. Natl. Acad. Sci. USA, 73: 2424-2428, 1976.
4) Brautigam, M., Dreesen, R., and Herken, H.: Journal on Neurochemistry, 42: 390-
396, 1984.
5) Wessels-Reiker, M., Haycock, J.W., Howlett, A.C., and Strong, R.: The Journal of
Biological Chemistry, 266: 9347-9350, 1991.
6) Suzuki H, Nakanishi N, Yamada S: Biochem. Biophys. Res. Comm., 153: 382-387,
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8) Blau, N. and Niederwieser, A.: Anal. Biochem. 128: 446-452, 1982.
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Exp. Ther., 242: 514-522, 1987.
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1224, 1990.
11) Bradford M.M.: Analyt. Biochem. 72, 248-254, 1976.

230
LONG-TERM TREATMENT OF PC12 PHEOCHROMOCYTOMA WITH
DIDUTYRYL CYCLIC AMP INCREASES BIOPTERIN CONTENT IN THE CELLS
BUT DECREASES THAT IN THE MEDIUM

Nobuo Nakanishi, Satoshi Onozawa, Akiyoshi Isono, Masayuki Hara,

Hiroyuki Hasegawa', and Shozo Yamada

Department of Biochemistry, Meikai University School of Dentistry, Sakado,

Saitama 350-02, Japan, and 'Department of Bioscience, Nishi-Tokyo
University, Uenohara, Yamanashi 409-01, Japan.

INTRODUCTION

Pheochromocytoma PC12 is known to be a nerve growth factor (NGF)-responsive

cell line: upon the addition of NGF, cells are induced to differentiate into sympathetic
neuron-like cells from the chromaffin cell-like phenotype'. Tetrahydrobiopterin (B~) and
total biopterin (BP) contents and GTP cyclohydrolase activity in PC12 and PC12h cells, a
subclone of PC12, were increased by the treatment with NGF2-5• Other agents which
elevate the intracellular cAMP such as dBcAMP, forskolin and cholera toxin were also
able to show the similar effect as NGF2-6 • Although both NGF and dBcAMP increased the
cellular BP content and the cyclohydrolase activity, they showed the quite different effect
on DA level of PC12h cells: dBcAMP stimulated the DO production but unexpectedly
decreased the cellular DA level of the subclone7•8• In both PC12 and PC12h cells, DA
content in the medium was extremely increased by dBcAMP treatment. We found that in
both cell lines vesicular membrane transport of monoamines was inhibited by the elevation
of intracellular cAMP thus increasing the outward flow ofDA and inhibiting its reuptake 8·*.
An aim of the present study is to test whether cAMP also affects on outward or
inward flow of BP4 through plasma membranes. We examine effect of dBcAMP on both
the cellular and medium BP levels. BP content in the medium of PC12 cells was decreased
by dBcAMP treatment in spite of the increase in its cellular content, indicating that the
agent somehow interferes the BP efflux, and suggesting a possibility that cAMP is the
messenger for regulating both the intracellular and extracellular BH4 levels in vivo.

METHODS

PC12 cell culture: PC12 cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 7% fetal bovine serum, 7% horse serum, 100 units/ml penicillin, and
100 J..l.g/ml streptomycin (DMEM(+++)l. For the experiment cells were seeded in 12

*Nakanishi, N., Onozawa, S., Matsumoto, R., Hasegawa, H., and Yamada, S. (manuscript in preparation)

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 231
well-plates and dBcAMP treatment was started one or two days after seeding.
BP measurement: BP in PC12 cells cultured in 12-well plates were extracted with
250 )ll of 0.2 M perchloric acid (PCA). Protein was precipitated by centrifugation (15,000
rpm, 15 min). Total BP in the supernatant was detemined according to Fukushima and
Nixon10• The precipitated protein was dissolved in 1 M NaOH and used for protein
determination by Bradford's method11 • To measure the medium BP content, 400 )ll of the
culture medium was taken from a well and briefly centrifuged (10,000 rpm, 30 sec) to
remove floating cells. A 300 )ll aliquot of the supernatant was mixed with 75 )ll of 1 M
PCA. Precipitated protein was removed and the supernatant was used for BP determination.
DA and DO measurement: DA and DO in PC12 cells were determined by using
HPLC/electrochemical detector (Coulochem Model5100A) as previously described7•8• DA
and DO in the medium were analyzed after partially purified and concentrated by using
alumina oxide according to Koshimura et al. 12

RESULTS AND DISCUSSION

We examined the effect of dBcAMP treatment on BP levels in PC12 cells and in

the medium (Fig. 1). Cellular BP content was increased by the long-term dBcAMP
treatment. On the other hand, dBcAMP unexpectedly decreased the BP content in the
culture medium (Fig. 1-a). At the early phase in the time course of dBcAMP treatment
both the cellular and medium BP contents were higher in the treated culture than in the
control culture. However, cells were incubated with dBcAMP for more than 3 to 5 hours,
the medium BP content in such culture became lower than that in the control culture,
although the cellular BP content in dEcAMP-treated culture was still increasing (not
shown). The results suggest that dBcAMP somehow inhibits BP transport (or leakage) out
to the extracellular fluid.

**** ****
300 1200

'2' '2
'Cii
·a;
e
a_
e
a_
Cl
Cl
E 200 E
..... 800
.....
.s
Cl
.s
Cl

E E
l!:l 2c:
c: 0
0 (,)
(,)
<(
a.. 100
Cl
400
co

................
Cant dB
Cell
..............
Cant dB
Medium
. Cant dB
Cell
Cant
Medium
dB

Figure 1. Effect of dBcAMP treatment of PCI2 pheochromocytoma on BP (a) and DA (b) contents in the
cells and medium. PC12 cells in 12 well plates were cultured for 16 hrs in DMEM (+++) (1 ml/well) in the
absence (Cant) or presence of 1 mM dBcAMP (dB). Values are the means±S.E. (n=6). Different from each
control value at ****P<O.OOl and *P<0.05 (Student's t-test).

232
 Since dEcAMP treatment of PC12 cells resulted in the great increase in dopamine
(DA) in the medium by more than 40-fold of control level (Fig. 1-b), and furthermore
aromatic amino acid precursors of monoamines were thought to have an effect on the
regulation of EP biosynthesis 13, we examine the effects of Tyr, Trp, Phe, dopa (DO), DA,
norepinephrine (NE), and epinephrine (EP) (100 11M each) on EP levels of PC12 culture.
None of those gave significant change in the cellular EP, while the medium EP level was
lowered by DO and catecholamines. DA was most potent among those and DO was less
effective than catecholamines. In this experiments, DA content in the cells incubated with
DO was far higher than that in the cells incubated with DA itself, suggesting that DA in
the extracellular fluid rather than intracellular one is crucial for inhibiting EP transport out
from the cells. As shown in Fig. 2, DA concentration-dependently decreased the medium
EP content without affecting its cellular content. These results suggest that dEcAMP
effect lowering the medium EP content of PC12 cells is, at least partially, due to its action
to raise the DA (and DO) content in the medium.
However, molar concentrations of DA in the medium of control and dEcAMP-treated
cells in the experiments for Fig. 1 were calculated to be 0.037 11M and 1.5 11M, respectively.
These values seemed to be somewhat smaller to fully explain the dEcAMP effect with the
consideration of the concentration-dependency curve of DA effect (shown in Fig. 2). Thus
there is a possibility that dEcAMP-induced decrease in the medium EP is also caused by
other mechanisms. We examined a presence of EP binding protein(s) which would be
induced by the dEcAMP treatment of PC12 cells and would explain the dEcAMP effect on
the medium EP. We have not obtained yet positive results supporting the presence of such
protein. Alternatively, it is also possible that the decrease in medium EP is caused by
some metabolite(s) derived from catecholamines or DO which may be produced much
more by the dEcAMP-treated culture than the culture incubated with catecholamines.

120

100

~
~

-
E so
Q)
1:
8
a.. 60
CD
Q)
>
'fij 40
Qj
0:

20

0
0 10 100 1000
DA concentration (!lM)

Figure 2. Effect of DA concentration on the cellular and medium BP contents of PC12 cells. PC12 cells
cultured in 12-well plates were washed twice with 0.5 ml of Hanks balanced salt solution (HBS), and then
the cells were incubated with 0.5 ml of HBS containing various concentrations of DA for 60 min in a C02
incubator. Values are the means±S.E. of triplicate experiments, and represented as the percent of each
control value. The cellular and medium BP contents obtained in the absence of DA (control value) were
104±2.68 ng/mg protein and 20.0±0.801 ng/mg protein, respectively.

233
 On the other hand, whether catecholamines influence the efflux of BP should also
be proven, since unlike dBcAMP they do not give significant changes in the cellular BP
content. Therefore, it seems unlikely but can not be neglected a possibility that catecholamine
does not affect BP flux but inhibits its synthesis needed to keep the medium BP level
without affecting the cellular content. Contrarily, it is also interesting that the cellular BP
content tends to be maintained in a certain level when the medium content is decreased by
the catecholamine addition to even 20% or less (Fig. 2).
Although mechanism by which dBcAMP lowers the outward flow of BP from the
cells is yet to be elucidated, whatever it is cAMP effect can influence both the intracellular
and extracellular levels of BP. This is important because on the one hand cAMP is one of
the major second messengers for the regulation of cell physiology by various signaling
molecules and on the other hand it has been reported new functions for tetrahydrobiopterin
such as serving as cofactor for nitric oxide synthase14' 15 , stimulating release of various
neurotransmitters in brain16•17 , and stimulating proliferation of erythro-leukemia cells 18' 19,
suggesting the actions of BH4 from the extracellular fluid and that of BH4 in cells not
producing BH4 by themselves.

REFERENCES
1. Greene, L. A. and Tischler, A. S. Adv. Cell. Neurobiol. 3:373 (1982)
2. Suzuki, H., Nakanishi, N. and Yamada, S. Biochern. Biophys. Res. Cornrnun., 153:382
(1988)
3. Nakanishi, N., Onozawa, S., Iwanaga, M., Akatsuka, I., Sawada, E., Asaumi, R.,
Hasegawa, H. and Yamada, S. 1.Meikai Univ.Sch.Dent. 19:197 (1990)
4. Nakanishi, N., Suzuki, H., Aono, K. and Yamada, S. in: "Pterins and Biogenic Amines
in Neurology, Pediatrics and Immunology" N. Blau, H. C. Curtius, R. A. Levine, and
G. R. H. Cotton, eds., Lakeshore Publishing, Grosse Pointe, pp. 101-108 (1991)
5. Nakanishi, N., Aono, K., Suzuki, H. and Yamada, S. in: "Pterins and Biogenic Amines
in Neurology, Pediatrics and Immunology" N. Blau, H. C. Curtius, R. A. Levine, and
G. R. H. Cotton, eds., Lakeshore Publishing, Grosse Pointe, pp. 109-117 (1991)
6. Woolf, J. H., Nichol, C. A., and Duch, D. S. (1986) in "Chemistry and Biology of
Pteridines 1986" B. A. Cooper, and V. M. Whitehead, eds., Walter de Gruyter, Berlin
New York, pp. 283-286 (1986)
7. Nakanishi, N., Onozawa, S., Matsumoto, R., Nakamura, M., Imagawa, S. and Yamada,
S. Pteridines 3:67 (1992)
8. Nakanishi, N., Onozawa, S., Kamei, M., Kawai, K., Hasegawa, H., and Yamada,S.
Pteridines (in press)
9. Nakanishi, N., and Guroff, G. 1. Biol. Chern. 260:7791 (1985)
10. Fukushima, T., and Nixon, J. C. Anal. Biochern. 102:176 (1980)
11. Bradford, M. Anal. Biochern. 72:248-254 (1976)
12. Koshimura, K., Miwa, S., Lee, K., Fujiwara, M., and Watanabe, Y. 1. Neurochern.
54:1391 (1990)
13. Yamaguchi,T., Nagatsu,Y., Sugimotomoto,T., Matsuura,S., Kondo,T., Iizuka,R., and
Narabayashi, H. Science 219:75 (1983)
14. Tayeh, M.A., and Marietta, M.A. 1. Biol. Chern. 264:19654 (1989)
15. Kwon, N. S, Nathan, C. F., and Stuehr, D. J. 1. Biol. Chern. 264:20496 (1989)
16. Koshimura, K., Miwa, S., Lee, K., Fujiwara, M., and Watanabe, Y. 1. Neurochern.
54:1391 (1990)
17. Mataga, N., Imamura, K., & Watanabe, Y. Brain Res. 551:64 (1991)
18. Tanaka, K., Kaufman, S., and Milstein, S. Proc. Natl. Acad. Sci USA 86:5864 (1989)
19. Kerler, F., Ziegler, 1., Schmid, C., and Bacher, A. Exp. Cell Res. 189:151 (1990)

234
NICOTINIC CHOLINERGIC REGULATION OF TETRAHYDROBIOPTERIN
LEVELS IN BOVINE ADRENAL CHROMAFFIN CELLS

Jack C. Waymire1, June E. Ayling2 and Gale L. Craviso3

1Department of Neurobiology and Anatomy, University of Texas Medical

School, Houston, TX 77030, 2Department of Pharmacology, University of
South Alabama College of Medicine, Mobile, AL 36688 and 3Department
of Pharmacology, University of Nevada School of Medicine, Reno, NV
89509

INTRODUCTION

Catecholamines (CA) in adrenergic neurons are maintained at a relatively constant

level despite wide variations in neural activity 1• The mechanism of this regulation is the
modulation of the conversion of tyrosine to dopa, the rate limiting step in CA biosynthesis.
The enzyme catalyzing this step, tyrosine hydroxylase2 (TH; EC 1.14.16.2; !-tyrosine,
tetrahydropteridine:oxygen oxidoreductase, 3-hydroxylating), is subject to a variety of
regulatory mechanisms. Acutely, TH is regulated by negative feedback inhibition of TH
by intracellular CA3 and activation through phosphorylation of TH by four second
messenger-activated protein kinases4• At the chronic level the quantity of THis regulated
through trans-synaptic mediated increase in the synthesis of enzyme molecules5•
An area of investigation that has received less attention with respect to CA synthesis
regulation is the modulation of the level of the TH cofactor, tetrahydrobiopterin (BH4). TH
uses molecular oxygen and reduced BH4 to hydroxylate tyrosine. Because intracellular BH4
is below the concentration required to saturate TH, its modulation is a good candidate for
the regulation of CA synthesis6• In support of this idea are the observations that exogenous
cofactor enhances dopamine synthesis in rat striatum7•9, in the isolated hypogastric
nerve-vas deferens preparation 10, in isolated chromaffin cells 11 , and that the BH4 level in
sympathetic tissues increases during stress 12 •
In the course of examining the influence of cholinergic stimulation on the regulation
of CA synthesis capacity in isolated chromaffin cells we discovered that the BH4 level is
regulated by nicotinic cholinergic receptor stimulation. In the studies presented here we
report that: 1) In the absence of cholinergic receptor stimulation the BH4 level falls in
isolated chromaffin cells soon after the cells are placed in culture, 2) In the presence of
cholinergic stimulation the BH4 level is not only maintained, but increases several fold over
initial values, and 3) The cholinergic influence on BH4 is blocked by protein synthesis
inhibitors indicating that the mechanism of these changes requires the synthesis of
regulatory protein(s).

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 235
METHODS

Isolated bovine adrenal chromaffin cells were prepared, purified and maintained in
suspension culture as described previously13 • Cells were treated by adding concentrated
solutions of drugs directly to the culture medium. To analyze TH activity cells were lysed
by freeze-thawing in hypotonic phosphate buffer (pH 6.8) containing 0.1% triton X-100,
and an aliquot assayed as previously described14 • To analyze in situ dopamine synthesis
rate, cells were harvested, preincubated in a balanced salt solution for 60 min at 37• C and
assayed for dopamine synthesis rate by assessing the conversion of 14C-carboxyl labeled
!-tyrosine to dopamine and 14C02 as previously described15 • BH4 and biopterin were
measured by lysing the cells in argonated H20 using three freeze-thaw cycles and then
adding an equal volume of argonated 1 M trichloroacetic acid. Following centrifugation
at 12,000 x g for 10 min, aliquots were injected onto an Adsorbosphere C-18 column
equilibrated in 50 mM ammonium formate, pH 3.3, containing 600 mg/L octane sulfonic
acid. BH4 was quantitated by electrochemical detection (0.3V vs Ag/AgC1 2) and biopterin
by fluorescence detection (Ex/Em = 367/450 nm).

RESULTS AND DISCUSSION

We previously reported that chronic treatment of isolated chromaffin cells with the
non-metabolizable cholinergic nicotinic agonist dimethylphenylpiperzinium (DMPP) causes
between 1.5- and 2.5-fold increase in chromaffin cell TH protein and activity'4• This
induction of TH requires a ten fold lower DMPP concentration (1 J.IM) than is required for
stimulating CA secretion (10 J.IM) and is blocked by actinomycin D and cycloheximide.
These studies also demonstrated that in parallel with the increase in TH level, nicotinically
stimulated cells underwent a large increase in their capacity to endogenously hydroxylate
tyrosine to form DA. However the magnitude of the increase in hydroxylation capacity
was much greater than predicted by the magnitude of the change in the TH level'\ implying
that the induction of TH was only one of the mechanisms by which cholinergic stimulation
influences the CA synthesis capacity of the chromaffin cell. As neither the affinity of TH
for BH4 cofactor nor the activity of dopa decarboxylase were changed by the nicotinic
stimulation14 , an alternative explanation for the large increase in dopamine synthesis
capacity was that cholinergic stimulation influences the level of the TH cofactor, BH4•
We conducted a series of experiments to test the hypothesis that nicotinic stimulation
increases chromaffin cell BH4 levels. First we examined whether supplying the cells with
excess cofactor would negate the difference in fold stimulation of in situ dopamine
synthesis rate and in vitro TH level produced by chronic stimulation with 1 J.IM DMPP.
By pre-incubating the cells with the lipophilic analog, 6-cyclohexyltetrahydropterin prior
to assaying DA synthesis, we found that the difference in DA synthesis rates between
control and chronic DMPP-treated cells was reduced to a value close to the change in TH
level (the DMPP-induced fold increase without added pterin is 5.3 ± 0.78, whereas the
DMPP-induced fold increase with pterin present was 1.65 ± 0.13).
To directly test the hypothesis that BH4 level is regulated by cholinergic stimulation
we analyzed the level of biopterin and BH4 in control and cholinergically treated cells and
compared these results with changes in TH activity and dopamine synthesis capacity in
parallel cultures. As shown in Figure 1 A. and B. TH level and DA synthesis rates were
stable over the time in culture and DMPP caused a two fold increase in TH activity and
nearly 7-fold increase in dopamine synthesis as previously reported14•

236
 A
c:
] 80
ea. 0 Control
0>
E 60 e 1 f"M OMPP

" 0
a.
0
"0

0
40
o-•~
•----------·
E ----0
8 0 0
20
.P
·;;
:;:;
<t " 0
I 0
f- 2 4 6 8

Days In Culture.
c:
.E
B
"q;
18
!12
0 Control
"c: 15

~!
~ 12
e 1 f"M DMPP
.E

/f _________,
'::::- 9
0
E
8 6
(IJ
'in
Q) 3
:5c:
,._
o-o 0 0
(f)
0
0 4 6

,___,_, ,
<t
2 8
0
Days in Culture

c
!12
q;
u
80
0 Control ___
~
c:
60 • 1 f"M DMPP
.E
'::::- /
Q~~
0
E 40
8

-~o-~------2-9
(IJ
q; 20
>
Q)

...
_J

I
([)
0
0 2 4 6 8

Days in Culture

Figure 1. Time course analysis of the effect of nicotinic stimulation of bovine chromaffin cells on A. TH
activity, B. DA synthesis capacity and C. BH4 level. DMPP at a final concentration of 1 pM was added
to chromaffin cells at day 2 in culture. Data are mean ± S.D. of four cell culture samples

237
 With respect to BH4, two important observations were made. First, as shown in Figure
1 C. in the absence of 1 J.lM DMPP, the level of BH4 gradually decreased with time in
culture. Second, upon addition of DMPP, not only did BH4 fail to decrease, but BH4
content underwent a marked rise so that by day 5 of treatment the level was 2.5 fold
greater than when cells were placed in culture and 7-fold greater than non treated cells at
the same time point. Pre-treating the cells with cycloheximide abolished this effect (data
not shown), indicating that the mechanism involves ongoing protein synthesis. Further
analysis of basal BH4 levels showed that in freshly isolated cells levels are 30-50 pmol/106
cells and by day 9 this level falls to less than 5 pmol/106 cells. This decline was not
associated with a rise in either biopterin or BH2•
These results show that BH4 levels are regulated by nicotinic stimulation of chromaffin
cells and that the nicotinic influence on cofactor content could be responsible for the
marked increase in the rate of dopamine production in response to prolonged DMPP
stimulation. The observation that isolated chromaffin cells maintain a constant
concentration of TH, but not BH4, raises the possibility that basal levels of TH protein and
its cofactor are differentially regulated in vivo and that the maintenance of BH4 is highly
dependent on synaptic activity. The observation that dopamine synthesis in the cultured
chromaffin cells is constant with time in culture even though BH4 level drops is an
unexpected finding in these studies that remains to be explained.

ACKNOWLEDGMENTS

Supported by NIH grants NS11061, NS26662, and NS27550.

REFERENCES

1. U. S. von Euler, R. Luft, and T. Sundin, Acta Physiol. Scand. 34, 169 (1955).
2. T. Nagatsu, M. Levitt, and S. Udenfriend, J. Bioi. Chem. 239, 2910 (1964).
3. S. Spector, R. Gordon, A. Sjoerdsma, and S.Udenfriend, Mol. Pharmocol. 3, 549 (1967).
4. J. W. Haycock, J. Bioi. Chern. 265, 11682 (1990).
5. R. A. Mueller, H. Thoenen, and J. Axelrod, J. Pharmacol. Exp. Ther. 196, 74 (1969).
6. R. A. Levine, L. P. Miller, and W. Lovenberg, Science 214, 919 (1981).
7. R. Kettler, G. Bartholini, and A. Pletscher, Nature (Lond. ) 249, 476 (1974).
8. S. Miwa, Y. Watanabe, and 0. Hayaishi, Arch. Biochem. Biophys. 239, 234 (1985).
9. N. Mataga, K. Imamura, andY. Watanabe, Brain Res. 551, 64 (1991).
10. M. Boadle-Biber, and R. H. Roth, Br. J. Pharmacal. 46, 696 (1972).
11. M. B. Abou-Donia, S. P Wilson, T. P. Zimmerman, C. A. Nichol, and 0. H. Viveros, J.
Neurochem. 46, 1190 (1986).
12. 0. H. Viveros, C. -L. Lee, M. M. Abou-Donia, J. C. Nixon, and C. A. Nichol, Science 213, 349
(1981).
13. J. C. Waymire, W. F. Bennett, R. Boehme, L. Hankins, K. Gilmer-Waymire, and J.W. Haycock, J.
Neurosci. Meth. 7, 329 (1983).
14. G. L. Craviso, V. B. Hemelt, and J. C. Waymire, J. Neurochem. 59, 2285 (1992).
15. J. C. Waymire, J.P. Johnston, K Hummer-Lickteig, A. Lloyd, A. Vigny, and G.L. Craviso, J. Bioi.
Chem. 263, 12439 (1988).

238
INTER-RELATIONSHIPS BETWEEN PTERINS AND CYTOKINES PRODUCED
DURING ALLOGENEIC IMMUNE REACTIONS AND POSSmLE USE AS EARLY
MARKERS OF IMMUNE ACTIVATION

J.J. Rippin and D.C. Henderson

Department of Immunology, Chacing Cross & Westminster Medical School

University of London, 369 Fulham Rd, London SWlO 9NH, UK

ABSTRACT

The pterins neopterin and xanthopterin have been measured together with the
cytokines IFN-y, TNF-a and TNF-~ in the supernatants of 20 allogeneic mixed lymphocyte
reactions. We have calculated rank correlation coefficients between these analytes and the
immune response. Our fmdings confirm that neopterin is a sensitive early marker of
immune activation and show that another pterin, xanthopterin could be used likewise.
Cytokine production was also proportional to immune activation but none could be used
consistently as early markers of immune activation.

INTRODUCTION

The pterin D-neopterin has proved to be a useful marker of immune activation, both
clinically and experimentally1• In vitro, monocytes have been identified as the principal
source, secreting D-neopterin in response to stimulation with the cytokine interferon-y
(IFN-y) 2•3• D-neopterin is a pterin metabolite of guanosine triphosphate in the synthetic
pathway of biopterin which is an essential cofactor in neurotransmitter synthesis4• D-
neopterin has been measured routinely by radioimmunoassay, however this method does
not permit the measurement of other pterins. To investigate the production of pterins during
immune activation we have developed a High Performance Liquid Chromatography (HPLC)
method for the measurement of pterins, including neopterin in serum and cell-culture
supernatants. We have used the in vitro, allogeneic mixed lymphocyte response (MLR) as
our model of immune activation. Cytokines are known to be intimately involved in the
initiation, development and augmentation of allogeneic immune responses5•6• IFN-y may
be involved in augmenting the allogeneic response through cell recruitment, activation and
proliferation, particularly macrophages, cytolytic T cells and natural killer (NK) cells, and
through the up-regulation of HLA class II expression7•8 • TNF is a major cytokine involved
in tissue damage, and appears to be involved in cell recruitment and activation.
In this study we have investigated neopterin and xanthopterin produced during in vitro
allogeneic immune reactions together with the production of IFN-y, TNF-a and TNF-b.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 239
MATERIALS AND METHODS

PBMC preparation

Heparinized human blood from healthy volunteers was centrifuged to obtain plasma
and the cells resuspended to their original volume in RPMI 1640 (Flow Laboratories).
PBMC were obtained by centrifuging the cells over lymphoprep (Nycomed, Birmingham,
UK) at 200g for 35 minutes at room temperature. The PBMC recovered were washed three
times in RPMI 1640 and resuspended in TCM at 5 x 106 viable cells/ml. Viability was
assessed by dye exclusion.

Mixed lymphocyte cultures

Four groups of five one-way MLR were performed by mixing equal numbers of
PBMC from one volunteer (responder) with X-irradiated (30 Gy) PBMC from a second
volunteer (stimulator) in 10 m1 cultures in TCM. Each group contained a control of a
mixture of the responder cells and the responder's own irradiated cells. The MLR's were
confirmed by thymidine incorporation (0.5 J!Ci added for final 18 hours in culture) in
parallel 200 J!l volume cultures in 96-well plates at 6 days. 2 m1 alliquots of the supernatant
of each MLR (including controls) were taken at 0, 2, 3, 4 and 5 days after culture,
centrifuged at 2500 rpm and supernantants removed and stored at -70°C. Alliquots were
later analysed for IFN-y, TNF-a, TNF-~ and pterin profile.

Cytokine assays

IFN-y ,TNF-a and TNF-~ were measured by ELISA using paired monoclonal and
polyclonal antibodies and standards kindly donated by Dr A Meager, NIBSC, Herts, UK.

HPLC analyses of pterins

Pterins were measured by HPLC as described previously9 with the following

modifications. Mobile phase was 30 mmol/1 potassium phosphate buffer pH 7.25 with a
flow-rate of 0.8 ml/min: 100 J!l 80 nmol/1 6-biopterin was used in place of 6-methyl pterin
as internal standard and 50 J!l extract was injected onto the column.
Pterins are light-sensitive and thus controls, standards and samples were kept in the
dark and stored at -70° C.

RESULTS

MLR: Thymidine incorporatation at 6 days showed that positive MLR were obtained
between all four responders and each stimulator combination. For members of each group,
an MLR stimulation index (si) was derived using the formula:

si = thymidine uptake of MLR on day 6 of MLR

thymidine uptake of group control on day 6 of MLR

Cytokine production: Cytokine levels showed a progressive increase over the culture
period with MLR levels exceeding that of their controls.

Pterin production: Neopterin production mirrored that of xanthopterin in the the MLR
with levels consistently greater than that of the the controls.

240
'Analyte indices' (AI) were calculated for each analyte at time points 3, 4 and 5 days
where:

analyte index = level of analyte in MLR at time t

level of analyte in group control at time t

Mean levels and one standard deviation of all AI's were derived for each MLR at each
time point, and are shown in Table 2. Spearman's rank correlation were calculated for each
analyte at 120 hours against the corresponding stimulation index (Table 3). All statistics
were calculated using SPSSx version 4.0 (SPSS inc.) run on a SUN 4/670.

Table 1: Stimulator I Responder table for MLRs.

STIMULATOR Volunteer 1 Volunteer 2 Volunteer 3 Volunteer 4

RESPONDER

Volunteer 1 1.0 183.6 52.6 3.3

Volunteer 2 3.3 1.0 17.2 3.5

Volunteer 3 2.3 104.2 1.0 3.0

Volunteer 4 1.9 120.6 41.6 1.0

Volunteer 5 2.7 171.3 55.6 4.6

Table 2: Mean AI and one standard deviation for each analyte at all time points of MLR.

Time 0 days 2 days 3 days 4 days 5 days

Analyte Mean 1 SD Mean 1 SD Mean 1 SD Mean 1 SD Mean 1 SD

AI AI AI AI AI

IFN-y - - 1.08 0.19 4.11 4.20 6.70 8.20 6.60 8.70

TNF-a - - - - 1.30 0.81 3.80 4.64 11.35 13.60

TNF-~ - - - - 103 0.12 110 0.26 1.68 0.91

Neoptenn - - - - - - 2.80 0.99 2.28 1.30

Xanthoptenn - - 1.12 0.34 1.70 0.41 1.85 0.42 2.00 0.71

Table 3: Spearmans rank correlation (R) and P value for cytok:ine

and pterin levels at day 5 of MLR vs stimulation index (day 6).

Analyte vs si R p
INF-y vs si 0.3196 0.085

TNF-a vs si 0.0560 0.407

TNF-~ vs si 0.5417 0.007

Neopterin vs si 0.7780 0.001
Xanthopterin vs si 0.7044 0.004

241
DISCUSSION

Cytokines are powerful immunomodulatory peptides, mostly acting at the cell to cell
level and have short half lives in the circulation (minutes). Cytokine levels in the MLR
culture supernatants represent a complex dynamic equilibrium between the level of
production and consumption by increased levels of cell-membrane associated and soluble
receptors. This could account for the poor correlation between cytokine levels and MLR
si, and for the lack of consistent differentiation from group controls.
Conversely neopterin and xanthopterin are stable molecules which accumulate as non-
biogenic analytes in an in vitro closed system such as an MLR. Pterin levels consistently
differentiated from their group controls relatively early in the MLR, (xanthopterin 3 days
and neopterin 4 days) compared with the cytokines although the AI for xanthopterin at 3
days did not show a significant rank correlation with immune activation (si) on day 6 (r
= 0.26, P = 0.012). Xanthopterin levels could thus only give a qualatative value for
immune activation in the MLR at this time. Pterin levels at 5 days gave a more significant
rank correlation with stimulation index thus reflecting more accurately, levels of immune
activation. 7,8 dihydropterin derivatives are converted to 7,8 dihydroxanthopterin10 which
in turn is oxidised to xanthopterin under our assay conditions. Our results show a greater
yield of xanthopterin over neopterin, which appeared to follow neopterin levels
stoichiometrically.
Our results confirm that neopterin is an early in vitro marker of immune activation
and indicate that xanthopterin which is produced in greater quantities could be more
sensitive.

REFERENCES

1. C.Huber, D.Fuchs, A.Haussen, R.Margreiter, G.Reibnegger, M.Speilberger and H.Wachlter,

Pteridines as a new marker of to detect human T cells activated by allogeneic of modified self-
histocompatibity complex determinations, J.Immunol. 130:1047-1050(1983).
2. J.Troppmair, K.Nachbaur, N.Herold, W.Aulitsky, H.Tilg, G.Gastl, P.Bieling, B.Kotlan, R.Flener,
R.Mull, O.Aulitsky, H.Rokos and Ch.Huber, In vitro studies and in vivo studies on the induction of
neopterin biosynthesis by cytokines, alloantigens and lipopolysaccarides, Clin Exp Immunol.
74:392-397(1988).
3. D.C.Henderson, J.Sheldon, P.Riches and J.R.Hobbs, Cytokine induction of neopterin production,
Clin Exp Immunol. 74:392-397(1991).
4. H.Rembold, W.L.Gyure, Biochemistry of Pteridines, Angew Chern. 84: 1088(1972).
5. Da.Morgan, F.W.Ruscetti, R.Gallo, Selective in vitro growth of T-lymphocytes from human bone
marrow, Science. 193:1007-1010(1976).
6. F.R.Balkwill, Interleukin-2 and lymphokine activated killer cells, in F.R.Balkwill. Cytokines in
Cancer Therapy, Oxford Press. 88-110(1989).
7. S.Gillis, M.M.Ferm, W.Ou, K.A.Smith, T cell growth factor: parameters of production and a
quantative microassay for activity, J Immunol. 120:2027-2033(1978).
8. G.Trichieri, B.Perussia, Immune interferon: a pleiotropic lymphokine with multiple effects,
Immunology Today. 6:131-136(1985).
9. J.J.Rippin, Analysis of fully-oxidised neopterin in serum by High Performance Liquid
Chromatography, Clin Chern. 38:(9)1722-1724(1992).
10. H.Rembold, H.Metzger, W.Gutensohn, Catabolism of pteridine cofactors, Biochim Biophys Acta
230: 117-126(1971 ).

242
AN EXAMPLE OF THE DETECTION OF AN ESOPHAGEAL CARCINOMA
IN ITS VERY EARLY STAGE BY URINARY XANTHOPTERIN
DETERMINATION.

Teruhiko Iinol, Hiroe Watanabe\ W.L.Gyure2 , Toshio Mazda 3 , Hiroyoshi Mieno4 and
Motoo Tsusue5

1Department of General Education, Nihon University, Setegayaku, Tokyo, Japan

2Seton Hall University, South Orange, N.J., U.S.A.
3Japanese Red Cross Tokyo Metropolitan Blood Center

4School of Medicine, Kitasato University, Sagamihara City, Japan

5 School of Liberal Arts, Kitasato University, Sagamihara City, Japan

INTRODUCTION

Neopterin (NP) has been used as a biochemical marker of the activated state of
cell-mediated immunity and to monitor and screen for some clinical disorders1 - 4• In previ9us
papers s- 6 , we reported that xanthopterin (XP) also reflects the same type of immune status as
well as various types of liver disease. In the course of our study on the determination of
urinary pteridines, one of the authors (M. T.) happened to find that his urinary XP level was
higher than the normal range. Although his other tumor indicators such as CEA, SCC and
CA19-9 showed normal values, an abnormality was found in the squamous cells of his
esophagus after endoscopic inspection. In the present paper we compare longitudinal
changes in his urinary excretion levels of XP and NP for a period of eleven months before
and after surgery.

MATERIALS AND METHODS

Collection ofurines.
Urine samples were collected from healthy individuals and the patient at early morning
hours and kept frozen at -4o·c until analyzed.
Blood sample analysis.
Blood samples were taken from the patient at the time of urine collection. Serum tests
for liver disease and tumor markers were performed for the following; r -GT, AST, ALP,

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 243
CEA(DAINABOT), CA19-9 and SCC.
Urinary XP and NP assay
Urinary XP was measured applying the isolation method of Plasner and Kalckar 7 and
HPLC according to the method described by Mazda et al 5 • Urinary NP and biopterin (BP)
were assayed after iodine oxidation by the method of Nakagoshi et al 8 The values were
expressed as J..L mole pteridines/mole creatinine.

RESULTS AND DISCUSSION

Blood sample analysis.

Values of several indicators in the serum of this patient were measured for a period of
eleven months. Liver disease marker values such as -r-GT, AST and ALP were 21-35
IU/L (normal value 6-60), 16-24IU/L (normal value 8-28) and 211-262 IU/L (normal value
73-248), respectively.

Table 1. Tumor Marker Values in Patient.

Oct.ll Jan.l7 Jan.24 Feb.24 Mar.l3 May 28 Aug. 6 Sep. 4

CEA 3.17 1.5 2.2 1.6 2.17

CA 19-9 17.4 10 7 5

sec 0.7 <0.5 <0.5

CEA : Carcinoembryonic Antigen (normal value <2.5 ng/ml )

CA 19-9 : Carbohydrate Antigen (normal value <37 U/ml)
SCC : Squamous Cell Carcinoma related Antigen (normal value <1.5 ng/ml )

Fig. I Resected specimen of esophagus carcinoma (Lugol's stain) with uneven surface measured 1.5 x 1.2
em (left). Microscopic appearance with clear nucleolus (indicated with arrow) in squamous cell carcinoma of
the esophagus ( HE stain. x 125, right).

244
 Although tumor markers showed normal levels ( except for the CEA value in Oct. Table
I ), an abnormality was found in the squamous cells of the patient's esophagus after endoscopic
inspection. A biopsy revealed the presence of well-differentiated squamous cell carcinoma.
After surgery (28 Jan.), we found that the squamous cell carcinoma measured 1.5 x 1.2
em and was unstainable by Lugol's solution ( Fig.lleft ). On histomorphological observation,
the nucleolus was sufficiently clear ( Fig. I right )
Urinary excretion levels ofXP and NP.
In the control group ( 54 healthy individuals ), the urinary excretion levels of NP and
XP were 435 ±71 JJ.mole/mole creatinine (Mean ± S.D.) and 661 ±119, respectively.
Although the usual liver disease markers mentioned above and tumor markers showed normal
levels, the urinary excretion levels of NP and XP for this patient were 789 and 1121,
respectively, two months before surgery. In order to determine whether excretion levels
of pterins could be used tQ follow the course of the disease, a longitudinal study was
conducted on the patient with the esophageal carcinoma. Urines were coHected for a period of
eleven months from this patient and analyzed for their pterin content.

3000
Normal value
(Mean +2SD)
2500
a.: • XP 899 11mol/mol Cr.
u 0 NP 577 11mol/mol Cr.
0 2000
e
-
~
~
1500

0
e::1. 1000

500

0
,....
c"
"'
...,

Date
Fig. 2 Change in the urinary excretion levels of XP and NP in patient as a function of time

Fig. 2 shows the change in the excretion levels of XP and NP as a function of time for
this patient with esophageal carcinorma. In spite of the fact that tumor markers such as CEA,
CA19-9 and SCC showed normal levels throughout the observation, the high levels of
urinary pteridines of the patient led to the finding of the abnormality in his esophagus by
endoscopic inspection. Urinary excretion levels of pteridines rose to high values and were
2-3 times higher than the normal level just before surgery. In February, we did not collect

245
a urine sample from this patient, because of the complication of acute pneumonia just after
surgery, which took place in January. The slight increase in the XP and NP levels observed
in the 2 months after the operation may be due to this disease. However, the nine weeks
after surgery, XP and NP levels gradually decreased and reached normal levels within two
months.
The elevation in excretion levels of XP and NP in cancer patients is not easily explained.
Several researchers have reported that in general, increased urinary excretion of pterins could
result from an increase in folate catabolism by cancer cells and/or an increase in
tetrahydroneopterin and tetrahydrobiopterin catabolism 9' 10 • Recently, we reported that the
XP concentration was found to be 1.3-1.8 times the basal concentration when solutions of
dihydro-XP, dihydro-NP or BH 4 were mixed with urine and assayed using this method6 •
Zeitler et al . reported that 20-40% of reduced forms of NP, BP and other reduced pterin
derivatives present in urine are decomposed to XP during activated charcoal treatment 11 •
Clarification of the these points will require further research.
The present data shows results for only one patient, however, the higher excretion
levels of urinary pteridines alone revealed the carcinoma cells in their very early stage while
the usual tumor markers were all normal. The present paper suggests that the determination
of urinary pteridines levels might possibly be a useful marker for the early detection of
cancerous malignancies.

REFERENCES
1. Wachter, H., Hausen, A., and Grassmayr, K., Hoppe-Seyler's Z. Physiol. Chern., 360, 1957-1960,
( 1979 ).
2. Huber, C., Batchelor, J.R., Fuchs, D., Hausen, A., Lang, A., Niederwieser, D., Reibnegger, G., Swetly,
P., Troppmair, J., and Wachter, H., J. Exp. Med., 160, 310-316, ( 1984 ).
3. Fuchs, D., Hausen, A., Reibnegger, G., Werner, E.R., Dierich, M. P., and Wachter, H., Immunol.
Today, 9, 150-155, ( 1988 ).
4. Hausen, A., Fuchs, D., Reibnegger, G., Werner, E.R., and Wachter, H., Pteridines, 1, 3-10, ( 1989 ).
5. Mazda, T., Ogasawara, K., Fukuda, A., Gyure, W.L., and Tsusue, M., in: Chemistry and Biology of
Pteridines 1989 ( Eds. Curtius, H.C., Ghisla, S. and Blau, N. ) pp. 579-582, Walter de Gruyter,
Berlin-New York, ( 1990 ).
6. Fukuda, A., Mazda, T., Gyure, W.L., Iino, T., Harada, H., Yakura, M., Kamitsukasa, H., Ohbayashi, A.,
Oka, T., and Tsusue, M., Eur. J. Clin. Chern. Clin. Biochem., 31 in press, ( 1993 ).
7. Plesner, P., and Kalckar, H.M., Meth. Biochem. Analysis, 3, 97-110, ( 1956 ).
8. Nakagoshi, M., Tak1kawa, S., Negishi, S., and Tsusue, M., Bioi. Chern. Hoppe- Seyler, 373, 1249-1254,
( 1992 ).
9. Stea, B., Backlund, P.S., Jr., Berkey, P.B., Cho, A.K., Halpern, B. C., Halpern, R. M., and Smith, R.
A., Cancer Res., 38, 2378-2384, ( 1978 ).
10. Rokos, H., Rokos, K., Frisius, H., and Kirstaedter, H.J., Clin. Chim. Acta, 105, 275-286, ( 1980 ).
11. Zeilter, H.J., and Andodonskaja-Renz, B., Bioi. Chern. Hoppe-Seyler, 373, 972-973, ( 1992 ).

246
NEOPTERIN IN SUBACUTE SCLEROSING PANENCEPHALITIS

Haruo Shintaku, 1 Ryousuke Murata, 1 Hideji Hattori/ Osamu Matsuoka/

Tatsuo Nakajima/ Takuji Imamura/ and Yoshitomo Sawada2
1Dept. of Pediatrics, Osaka City University Medical school, Osaka 545, Japan
2Dept. of Pediatrics, Juso Citizens' Hospital, Osaka 532, Japan

INTRODUCTION
Subacute sclerosing panencephalitis (SSPE) has a very poor prognosis. In recent
years, the introduction of various treatments including inosiplex and interferon has somewhat
improved the survival rate1.2• However, there are large variations in the response of patients.
It is difficult to forecast the response and prognosis on the basis of clinical symptoms
alone, but sequential recordings by computed tomography (CT) and magnetic resonance
imaging (MRI) can be used to identify the lesions and severity of the disease, providing
information that may be correlated with changes in the neurological symptoms3 • A number
of laboratory tests have been used to define the clinical stage of patients with SSPE. Here,
we report that sequential monitoring of neopterin, ferritin, and creatine kinase (CK) in the
cerebrospinal fluid (CSF) of two patients with SSPE was useful as an index of the progress
of the disease.

PATIENTS
Case 1 was an 11-year-old boy. At the end of March 1988, his achievement at
school decreased, loss of memory developed and he experienced recurrent falls. By the
middle of May, he became less attentive and indifferent to his surroundings; personality
changes, a speech disorder, and myoclonus followed. An EEG showed typical periodic
slow-wave complexes. Measles antibody titers in the serum and CSF were 1:1024 and 1:16,
respectively, by complement fixation (CF). Based on these findings, the diagnosis was of
SSPE. Treatment with inosiplex and antiepileptic drugs improved the patient's condition
enough so that he could have simple conversations, but in the middle of July he began to
suffer from tremor and uncontrollable restlessness; at the beginning of August, right
hemiparesis developed and he was unable to walk. In the middle of August, generalized
tonic seizures appeared, followed by deterioration of consciousness. At the end of August,
administration of interferon a (INF- a ) intrathecally was started in addition to inosiplex,
but the only result was the development of spasticity, which worsened during September.
The patient also received intravenous interleukin 2, but his level of consciousness further
decreased, leading to dysphagia, so that nutrition by a nasal tube became necessary. Thereafter,
the progression of symptoms was arrested at stage IV according to Jabbour's classification.4
Case 2 was a previously healthy girl who began to have myoclonic seizures,
cerebral dysfunction, slow speech and parkinsonian gait at the age of 17, in 1985. Measles
antibody titers in the serum and CSF were 1:512 and 1:128, respectively, by hemagglutination
inhibition (HI), and there were typical periodic slow-wave complexes on the EEG, confirming
the diagnosis of SSPE. Inosiplex and anticonvulsants were given and the patient recovered

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 247
gradually.' She was in Jabbour's stage I for about 6 years. Speech deteriorated starting in
May 1991. She became unable to perform activities of daily living, and lost an ability of
walking without help in December 1991. She is now nearly in Jabbour's stage III.

METHODS
The diagnosis of SSPE was based on the clinical manifestations, EEG findings, and
elevated serum and CSF measles antibodies. Patients were assessed by a neurological
disability index (NDI) score. 1
The MR images were obtained with a 0.5 Tesla superconducting magnet (Picker-
International Co).
CSF collected from the two patients was frozen and preserved until the various
markers listed below were measured. Protein, glucose, numbers of cells, measles antibody
titers by HI or by CF, and measles IgG by enzyme immunoassay (EIA) in the CSF were
measured on the day when the CSF was collected.
Ferritin, CK, and neopterin in the CSF were studied. Ferritin were measured by
radioimmunoassay, and neopterin were measured by reverse-phase high-pressure liquid
chromatography 6•

RESULTS
In case 1, the protein, glucose and number of cells in the CSF remained within
normal ranges throughout the course of treatment between May 1988 and February 1989.
The normal level of neopterin is 18.5 ± 10.4 pmol/ml. When first measured, neopterin
was 111.5 pmol/ml. Neopterin remained at high levels in the CSF throughout the course of
treatment. Immediately before the time when ferritin and CK increased to abnormal levels,
neopterin was particularly high: 180.8 pmol/ml.

Table 1. Results of various tests of CSF.

Patient Date Titer IgG Neopterin CK Ferritin
byCF byEIA (pmol/ml) (IU/L) (ng/ml)

easel '88 5.30 16 14 x 104 111.5 0 2.1

8.26 11.5x10 4 90.6 2 5.2
8.31 14.5 X 10 4 180.8 3 4.3
9.19 16 13.0 X 10 4 91.0 8 7.0
9.22 9.1 X 10 4 87.5 5 7.3
9.29 16 9.9 X 10 4 87.8 7 6.0
10.17 16 12.4 X 10 4 100.2 6 6.6
'89 2.06 8.4 x 10• 29.1 1 2.4

Case2 '91 5.10 19.7 4.5

5.22 15.4 4.0
12.13 64 87.8 2.3
-:not measured

Ferritin was at the level of 2.1 ng/ml when first measured, but it increased as the
neurological symptoms worsened, eventually exceeding the upper limit of normal, 6.0
ng/ml. When the NDI became constant with the stabilization of symptoms, ferritin decreased
again to 2.4 ng/ml, which was comparable to the level in May. CK showed a similar pattern
of changes; it exceeded the normal upper limit of 3.8 IU/L in September and October,
reached the level of 8 IU/L, and then returned to normal. Changes in ferritin and CK
roughly corresponded with changes in the extent of the high-intensity areas in the white
and gray matter observed by MRI. Fig 1 shows changes in neopterin, ferritin, CK, NDI and
the high-intensity areas of the white and gray matter together with details of treatment.
Changes in the MRI are shown based not on measurements but on an overall impression.
In case 2, the CK and ferritin values were in normal ranges at all points of measurement.

248
 pmoVml
Neopterin & Biopterin
200

100

Neopterin
Biopterin

lUll CK & ferritin nglml

10 8
ferritin

NOI
80

60

40

20

3 4 5 6 7 8 9 10 11 12 1 2 3 4
1988 1989

INOSIPLEX r-=:-=---:372oo:-:'."44
':'~oo::-m------
g
~ l!!f -
I
INTERFERON IT 150·900x 10'units
IV 1200 X 10'uniiS m
INTERLEUKIN2 aB I 75-500 units

MRI

Fig 1. Changes in CSF levels of neopterin and ferritin, the NDI, and the high-intensity areas of the white and
gray matter by MRI, together with details of treatment. Antiepileptic drugs given are not listed.

On the other hand, although neopterin levels were in normal ranges when measured in
May, neopterin was high (87.8 pmol/ml) in December 1991.
MR images in case 1 were taken sequentially. At the end of May, high-intensity
areas in T2-weighted images were detectable, but barely so, in the white and gray matter.
From August to October, high-intensity areas appeared over an extensive region, including
the white matter and cortex of the posterior lobe and the white matter around the ventricles.
Afterwards, the area of the T2-weighted images in the lesions grew smaller, making the
dilatation of the ventricles more conspicuous, indicating the progression of cerebral atrophy.
MR images in case 2 obtained in 1985 showed prolonged T1 and T2 relaxation
times in both lentiform nuclei and the white matter adjacent to the lateral ventricles. In the
autumn of 1986, the lesions in the lentiform nuclei had almost disappeared, but in 1991,
MR revealed severe atrophic changes. In that year, there was little further change.

DISCUSSION
We reported for the first time an association between neopterin levels in the CSF of
patients with SSPE and the clinical stage. Neopterin are synthesized from guanosine

249
triphosphate (GTP) in the synthetic pathway of tetrahydrobiopterin, which act as a cofactor
in neurotransmitter synthesis. GTP cyclohydrolase I is present in macrophages, and neopterin
is formed as a metabolite of dihydroneopterin triphosphate produced from GTP by GTP
cyclohydrolase I. The general assumption has been that there is no enzyme synthesizing
biopterin in macropha.pes and that only neopterin is produced there, but the enzyme does
exist in macrophages . GTP cyclohydrolase I in macrophages is activated by INF- y .
Neopterin is a sensitive marker of activation of the cellular immune system, and it is
released from macrophages stimulated by INF- y produced by activated T lymphocytes8•
Elevated levels of neopterin are correlated with and mark previous or ongoing stimulation
of macrophages by INF- y. Neopterin in the CSF is elevated in various infections 9,
malignancies and immune-mediated diseases 10• An increased amount of neopterin is present
in the CSF during direct viral infection of the central nervous system and during exacerbations
of multiple sclerosis 11 • In addition, neopterin is increased in the CSF during postmeasles
encephalomyelitis 11 • The encephalomyelitis that occurs as a complication of measles is an
autoimmune demyelinating disease triggered by the measles virus.
Intracerebral inflammation and subsequent nerve tissue damage seemed to be
progressing rapidly in case 1, to judge from the expansion of high-signal areas in T2-weighted
images on MRI and their relation to biochemical parameters. On the other hand, that
neopterin levels rose slightly earlier than the changes in CK and ferritin levels may be
because of stimulation by INF- y derived from T cells in the acute exacerbation stage of
inflammation and consequent acceleration of macrophage metabolism and neopterin release.
Three courses of INF- a intrathecally or intravenously were used in case 1. The neopterin
level did not rise with the second and third INF- a administrations, so INF- y production
seems not to rise with INF- a administration. Thus, it does not lead macrophage activation
and neopterin production.In case 2, there was a relatively mild course for 6 years, with
gradual clinical exacerbation in 1991. Neopterin levels in CSF were in the normal range
when the patient was in Jabbour's stage I and stable except for disturbance of intellectual
function. The level rose at the end of December 1991 when gait disturbance developed.
These results suggest that assay of the neopterin level in CSF would provide an
index of the intensity of inflammation and possibly an index of the progress of intracerebral
lesions before they can be detected by imaging or by changes in other biochemical parameters.
In our cases, an increase in neopterin excretion in the CSF preceded changes in the clinical
condition and was correlated with the findings of MRI. If this index is used, treatment
could be started earlier when neopterin begins to increase.

REFERENCES

1. P.R. Dyken, A. Swift, R.H. DuRant, Long-term follow-up of patients with subacute sclerosing panencephalitis
treated with inosiplex, Ann Neural. 11:359 (1982).
2. K. Yalaz, B. Anlar, F. Oktem, eta!., Intraventricular interferon and oral inosiplex in the treatment of
subacute sclerosing panencephalitis, Neurology. 42: 488 (1992).
3. G.B. Lum, J.P. Williams, P.R. Dyken, eta!., Magnetic resonance and CT imaging correlated with clinical
status in SSPE, Pediatr Neural, 2: 75 (1986).
4. J.T. Jabbour, J.H. Garcia, H. Lemmi, J. Ragland, D.A. Duenas, J.L. Sever, SSPE: a multidisciplinary
study of eight cases, JAMA, 207:2248 (1969).
5. R. Murata, 0. Matsuoka, S. Nakajima, eta!., Serial magnetic resonance imaging in subacute sclerosing
panencephalitis, Jpn J Psychiatr Neural, 41:277 (1987).
6. T. Fukushima, J.C. Nixon, Analysis of reduced forms of biopterin in biological tissues and fluids, Anal
Biochem, 102: 176 (1980).
7. J. Guzman, N. Blau, 6-Pyruvoyl tetrahydropterin synthase in human tissues and cell lines, Pteridines 3: 43
(1992).
8. S. Huber, J.R. Batchelor, D. Fuchs, et a!., Immune response-associated production of neopterin: release
from macrophages primarily under control of interferon-gamma, J Exp Med, 160: 310 (1984 ).
9. H. Shintaku, A. Nishimura, R. Murata, Neopterin and biopterin levels in cerebrospinal fluid in children
with meningitis, Bioi. Chern. Hoppe-Seyler, 369:542 (1988).
10. D.E. Griffin, J.C. McArthur, D.R. Cornblath, Neopterin and interferon-gamma in serum and cerebrospinal
fluid of patients with HIV-associated neurologic disease, Neurology 41:69 (1991).
11. S. Fredrikson, P. Eneroth, H. Link, Intrathecal production of neopterin in aseptic meningo-encephalitis
and multiple sclerosis, Clin Exp Immunol, 67:76 (1987).

250
THE 7-DEAZAGUANINE DERIVATIVE, QUEUINE,
REGULATES MAMMALIAN CELL PROLIFERATION
DEPENDING ON THE METABOLIC STATE

Werner Langgut, Marianne Haupt, and Thomas Reisser

Institut fiir Biochemie, medizinische Fakultiit

Universitat Erlangen-Niirnberg
Fahrstrasse 17, D-8520 Erlangen, Germany

INTRODUCTION

The highly modified nucleoside, queuosine (Q), a 7-deazaguanosine derivative,

occurs in position 34 of the anticodon of tRNAs accomodating for the amino acids asn,
asp, his, and tyr in place of guanosine (G) 1•2. The synthesis of Q in E. coli starts from
GTP and shares similarity with the synthesis of pteridines and riboflavin. The precursor of
Q, 7-aminomethyl-7-deazaguanine, is inserted into tRNAs in exchange for guanine by the
tRNA guanine transglycosylase (EC 2.4.2.29) without breakage of the sugar phosphate
backbone3. The synthesis of Q is completed at the level of tRNAs. Eukaryotes cannot
synthesize Q, but Q-tRNAs are present in all organisms examined thus far, yeast being the
only known exception. Mammals obtain the free base, queuine (7-(((4,5-cis-dihydroxy-2-
cyclopenten-1-yl)-amino)-methyl-7 -deazaguanine), from their diet or their intestinal flora.
The eukaryotic tRNA guanine transglycosylase uses queuine (q) instead of7-aminomethyl-
7-deazaguanine as a substrate to replace guanine in the corresponding tRNAs irreversibly.
Pteridines, especially 6-aminomethyl-tetrahydrobiopterin, are competitive inhibitors of this
inserting enzyme4. Increased levels of tetrahydrobiopterin and derivatives may be related to
hypomodification of Q-tRNAs and accumulation of the q-base found in tumor cells 1•2•5.

RESULTS AND DISCUSSION

HeLa-S3 cells were grown in medium supplemented with q-free horse serum and
subsequently lacked the modified nucleoside Q in tRNAs (Q-deficient cells). Addition of
chemically synthesized q-base (0.3 J.!M) to these cells resulted in a significant stimulation
of their proliferation (table 1). When similar experiments were performed under conditions
of hypoxic stress, i.e. 7% oxygen instead of 21% for aerobic conditions, the addition of q
to the culture medium resulted in an inhibition of proliferation (table 1). A detailed analysis
revealed that oxygen limitation in the case of HeLa-S3 cells caused an induction of the ldh a

Chemistry and Biology of Pteridines and Palates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 251
gene encoding lactate dehydrogenase (the terminal enzyme of anaerobic glycolysis in
mammals), an increase in the amount of LDH A protein, an increased activity of the muscle
type isoenzyme LDH A4 (M4), and also an increase in total specific LDH activity,
confmning that these cells had shifted their energy metabolism from a respiratory to a more
glycolytic state6. Thus, q apparently favours the proliferation of oxygenated, but inhibits
the proliferation of glycolytic HeLa cells.
To answer the question as to whether the modulating activity of queuine on cell
proliferation might be of general significance for mammalian cells, we investigated the
influence of q on the proliferation of a variety of other cell lines, using analogous
experiments as in the case of HeLa cells. The addition of q (0.3 JlM) to the culture medium
of all non-transformed cells used in this study (AV-3, Chang Liver, Swiss-3T3, NIH-3T3,
and Wi-38) stimulated proliferation (table 1), corroborating that q is favourable for the
proliferation of aerobically growing mammalian cells. However, the extend of stimulation
varied between different cell lines, fibroblast-like cells being more responsive than
epithelial-like cells. The response of Q-deficient cancer-derived cells to the addition of q
falls into two categories: (i) a stimulation of proliferation was observed using HeLa-S3
cells, human A-431 epidermoid carcimoma cells, and human HL-60 promyelocytic
leukemia cells. (ii) Proliferation after addition of q was inhibited using human Colo-
DM320 adenocarcinoma cells, rat PC-12 phaeochromo-cytoma cells, and mouse Ascites
tumor cells. The results indicate that the modulating effect of q on the proliferation of
mammalian cells in culture might be of general significance. Obviously, q can act as a
growth promotor on some cell lines but as a growth inhibitor on other cell lines. Non-
transformed cells in culture apparently react to q-addition like aerobically growing HeLa
cells, whereas cancer cells may either respond like oxygenated or like glycolytic HeLa
cells, depending on the origin of the cell line.
In another series of experiments, the effect of q-addition to normal NIH-3T3
fibroblasts, and to NIH-3T3 cells transformed by the activated cellular oncogenes c-Ha-
ras and raj respectively was compared. Addition of q to normal, Q-deficient NIH-3T3
cells stimulated proliferation under aerobic conditions (table 1). However, proliferation of
NIH-3T3 cells transformed by the activated c-Ha-ras oncogene was inhibited on addition
of q. In contrast, the proliferation of raJ-transformed NIH-3T3 cells was not inhibited by
q. These results suggest that activation of some (but not all) oncogenes may create
conditions that change the response of the cell to q from growth stimulation to growth
inhibition.
Based on the result that a stimulation of proliferation on addition of q was
observable with oxygenated and inhibition with hypoxically growing HeLa cells, we
propose that growth modulation by q depends on the metabolic state of cells. As first
proposed by Warburg, many tumor cells are characterized by a high rate of aerobic
glycolysis7 . This has been shown for instance in EAT cells used in this study8. In
agreement with this observation, EAT cells respond to q like glycolytic HeLa cells even
under aerobic conditions. It has been shown by Racker et al. that ras-transformed
fibroblasts exhibit a higher rate of aerobic glycolysis compared to their non-transformed
counterpart9, suggesting that the metabolic state of tumor cells is determined by the
activation of discrete oncogenes. This fits well with the observation that non-transformed
NIH-3T3 cells respond to q like aerobically growing HeLa cells, and ras-transformed
NIH-3T3 cells like hypoxically growing HeLa cells. In conclusion, our results suggest that
q stimulates proliferation of oxygenated cells, but inhibits proliferation of those cells that
have shifted their energy metabolism from respiration towards glycolysis, either as a
consequence of oxygen limitation, or as the result of the activation of discrete oncogenes.

252
 Table 1. Modulation of mammalian cell proliferation by queuine.

cell line relative increase1 hours after number of origin and

A+q/A-q addition of q experiments morphology

AV-3 1.2 - 1.5 24-48 3 human, normal amnion cells, epithelial like
Chang Liver 1.3- 2.0 48 4 human, normal lung cells, epithelial like '
Swiss-3T3 1.8- 2.9 24-48 4 mouse, normal embryonic tissue, fibroblast-like
NIH-3T3 1.4- 2.0 24-48 5 mouse, normal embryonic tissue, fibroblast-like
Wi-38 2.8- 3.7 24-48 3 human, normal embryonic lung, fibroblast-like

HeLa-S3 1.6- 4.5 48-72 5 human cervical carcinoma, epithelial-like

HL-60 2.2 - 2.7 96 2 human promyelocytic leukemia, lymphoblast-like
A-431 1.6- 4.7 24 3 human epidermoid carcinoma, epithelial-like

HeLa-S3 hypoxic2 0.8 - 0.3 48 6

Colo-DM320 0.8-0.3 48-96 3 human adenocarcinoma, colon, rounded and refractile
PC-12 0.7-0.3 24-48 5 rat adrenal pheochromocytoma, responsive to netve growth factor
EAT 0.7- 0.4 24-48 3 mouse Ehrlich-Lettre Ascites carcinoma, epithelial-like

Ha-ras-3T3 0.7- 0.4 24 3 NIH-3T3 cells transformed by the activated human Ha-ras gene
raf-3T3 0.9- 1.6 24 3 NIH-3T3 cells transformed by the activated rqf gene

1All cells were maintained in medium supplemented with q-free horse serum. Q-deficient cells were precultivated for 2 - 3 days followed by addition of chemically synthesized q to the
culture medium. The increase in cell number after addition of q was determined and is expressed as fold increas in the cell number of q-free cultures within the same period (relative
increase). The point of maximal response to q was chosen from each experiment The minimal and maximal values found in repeated experiments are listed.
2For cultivation under hypoxic conditions, HeLa cells were grown in an incubator in which the atmosphere was replaced with nitrogen to reduce the oxygen content to 7%.

~
w
 The molecular basis for the growth modulating activity of q remains to be
elucidated. However, we have gained substantial evidence that both the free q-base and Q-
tRNAs are involved in this process. The free base probably interacts with specific
phosphoproteins involved in growth control, and thereby influences mitogenic signalling
by growth factor receptors 10. On the other hand, q and Q-tRNAs might be necessary for a
proper adaptation of the cell metabolism to the enviromental oxygen tension6. Both of these
queuine-mediated events may finally contribute to an altered growth behaviour of
mammalian cells in culture. The modulating effect of q may be of significance for rapidly
proliferating tumor cells, which are characterized by a deranged growth control and an
altered metabolic state. Increased levels of pteridines may be connected with Q-deficiency
of tRNAs and accumulation of the free q-base in tumor tissues. Fluctuations in the amount
of free q and of Q-deficient tRNAs may influence the proliferative capability of
neoplastically transformed cells and may contribute to the malignant phenotype of tumor
cells in vivo. A detailed analysis of the state of modification of tRNAs in the various cell
lines is currently in progress.

ACKNOWLEDGEMENTS

Queuine was a kind gift from Dr. S. Nishimura and was chemically synthesized by Drs.
H. Akimoto and N. Nomura of the Central Research Laboratories, Takada Chemical Ind.
Ltd., Osaka, Japan. We are greateful to Drs. Th. Dingermann and A. Ogilvie at our
institute for providing various cell lines, and to Dr. Sakiya and Dr. Nagao of the National
Cancer Center Research Institute, Tokyo, Japan for providing the ras -and raj-
transformed fibroblasts respectively. This work was supported by the Johannes and Frieda
Marohn Foundation, Germany.

REFERENCES

1. Kersten, H. and Kersten, W. Chromatography and modification of nucleosides part B:

biological roles and function of modification, in: "Journal of Chromatography
Library", 45B, Gehrke, Ch. W. and Kuo, KC.T., eds., Elsevier, Amsterdam
(1990).
2. Nishimura, S., Prog. Nucleic Acid Res. Mol. Bioi. 28: 49 (1983).
3. Farkas, W.R., Nucleosides & Nucleotides 2: 1 (1983).
4. Farkas, W.R., Jacobson, KB., and Katze, J.R., Biochim. Biophys. Acta 781: 64
(1984)
5. Emmerich, B., Zubrod, E., Weber, H., Maubach, P.A., Kersten, H., and
Kersten, W., Cancer Res. 45:4308 (1985).
6. Reisser, Th., Langgut, W., and Kersten, H., Biological Chemistry Hoppe-Sayler
373: 814 (1992).
7. Warburg, 0., Science 123: 309 (1956).
8. Scholnick, P., Lang, D., and Racker, E., J. Bioi. Chern. 248: 5175 (1973).
9. Racker, E., Resnick, R.J., and Feldman, R., Proc. Nat!. Acad. Sci. USA 82:3535
(1985).
10. Langgut, W., Reisser, Th., Eicher, A., and Nishimura, S., Biological Chemistry
Hoppe-Sayler 373: 792 (1992)

254
TETRAHYDROBIOPTERIN DEFICIENCY AND AN INTERNATIONAL
DATABASE OF PATIENTS

N. Blaul and Jean-Louis Dhondt2

I Division of Clinical Chemistry, Department of Pediatrics, University of

Zurich, Steinwiesstrasse 75, 8032 Ziirlch, Switzerland
2Laboratoire de Biochimie, Faculte Libre de Medecine, 56 rue du Port,
59046 Lille, France

INTRODUCTION

Tetrahydrobiopterin (BH4) deficiency comprises a group of rare autosomal

recessively inherited diseases characterized by progressive neurological symptoms
unresponsive to treatment with low-phenylalanine dietl. 6-Pyruvoyl tetrahydropterin
synthase (PTPS) deficiency, the most common form of BR4 deficiency, occurs in various
clinical forms which are sometimes hard to distinguish. This complicates the screening of
newborns, prenatal diagnosis, the determination of heterozygote carriers, and treatment of
patients. Besides severe, atypical (peripheral and/or partial), and transient forms there
might be other variants only marginally characterized2. In dihydropteridine reductase
(DHPR) deficiency, the second most common form of BH4 deficiency, various point
mutations have been observed. Recently a new form of hyperphenylalaninemia,
primapterinuria, presumably due to deficiency of the phenylalanine-4a-hydroxylase
stimulating protein pterin-4a-carbinolamine dehydratase (PCD) was described3. PCD
deficiency and the GTP cyclohydrolase I (GTPCH) deficiency are less frequent forms of
BH4 deficiency. The metabolic pathway of BH4 as well as the known enzyme defects
leading to hyperphenylalaninemia are shown in Figure 1.
As a result of screening carried out in more than 50 Departments of Pediatrics and
of Biochemistry worldwide in the last 18 years, 263 patients with BH4 deficiency were
discovered. Of these 263 patients 150 suffer from PTPS deficiency, 86 from DHPR
deficiency, 9 from PCD deficiency, and 8 from GTPCH deficiency4,5.
The database BIODEF we are establishing is an informative resource and retrieval
system that includes biochemical and clinical information on variants of
hyperphenylalaninemia as well as a physicians' and researchers' directory. A complete
bibliography on BH4 deficiency will also be included. A hard copy and the copyright-free
software should help physicians in managing the patients, paying special attention to long-
term protocols.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 255
 GTP
~ ~GTPCH
Dihydroneopterin triphosphate
~ ~PTPS
6-Pyruvoyl-tetrahydropterin
SRorCR + __.SR + SR

SR + +- t SRorAR

'\ 6
Tetrahydrobiopterin ~02

Phenylalanine
4a-Peroxy-tetrahydrobiopterin
~ DHPR PAH\ ~

··""""""''"""'-"'""''":;:j~'·"'''"'
q-Dihydrobiopterin ..-.;( ~

Figure 1. Biosynthesis and regeneration of tetrahydrobiopterin. Metabolic defects causing

hyperphenylalaninernia are marked. PAH: phenylalanine-4-hydroxylase; SR: sepiapterin
reductase; CR: carbonyl reductase; AR: aldose reductase.

RESULTS AND DISCUSSION

Screening for tetrahydrobiopterin deficiency

The following investigations should be performed in all newborns with even slight
hyperphenylalaninernia6:

1. Analysis of pterins in urine7,8_

2. Measurement of DHPR activity in blood from Guthrie cards9.
3. Analysis of phenylalanine in plasma before and after BR4loading testlO.

The first two tests are essential and enable to differentiate between all variants of
BH4 deficiency. The BR4 loading test is an additional useful diagnostic tool for the fast
discrimination between the classic phenylketonuria (PKU) and BH4 variants.

Pterin analysis. This test must be performed at elevated plasma phenylalanine

levels (not under low-phenylalanine diet). Either a native urine or urine dried on filter
paperll may be used for the selective screening. The characteristic urinary pterin pattern
makes it possible to identify all four variants of BR4 deficiency.

256
 Loading test. The loading test with BH4 allows the detection of all variants w'l.th a
defect in B~ biosynthesis, even if the defect is only partial. A decrease of the plasma
phenylalanine occurs within 4 to 8 hours after administration of B~. The original test at a
dose of 7.5 mg/kg bw was modified by increasing the loading dose to 20 mg BR4fkg bw.
However, one patient was described as a non-responder even at this high dose of BH4 and
died despite an early diagnosis and treatrnent12.

Combined loading test. Another procedure for the screening of

hyperphenylalaninemias is the combined phenylalanine (100 mg/kg bw) and B~ (20
mg/kg bw) loading test13. This test can be performed while patients are on a phenylalanine-
restricted diet, and/or when pterin analysis is not available. This test also has to be
combined with the measurement of DHPR activity in blood. The normalization of serum
phenylalanine 4 and 8 hours after B~ administration (7 and 11 hours after phenylalanine
administration) allows differentiation between variants of hyperphenylalaninemia.

Clinical manifestation

Typical forms. The clinical course of the illness in untreated patients is similar in
typical PTPS, DHPR and GTPCH deficiencies14. The median age at which clinical signs
become evident is 4-5 months, but symptoms do not necessarily correlate with age of
diagnosis, even in the same family. However, when information on the neonatal period is
available, abnormal signs (poor sucking, decreased spontaneous movements, floppy baby)
can be noticed during the neonatal periodS.
The most common symptoms are mental retardation, convulsions (grand mal or
myoclonic attacks), disturbances of tone and posture, drowsiness, irritability, abnormal
movements, recurrent hyperthermia without infection, hypersalivation, swallowing
difficulties. Microcephaly is observed in both diseases but with a higher incidence in PTPS
deficiency (52%) than in DHPR deficiency (33 %). However, in 13 of 25 DHPR deficient
patients for whom repeated measurement with increasing age were available, decrease of
head circumference to progressive microcephaly was observed, whether patients were
treated or not.

Atypical Forms. Atypical PTPS deficiency: The absence of clinical signs theoretically
defines atypical forms. However, in 2 cases neonatal hypotonia was noticed, 2 were
mentally retarded, 1 had focal signs on EEG at 9 years of age and 1 presented with acute
but transient symptoms (behaviour problems, neurovegetative signs, sleeping difficulties).
Atypical DHPR deficiency: Some authors have suggested that partial deficiency of
DHPR activity may also exist. That may explain a few reports on patients who were
unusual in some aspects.
Primapterinuria: Although this form seems to have no serious consequences, minor
signs can be observed. In the first easelS, slight tremors of upper limbs after stimulation
and a moderate tendency to hypertonia were noticed during the neonatal period. With
dietary control of blood phenylalanine, neurological development normalized. In the case
reported by Blaskovics and Giudici16, transient hypotonia and motor delay were also
noticed.

257
Treatment

Neurotransmitter replacement. L-Dopa/Carbidopa and 5-hydroxytryptophan (5-HT)

administration represents a common therapeutic approach to BH4 deficifncies. The
monitoring of treatment represents the crucial point. The analysis of neurotransmitter
metabolites in CSF represents the best way to evaluate the efficiency of the treatment
(Figures 2-4).
Control of blood phenylalanine levels. Although Bli4 deficient subjects exhibit a
higher dietary phenylalanine tolerance than classical PKU patients, a limiting factor in the
response to neurotransmitter precursor therapy might be the plasma phenylalanine
fluctuations. The control of blood phenylalanine has to be stricter than in other HPAs;
some patients on neurotransmitter treatment have had neurological problems with
phenylalanine concentrations as low as 360 J.Lmol/1.
Tetrahydrobiopterin administration. In most cases BH4 monotherapy failed to
restore a normal level of cerebral neurotransmitter synthesis as confirmed by low
concentrations of 5IDAA and HVA in the CSF (Figure 2). On the other hand, relatively
low doses of BH4 normalize blood phenylalanine levels and offer an alternative to a
phenylalanine-restricted diet.
Folinic acid supplementation. Low CSF folate levels and the frequent occurrence of
calcifications in the basal ganglia similar to those described in congenital folate
malabsorption or in methotrexate toxicity support the hypothesis of a gradual CNS
deficiency of folate in patients with DHPR deficiency. These patients benefit from folinic
acid supplementation.

a
R•0 .31

10 Q

0 0 a

1000 AGE Cda~~5> 10000 10 100 1000 AGE Cdays> 10000

1000~~~~~~~~~;;~==~

i tM-~·-:i?·tf-~1~1;'-F
"'
:. H%~H~:f+··i¥~~t.%i;
.. 100 6 ail 0 : ""

0
• • ' .'

..
0
10 10

o on nRWotrMVnitt•r o on ntu.otro~~ nmi ncor

(CSF HI A) 6 on 1HB

I
• on nKrott.ann'IIIIH + THB
I
~ 6 on THB
• on IWU'otranmit
10 100 1000 AGE Cda~~S)IOOOO 10 100 1000 AGE Cdays>10000

Figure 2. CSF levels of 5IDAA and HVA in patients with typical (severe) PTPS
deficiency without treatment (upper) and under different treatment protocols; 0 on
neurotransmitters; A on BH4; • on neurotransmitter+ BH4, (lower).

258
 10 F\o 0.8C 10

• on n-l9 • on 1MB
@SFHIA)
~
I I
10 100 1000 AGE fda~ 10000 10 100 1000 AGE fdllysl 10000

Figure 3. CSF levels of 5HIAA and HVA in patients with atypical (peripheral, mild) PTPS
deficiency without treatment llSl and on BH4 treatment •.

1000

""~
c:

100

10 10
R-038

00

1 +---~--~~--~----~--~--~
10 100 1000 AGE Cdaysl 10000

...
•
10 10

• on ne-urouta~nmitl:tr • oni\IPurOtr.antri'l!'r
1+----------r--------~--------~
10 100 ' 10
+-------~-r--------~--------~
1000 AGE <o:laysl 10000 100 1000 10000
AGE fda~

Figure 4. CSF levels of 5HIAA and HVA in patients with DHPR deficiency without
treatment (upper) and on treatment with neurotransmitter • (lower).

259
BIODEF: An international database

Patients' data collected from over 50 Departments of Pediatrics and of Biochemistry

worldwide were collected during the last 18 years. The questionnaire distributed to the
clinics includes information on: (1) patient identification (e.g. birth date, ethnic origin,
consanguinity, birth weight and the clinical status at the birth, etc.), (2) screening data on
hyperphenylalaninemia (e.g. age at screening, phenylalanine loading test, diet, tolerance
etc.), (3) screening data on pterins, and related enzymes (e.g. pterins in urine, serum and
CSF, BI4 loading test, enzyme activity measurement in tissue or blood cells etc.), (4)
clinical symptoms before and on treatment (e.g. onset of symptoms, EEG, CT, MRI etc.),
(5) treatment, follow up and CSF status (e.g. therapy protocol, clinical examinations, CSF
status, blood phenylalanine etc.), (6) DNA analysis (e.g. type of mutation etc.), and (7)
clinic/physician's data.

Total 263 patients PTPS (150}

57%

PCD (9) 4%

GTPCH (8) 3%

? (10)
33%

DHPR (86)
Figure S. Summary of the patients registered in the international database of
tetrahydrobiopterin deficiencies.

The database we are establishing is an informative resource and retrieval system that
includes biochemical and clinical information on variants of hyperphenylalaninemia as
well as a physicians' and researchers' directory. A complete bibliography on BH4
deficiency (over 370 entries) will also be included. BIODEF will run both under MS-DOS
and as an Windows application on 100% IBM compatible media. Both interfaces will be
linked to the same data stored in dBASE format. It will be copyright-free and it will store
the patients' records (no patients' names) and will allow via network the possibility to
browse, edit, append, query, export, and print such data. A hard copy in a booklet form will
also be available.
So far 263 patients with BI4 deficiency have been registered. Of these 263 patients
150 suffer from PTPS deficiency, 86 from DHPR deficiency, 9 from PCD deficiency, 8
from GTPCH deficiency, and 10 are still not classified (Figure 5).

260
ACKNOWLEDGEMENTS

This work was supported by the Swiss National Science Foundation, project No.
31.33897.92 and by the Helmut Horten Research Foundation.

REFERENCES

1. N. Blau, Inborn errors ofpterin metabolism. Ann Rev Nutr. 8:185 (1988).
2. J.L. Dhondt, Strategy for the screening of tetrahydrobiopterin deficiency among
hyperphenylalaninemic patients: 15-years experience. J Inherit Metab Dis. 14:117
(1991).
3. N. Blau, H.C. Curtius, T. Kuster, A. Matasovic, G. Schoedon, J.L. Dhondt, P.
Guibaud, T. Giudici and M. Blaskovics, Primapterinuria: a new variant of atypical
phenylketonuria. J Inherit Metab Dis. 12/2:335 (1989).
4. J.L. Dhondt, Tetrahydrobiopterin deficiency. Lessons from the analysis of 90 patients
collected in the international register. Arch Fr Pediatr. 12:655 (1987).
5. J.L. Dhondt, Tetrahydrobiopterin deficiencies. Lessons from the compilation of 200
patients. Developmental Brain Dysfunction. 6:139 (1993).
6. N. Blau, Guidelines for the screening for hyperphenylalaninemia due to
tetrahydrobiopterin deficiency. Cro Med J. 33:17 (1992).
7. J.L. Dhondt, C. Largilliere, P. Ardouin, J.P. Farriaux and M. Dautrevaux, Diagnosis of
variants of hyperphenylalaninemia by determination of pterins in urine. Clin Chim
Acta. 110:205 (1981).
8. A. Niederwieser, W. Staudenmann and E. Wetzel, High-performance liquid
chromatography with column switching for the analysis of biogenic amine metabolites
and pterins. J Chromtogr. 290:237 (1984).
9. N. Arai, K. Narisawa, H. Hayakawa and K. Tada, Hyperphenylalaninemia due to
dihydropteridine reductase deficiency: diagnosis by enzyme assays on dried blood
spots. Pediatrics. 70:426 (1982).
10. A. Ponzone, 0. Guardamagna, S. Ferraris, G.B. Ferrero, I. Dianzani and R.G.H.
Cotton, Tetrahydrobiopterin loading test in hyperphenylalaninemia. Pediatr Res.
30:435 (1991).
11. N. Blau, L. Kierat, C.W. Heizmann, W. Endres, T. Giudici and M. Wang, Screening
for tetrahydrobiopterin deficiency in newborns using dried urine on filter paper. J
Inherit Metab Dis. 15:402 (1992).
12. W. Endres, H. lbel, L. Kierat, N. Blau and H. C. Curtius, Tetrahydrobiopterin and
"non-responsive" dihydropteridine reductase deficiency. Lancet. 2:223 (1987).
13. A. Ponzone, 0. Guardamagna, M. Spada, S. Ferraris, R. Ponzone, L. Kierat and
N. Blau, Differential diagnosis of hyperphenylalaninemia by a combined
phenylalanine-tetrahydrobiopterin loading test. Eur J Pediatr. in press (1993).
14. J.L. Dhondt, Tetrahydrobiopterin deficiencies: preliminary analysis from an
international survey. J Pediatr. 104:501 (1984).
15. J.L. Dhondt, P. Guibaud, M.O. Rolland, C. Dorche, S. Andre, G. Forzy and
J.M. Hayte, Neonatal hyperphenylalaninaemia presumably caused by a new variant of
biopterin synthetase deficiency. Eur J Pediatr. 147:153 (1988).
16. M. Blaskovics, and T.A. Giudici, A new variant ofbiopterin deficiency.
N EnglJ Med. 319:1611 (1988).

261
TETRAHYDROBIOPTERIN DEFICIENCY IN PORTUGAL: RESULTS OF THE
SCREENING FOR HYPERPHENYLALANINEMIA

I. T. de Almeidal, P. P. Leandrol, R. Portela2, A. Cabral2, F. Eusebio2, T.

Tasso2, A. Matasovic3, and N. Blau3

I centro de Metabolismos e Genetica (INIC) - Faculdade de Fanmkia,

1600 Lisboa, Portugal
2Unid. D. Met, Serv. Ped., H. Sta. Maria, Lisboa, Portugal
3Division of Qinical Chemistry, University Children's Hospital,
Steinwiesstrasse 75, CH-8032 Zurich, Switzerland

INTRODUCTION

Since 1974 variants of phenylketonuria (PKU) have been described in which

neurological deterioration occurred despite a low phenylalanine (Phe) diet. Subsequently
the lack of the cofactor, tetrahydrobiopterin (BR4), has been demonstrated to be the cause
of this deterioration.
At present, three/four different enzymopathies are known to affect BH4 metabolism:
(i) dihydropteridine reductase (DHPR) deficiency; (ii) 6-pyruvoyl-tetrahydropterin synthase
deficiency; (iii) GTP cyclohydrolase I deficiency and (iv) pressumed pterin-4a-
carbinolamine dehydratase deficiency I.
Although the incidence of BH4 deficiencies is low (1-3% among patients with
hypetphenylalaninemia) it is important to perform early differential diagnosis in order to
establish a specific treatment and to avoid irreversible brain damage 2.
Since 1990 a systematic investigation of pterin metabolism has been conducted in
Portugal. The strategy for screening tetrahydrobiopterin deficiency includes: (i) analysis of
pterins in urine and (ii) measurement of DHPR activity in blood from Guthrie cards.
We have studied an hypetphenylalaninemic (HPA) population and found two BH4
deficiencies due to an impaired DHPR activity. Here we report the clinical and biochemical
data from these two patients as well as the results of the screening performed on the HPA
population.

CASE REPORTS

Patients: A total of 41 patients with different forms ofHPA have been investigated, 27
of them were hypetphenylalaninemic newborns detected by neonatal screening (Group A),
11 were PKU children between 3-14 years old (Group B) and the others (Patient 1 and 2)
presented moderate hyperphenylalaninemia and were revealed to be BH4 deficient.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 263
 Patient 1, H.S., male, Negro race from Angola, was born of healthy and related
parents. Pregnancy and delivery were normal. He had not been submitted to the Guthrie
test for PKU mass screening. His mother reported a normal neonatal period. Mioclonic
attacks began at 2 months of age and one month later developmental delay became
obvious; the first neurological signs were noted and consisted of hypotonia of the trunk. At
the age of 11 months he was diagnosed with a West syndrome and therapy with ACTII was
started. Motor development regressed further and at the age of 3 years he was admitted to
the hospital. The clinical examination at this time revealed severe truncal hypotonia and
rigidity of the extremities, microcephaly and severe developmental delay
Amino acid analysis revealed a plasma Phe level of 924 JlmoVL. The finding of an
abnormal profile of urinary pterins (Table 1) and the deminished activity of DHPR (< 0,5
mU/mg Hb; normal= 2-5 mU/mg Hb) supported the diagnosis of DHPR deficiency. A low
Phe diet was introduced as well as the substitution with neurotransmitter precursors. His
follow-up was discontinued since his mother had to leave the country.

Patient 2, A.L., male, Caucasian race, born of consanguineous parents after an

uneventful pregnancy, showed high plasma Phe (499 J.LmoVL) in the fust days of life
(Guthrie test for PKU mass screening). At the age of 9 months irritability and abnormal
movements were noticed. At the age of 12 months the child was referred to the metabolic
unit. The most prominent findings were hypotonia of the trunk, hypertonia of the limbs,
microcephaly, hypersalivation and swallowing difficulties, poor head control, eczema and
lateral deviation of the eyes as well as severe psycho-motor delay.
At this time the plasma Phe level was 838 JlmoVL. According to the results of the
pterin analysis (Table 1) and that of the DHPR activity(< 0,5 mU/mg Hb; normal= 2-5
mU/mg Hb), a low Phe diet was supplied as well as therapy with L-Dopa/carbidopa (4
mg/k:g!day), folinic acid (15 mg/day) and 5-hydroxytryptophan (4 mg/k:g/day). A
reevaluation of BH4 metabolism was made as well as that of the neurotransmitter
metabolites in urine and in CSF during treatment. The patient's clinical condition had
improved but his developmental delay remained.

METHODS

Analysis of pterins in urine and CSF, after oxidation with Mn02, was performed by
HPLC3. The neurotransmitter metabolites - 5-hydroxyindoleacetic acid (5-HIAA) and
homovanillic acid (HVA)- were extracted from CSF and urine samples using Sep-pak C18
cartridges (Waters Assoc., Mildford, MA, USA), as previously described4. These
metabolites were then determined by reversed-phase HPLC in 10 mM acetate buffer :
methanol (90:10), pH adjusted to 3.3 with glacial acetic acid and fluorimetric detection
0.-ex= 280 nm; "-em= 320 nm). Plasma amino acids were determined as o-phtalaldehyde
(OPA)-derivatives by reversed phase HPLC, according to established methodology5.
DHPR activity was measured in erythrocytes obtained from dried blood spots of Guthrie
cards, using the method of Arai et a1.6.

RESULTS AND DISCUSSION

Our results of pterin determinations in urine confirmed that described in literature7. In

particular, we observed a decrease in the neopterin/biopterin ratio versus age (Table 1) and
a positive correlation between biopterin excretion and blood Phe levels in PKU patients.

264
No difference in DHPR activity between controls (2 - 5 mU/mg Hb) and HPA patients
(Group A and B: 4.2 ± 1.1 mU/mg Hb; N= 39) and no variation of enzyme activity versus
blood Phe levels were observed.
The clinical presentation of patients H.S. and A.L. suggested that the patients had a
deficiency of BH4. This was confirmed in both cases and shown to be due to a defect in
DHPR, the enzyme that converts quinonoid dihydrobiopterin to B~, the active form of
the coenzyme.
On admission pterin analysis of urine samples from both patients showed increased
biopterin levels and the percentage of biopterin (%B) with respect to the sum of biopterin
and neopterin was higher or equal to 80% (Table 1).

Table 1. Urinary pterins in the studied population and excretion of pterins and
neurotransmitter metabolites before and during therapy with L-Dopa/Carbidopa, 5-
hydroxytryptophan, and folinic acid in the two patients with DHPR deficiency.

Group Age PlasmaPhe Urinary Pterins Urinary neurotransmitter

JliiiOI/l mmol/mol creatinine metabolites (Jliilol/24 h)
N B %B HVA 5-HIAA
A < 1.5 m 605-3208 0.66-28.7 0.49-14.2 14-66 - -
B 3-14 y 363-1211 0.56-3.51 1.10-5.58 42-80 - -
C (patient H.S.) 3y 924 2.7 22.4 89 - -
C (patient A. L.) 8m 838 3.7 15.3 80 3.45 0.72
Treatment Dopa/Carbidopa (4 mg/kg/d); 5-0H-Trp (4 mg/kg/d); Folinic Acid (15 mg/d); diet
Patient A.L. lOrn 21 2.5 5.2 70 24.34 94.58
Controls 2-12m <70 1.1-4.0 0.5-3.0 18-63 17.6-41.7 7.4-16.8
3-14 y <60 0.2-1.7 0.5-2.7 44-77 - 12.1-35.5

Only in patient H.S., with a %B of 89, an increased level of neopterin was observed
(2.71 rnmol/mol creatinine) when compared with the values of aged-matched controls,
probably due to the lack of feedback inhibition of GTP cyclohydrolase. The DHPR
deficiency was confirmed through measurement of the enzyme activity in both patients. The
DHPR activity was also determined in the parents of A.L.. The samples from the father and
the mother showed 51 and 55%, respectively, of the mean enzyme activity of controls. The
adequate control of pterins and neurotransmitter metabolites was performed systematically
after therapy had been initiated in patient A.L.. At the onset, the monoamine metabolites
HVA and 5-HlAA were not detectable in CSF. The urinary excretion of the same
metabolites was very low (Table 1). After 2 months of therapy, HVA and 5-HlAA in CSF
increased to the normal range and an increased excretion in urine was also observed (Table
1). Although, the initial condition of the patient had improved, the severe developmental
delay remains. The late detection of the patient could account for the lack of better
improvement.
Nevertheless, these two case reports demonstrate, once more, the need for a careful
evaluation of tetrahydrobiopterin metabolism in all infants with neonatal
hyperphenylalaninemia, whether mild or transient.

265
 ACKNOWLEDGEMENTS

We are grateful to Dr. L. Vilarinho (Instituto de Genetica Medica, Porto) for sending
the samples. This work was supported in part by the Swiss National Science Foundation,
project No. 31.33897.92 and by the Helmut Horten Research Foundation (both to NB).

REFERENCES

1. N. Blau, CroMedJ. 33:17 (1992)

2. J. L. Dhondt, J Pediatr. 104:501 (1987)
3. A. Niederwieser, W. Staudenmann and E. Wetzel, J Chromtogr. 290:237 (1984)
4. I. T. de Almeida, P. P. Peralta and C. Silveira, in: "Progress in Tryptophan and
Serotonin Research", J. Bender and Kochen, eds., Walter de Gruyter, Berlin, New
York. pp.365 (1987)
5. I. T. de Almeida, Doctoral Thesis, University of Lisbon (1988)
6. N. Arai, K. Narisawa, H. Hayakawa and K. Tada, Pediatr. 70:426 (1982)
7. J. L. Dhondt, J.P. Farriaux and J. B. Hayte, Arch Fr Pediatr. 43:785 (1986)

266
A MICROTITRE PLATE METHOD FOR
MEASURING BIOPTERIN WITH
CRYOPRESERVED CRITHIDIA FASCICULATA

Robert J Leeming, SKate Hall, Helen Friday, Philip Hurley and Anne Green

Department of Clinical Chemistry

Childrens Hospital, Ladywood Middleway, Birmingham Bl6 8ET
United Kingdom

SUMMARY

The assay of biopterin derivatives in dried blood spots is used by us in initial screening
for inherited defects in tetrahydrobiopterin synthesis. The previously described method (1)
required aseptic technique and microbiological facilities. The modification detailed here has
the advantages of antibiotic cover, which overcomes these needs and microtitre plate
technology allowing the incubation time to be halved with precision and accuracy retained.
Data reduction facilities may be applied.

MATERIALS

Crithidia fasciculata (ATCC 12857)

0.2M Phosphate Buffer pH 5.0

26.89lg NazHP04.2HzO 0.425g

Made to 1 litre with distilled water. Stored in refrigerator at 4.0--6.0°C and discarded after 2
weeks.

Sterile 80% Glycerol

Glycerol 160m! Distilled water 40ml
Sterilized by autoclaving at 121 °C for 15 minutes.

Vitamin Mix

Adenine l.Og Biotin O.OOlg

Calcium pantothenate 0.3g Pyridoxine dihydrochloride O.lg
Riboflavin 0.06g Thiamine hydrochloride 0.6g
Ground together thoroughly and stored in a dry container in the refrigerator at 4.0-6.00C

Folic Acid

Folic acid 400ug/L (400mg/L folic acid in 25% ethanol diluted 1/1000 in distilled water)

Chemistry and. Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 267
Assay Culture Medium

L-Arginine hydrochloride l.Og L-Glutamic acid 2.0g

L-Histidine 0.6g DL-Isoleucine 0.2g
DL-Leucine 0.2g L-Lysine hydrochloride O.Sg
DL-Methionine 0.2g DL-Phenylalanine 0.12g
DL-Tryptophan O.l6g L-Tyrosine 0.12g
DL-Valine O.lg MgS04.7H20 1.3g
Ethylenediaminotetra-acetic acid 1.2g MnS04AH20 0.28g
K3P04.H20 0.3g ZnS04.7H20 O.lg
H3B03 O.OOlg CaCI2.2H20 O.OOlg
CoS04.7H20 0.005g CuS04.SH20 0.005g
(NI-f4)2S04.FeS04.6H20 0.002g Sucrose (Analar) 30.0g
Vitamin mix(see above) O.lg Folic acid(see above) S.Oml
Vitamin free Casamino acids (Difco) 20.0g Triethanolamine( Sigma) S.Oml
Haemin 5mg in 50%Triethanolamine lO.Ornl Chloramphenicol 0.16g
Distilled water to 1600.0rnl
This was adjusted to pH7.5 with concentrated sulphuric acid, boiled for 3 minutes in a cotton
wool stoppered flask, allowed to cool, bottled in 50ml amounts in 60ml bottles and stored
frozen at -2ooc.

Maintenance Medium

Assay culture medium(above) 160 ml 0.2M phosphate buffer (above) 25 ml

Biopterin 200ng/L 1 ml Distilled water 14 ml
Sterilized by autoclaving at 121 °C for 15 minutes.

Cryopreservation of Crithidia fasciculata

A culture of Crithidiafasciculata was grown for 48hours at 29°C in maintenance culture

medium of which 0.5 ml was added to 200m! maintenance medium and incubated at 29°C
for a further 48hours, with occasional shaking. After centrifuging at 2000rpm for 10 minutes
the supernatant was removed and replaced with 80ml assay culture medium and 20m! sterile
80% glycerol. It was mixed thoroughly, dispensed in l.Oml quantities in stoppered tubes
and frozen rapidly at -70°C in an upright position.

Stock Standard Biopterin

Biopterin (Schircks Laboratory) was dissolved in distilled water to give a concentration of
2mg/L. Smg/ml asc~rbic acid was added before storing at -200C in O.Sml quantities.

5% Cycloheximide

0.25g cycloheximide dissolved in 2.0rnl acetone.

3 ml distilled water was added.
Stored in a refrigerator at 4.0-6.00C

ASSAY PROTOCOL

A 50 ml bottle of assay culture medium and a 1 ml vial of cryopreserved Crithidia

fasciculata were thawed in an incubator at 370C. All the Crithidia fasciculata was added to the
medium with a sterile Pasteur pipette and mixed thoroughly.
300 ml phosphate buffer was boiled in a cotton wool stoppered flask for 3 minutes and
cooled to room temperature.
The stock standard biopterin was diluted 1/1000 in phosphate buffer (O.lml made to
lOOm!) to make an intermediate standard. Working standards were made by making 0, 20,

268
50, I 00, 150, 200, 250, 300, 400, 500, 700 and! OOOul of the intermediate standard to I 0 ml
with phosphate buffer
Samples were prepared by punching 8.0mm discs from blood spots into 2.0 ml of
phosphate buffer (calculated 1/120 of blood), 0.1 ml of plasma was added to 2.9 ml of
phosphate buffer (1/30) and 0.1 ml of whole blood added to 3.9 ml buffer ( 1/40) in glass test
tubes. The tubes were placed in a steamer for 10 minutes and the contents centrifuged and
filtered through 0.4 micron syringe filters into glass test tubes. Further dilutions were made if
necessary.
50 ul of standards and samples were placed in quadruplicate in flat bottomed wells in
microtitre plates followed by 200ul of culture medium. One well of each quadruplicate set
was used as a blank by adding 10 ul of 5% cycloheximide which inhibited growth without
altering the optical density significantly. A sterile lid was placed on each plate which was then
left in a humidity chamber at 290C for 48 hours. After mixing optical density was read at
595nm in a microtitre plate reader . Readings from samples were corrected for dilutions. A
typical standard curve is shown in figure 1.

400

300
0
0
0

X
200
0
0
100

0
0 100 200

Biopterin ng/litre
Figure 1 A Typical Standard Curve

SENSITIVITY

The lowest standard contained 0.2pg/50ul which was equivalent to 0.48ug/L in blood
spots or 0.12ug/L in plasma at the dilutions given above. Lower concentrations could be
measured by decreasing the dilutions of samples.

PRECISION

Multiple blood spots on Guthrie cards were spotted with blood from 6 normal adult
volunteers and from one phenylalanine hydroxylase deficient patient on a relaxed diet. 8mm
discs were punched out and divided between two assay runs.
The within batch coefficient of variation for normals was 8.8% and between batch
13.2% (range 1.4- 5.0 ug/L biopterin). The within batch coefficient of variation for the
phenylalanine hydroxylase deficient patient was 8.5% and between batch 12.7% (range 7.2-
10.6ug/l). The values were influenced by variability in the quality of the dried blood spots,
this was shown clearly when 7 preparations from whole blood and plasma samples were
divided into 57 aliquots and the measured biopterin concentrations gave a 4.8% coefficient of
variation.

269
COMPARISON OF MICROTITRE PLATE WITH TUBE METHOD
50 dried blood spot samples had biopterin measured by the tube method (1) and by
microtitre plate modification. Regression analysis gave a good correlation (R = 0.96) and
microtitre values were, on average, 15% higher than by the tube method. The blood spot
phenylalanine concentrations (available from requesting laboratories) showed only a modest
correlation (R =0.58) with biopterin, this was anticipated as red cell biopterin reflects chronic
hyperphenylalaninaemia, unlike plasma biopterin (R= 0.86) which reaches a peak 2.0 hours
after oral phenylalanine (3) and is therefore more closely related to the phenylalanine
concentration at the time of sampling. The blood spot biopterin concentrations grouped
according to phenylalanine concentrations are shown in figure 2 which includes specimens
from patients with tetrahydrobiopterin synthesis deficiency.

40
I

-
~
Q)
.....

~
30
•
• •
c •
-~
a.0 20 • •
I
I
iD I I
Q)
I • I
~
*".....0 I I
10
•
~
0

I
••
•
I •
X
0
2 3 4 5 6

Phenylalanine
1 = Normal Subjects, 2 = PKU<1 00, 3 = 100-200, 4 = 200-800, 5=800-1600, 6 = >1600 llmol/1

x = Tetrahydrobiopterin Synthesis Deficient

Figure 2 Microtitre Biopterin Measurements in 50 Blood Spots Grouped According
to Phenylalanine Concentrations

DISCUSSION
The microtitre plate method for measuring biopterin with Crithidia fasciculata is
economical in time and materials. As medium, culture and standard are accessible from cold
storage, the response time is minimal and results can be available in 48 hours.
Reproducibility is satisfactory for screening dried blood spots as there is such a clear
differentiation of biopterin synthesis deficiency from phenylalanine hydroxylase deficiency.
This modification is now used in conjunction with measurement of dihydropteridine
reductase (4) as the first line screening procedure for inherited tetrahydrobiopterin deficiency.

REFERENCES
l. R.J.Leeming, P.A.Barford,J.A.Blair and I.Smith .
Arch. Dis. Childhood 59: 58-61 (1984)

2. R.J.Leeming, S.K.Hall, I.M.Surplice and A.Green .

J. Inher. Metab. Dis. 13: 883-887 (1990)

3. R.J.Leeming , J.A.Blair,A.Green and D.N.Raine.

Arch. Dis. Childhood 51:771-777 (1976)

4. I.M.Surplice, P.D.Griffiths, A.Green and R.J.Leeming.

J. Inher. Metab. Dis. 13: 169-171 (1990)

270
ORAL ADMINISTRATION OF LIPOSOMALLY ENTRAPPED
TETRAHYDROBIOPTERIN

Yoshitomo Sawada\ Haruo Shintaku2 , Tatsuo Nakajima 2, Takuji Imamura 2,

Yuriko Tsubakio 1 , Chiyo Iwamura 1 , Gen Isshike, and Toshiaki Ohura3
1Dept. of Pediatrics Juso Citizens' Hospital, Osaka 532, Japan
2Dept. of Pediatrics Osaka City University Medical School, Osaka 545, Japan
3 0saka Municipal Rehabilitation Center for the Disabled, Osaka 547, Japan

INTRODUCTION
Tetrahydrobiopterin (BH4) has been used for the therapy of BH4-deficient patients.
However, BH4 is poorly absorbed from the intestine. The plasma level of biopterin that was
reached after oral administration of BH4 to a BH4-deficient patient was reported to be 1-2%
of the levels reached after either intravenous or subcutaneous administration1 • This poor
absorption from the intestine needs a large amount of BH4 for the oral therapy, leading to
high costs.
Recently, liposomes have been widely used as a drug delivery system. A substance
such as factor VIII is absorbed into the circulation when administered orally in a liposomally
entrapped form 2• To facilitate absorption of BH4 from the intestine, we used a liposome for
the first time.

MATERIALS & METHODS

BH4 was a gift from Suntory Limited, Japan. Distearoylphosphatidylcholine ((18:0)
DSPC) was purchased from Nihon Seika Limited, Japan. Cholesterol and dicetylphosphate
were purchased from Sigma. Liposomes were prepared as described by Patee with
modifications from the following lipid compositions: DSPC/cholesterol/dicetylphosphate
(10:10:1, molar ratio). These lipids were mixed with 20 ml of chloroform water in the flask
and evaporated to dryness by a rotary evaporator at 5YC, the transition temperature of
DSPC, forming a thin layer membrane of the inner surface of the flask. Multilamellar
liposomes containing BH4 were formed by shaking the flask vigorously on a vortex mixer
after adding BH4 dissolved in lOml of 5% glucose. Final concentrations of DSPC, cholesterol
and dicetylphosphate were lOOmM, lOOmM and lOmM, respectively. Concentrations of
biopterin in the liposomally entrapped BH4 were measured after destroying the liposomal
membrane by triton X.
Guinea-pigs (Hartley strain) were anesthetized with 20mg/kg of 5% nembutal. A
silicon catheter with a 0.5mm inner diameter was placed in the carotid artery for a blood
collection. 5mg/kg of liposomally entrapped form of BH4 or free BH4 was administered
orally with nasal tube to guinea pigs, separately. Blood was collected hourly from carotid
artery. Concentrations of plasma biopterin were measured by the method described by
Fukushima and Nixon 4 •

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 271
 RESULTS & DISCUSSION

The plasma biopterin level reached a maximum one hour after administration of both
liposomally entrapped BH4 and free BH4. The plasma biopterin level at peak was significantly
higher in the liposomally entrapped BH4 than that in the free BH4. Moreover, a plasma
biopterin level four hours after administration was also significantly higher in liposomally
entrapped BH4 than that in the free BH4 (Fig 1) .

Biopterin level (nM)

300 -

240

210

180

150

120

90

60

30

0 I
0 2 3 4
Time (hr)
Figure 1. Comparison of changes in plasma biopterin levels after oral administration of liposomal BH4 and
free BH4. (e:liposomal BH4, D :free BH4)

This result suggests that liposomes improved intestinal absorption of BH4 . If

liposomally entrapped BH4 is available for treatment of BH4-deficient patients, it will be
contributed to the reduction of BH4 dosage, leading to low costs.

REFERENCES

1. S. Kaufman, Hyperphenylalaninaemia caused by defects in biopterin metabolism (Raine Memorial Lecture),

J lnher Metab Dis. 8 Suppl.l, 20 (1985).
2. N.Sakuragawa, K. Niiya, S. Kondo, Oral administration of factor VIII concentrate preparation in Von
Willebrand's disease, Thrombos Res. 38.681(1985).
3. H.M.Patel, N.S. Tuzel, and R.W. Stevenson, Intracellular digestion of saturated and unsaturated phospholipid
liposomes by mucosal cells. Possible mechanism of transport of liposomaly entrapped macromolecules
across the isolated vascularly perfused rabbit ileum, Biochim Biophys Acta 839:40 (1985).
4. T.Fukushima, and J.C.Nixon, Analysis of reduced biopterin in biological tissues and fluids, Anal Biochem
102:176 (1980).

272
EXPERIMENTAL RESEARCH ON A FETAL TREATMENT FOR
TETRAHYDROBIOPTERIN DEFICIENCY

Haruo Shintaku\ Tatsuo Nakajima1 , Takuji Imamura\ Yoshitomo Sawada2,

Gen Isshiki 1 and Toshiaki Oura 3
1Dept. of Pediatrics Osaka City University Medical School, Osaka 545, Japan
2Dept. of Pediatrics Juso Citizens' Hospital, Osaka 532, Japan
3 0saka Municipal Rehabilitation Center for the Disabled, Osaka 547, Japan

INTRODUCTION
Tetrahydrobiopterin (BH4) synthase deficiency has a high incidence of low birth
weight, 1 and some of them had a mild mental retardation in spite of their early treatmene.
In this study we performed an intravenous loading of 2,4-diamino-6-hydroxypyrimidine
(DAHP) with a small amount of BH4, and successfully made a model of fetal BH4 deficiency.
We investigated the possibility of the fetal therapy of BH4 deficiency in this model by
measurements of phenylalanine(Phe ), tyrosine(Tyr), BH4, dopamine and catecholamines.

MATERIALS AND METHODS

Guinea-pigs (Hartley strain) in the third trimester of pregnancy were anesthetized
with 20 mg/k:g of 5% nembutal. A silicon catheter with 0.5 mm inner diameter was placed
in the carotid artery for a blood collection and another one was in the carotid vein for
infusion of DAHP (a potent inhibitor of guanosine triphosphate cyclohydrolase I) and
6R-L-5,6,7,8-tetrahydrobiopterin (R-BH4). Guinea-pigs were divided into three groups.
The intravenous loading of DAHP (60 mg/kg/hour) and small amount ofR-BH4( 40 f.lg/kg/hr)
were performed continuously by the injection pump in both of the first group (the DAHP
group) and the second group (the DAHP+BH4 group) for 72 hours. This condition would
keep the mothers' biopterin levels normal, but not the fetuses' levels because this small
amount of BH4 could not pass through the placenta. To the second group the same dose of
DAHP (60mg/kg/hr) and large amount ofR-BH4 (1mg/kg/hr) were injected moreover for 6
hours. The third group (the control group) was injected with half concentration of saline for
72 hours.
The blood samples of the mothers were obtained from arterial catheter and fetal
blood was obtained by the umbilical cord puncture or cardiopuncture. Phe, Tyr and BH4
levels of plasma, liver, adrenal and brain were measured by a high-performance liquid
chromatography (HPLC).
Every samples were added 0.5 M PCA and homogenized in cold water. After
centrifugation (15000 rpm, 10 min), the supernatants were injected directly in volume of
10f.ll to HPLC and dopamine and catecholamines were measured by electrochemical detector
(ECD). The detector was operated at a potential of 750 mV vs. Ag/AgCl. The mobile phase
consisted of 0.6 v/v % triethylamine, 8 v/v % acetonitrile, 0.1 mM EDTA-2Na and lOmM
heptanesulphonate-Na, and pH adjusted to 2.70 with orthophosphoric acid. The flow-rate

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 273
was maintained at 0.8 ml/min. The mobile phase was filtered through a 0.2 J.tm filter and
then degassed under vacuum before use 3•

RESULTS
Basal values of plasma BH4 were 56 nM in the mothers and 150 nM in their fetuses.
In DAHP group, plasma BH4 levels were 66 nM in the mothers and 11 nM in their fetuses.
While in DAHP+BH4 group, BH4levels were 1500nM in the mothers and 250 nM in their
fetuses. Basal values of plasma Phe and Tyr were 0.75 mg/dl and 0.87 mg/dl in the
mothers, and 1.31 mg/dl and 1.48 mg/dl in their fetuses, respectively. In DAHP group, Phe
and Tyr levels were 0.85 mg/dl and 0.54 mg/dl in the mothers, and 4.61 mg/dl and 1.75
mg/dl in their fetuses, respectively. While in DAHP+BH4 group Phe and Tyr levels were
0.77 mg/dl and 0.86 mg/dl in mothers, and 1.00 mg/dl and 1.66 mg/dl in their fetuses,
respectively. (Table 1.)

Table 1. Plasma biopterin, phenylalanine and tyrosine levels in BH4 deficiency

model of guinea-pig before and after DAHP and R-BH4loading. ( mean±SD).

Group Biopterin Phenylalanine Tyrosine

nM mg/dl mg/dl
Mother Control 56 ± 21 0.75 ± 0.26 0.87 ± 0.35
DAHP 66 ± 24 0.85 ± 0.49 0.54 ± 0.42
DAHP+BH4 1500 ± 400 0.77 ± 0.33 0.86 ± 0.33
Fetus Control 150 ± 62 1.31 ± 0.41 1.48 ± 0.53
DAHP 11 ± 4 4.61 ± 0.77 1.75 ± 0.21
DAHP+BH4 250 ± 91 1.00 ± 0.34 1.66 ± 0.58

Basal values of BH4 in liver were 2.3 nmol/g tissue in the mothers and 1.0 nmol/g
tissue in their fetuses. In DAHP group, BH4 levels were 1.4 nmol/g tissue in the mothers
and 0.07 nmol/g tissue in their fetuses. While in DAHP+BH4 group, BH4 levels were 22.1
nmol/g tissue in the mothers and 0.7 nmol/g tissue in their fetuses. Basal values of liver Phe
and Tyr were 8.0 J.lg/g tissue and 10.5 J.lg/g tissue in the mothers, and 11.7 J.lg/g tissue and
18.0 J.lg/g tissue in their fetuses, respectively. In DAHP group, Phe and Tyr levels were
12.5 J.tg/g tissue and 11.5 J.tg/g tissue in the mothers, and 29.7 J.lg/g tissue and 16.6 J.lg/g
tissue in their fetuses, respectively. While in DAHP+BH4 group Phe and Tyr levels were
7.8 J.lg/g tissue and 14.5 J.lg/g tissue in mothers, and 12.8 J.lg/g tissue and 21.4 J.lg/g tissue in
their fetuses, respectively. (Table 2.)

Table 2. Biopterin, phenylalanine and tyrosine levels in liver. (mean±SD).

Group Biopterin Phenylalanine Tyrosine

nmol/g tissue }lg/g tissue J.l g/g tissue
Mother Control 2.3 ± 0.6 8.0 ± 2.1 10.5 ± 3.3
DAHP 1.4 ± 1.5 12.5 ± 4.3 11.5 ± 4.7
DAHP+BH4 22.1 ± 6.3 7.8 ± 3.8 14.5 ± 6.6
Fetus Control 1.0 ± 0.4 11.7 ± 6.4 18.0 ± 7.5
DAHP 0.07 ± 0.02 29.7 ± 13.7 16.6 ± 6.1
DAHP+BH4 0.7 ± 0.3 12.8 ± 4.1 21.4 ± 6.2

Basal values of BH4 in adrenal glands were 610 nmol/g tissue in the mothers and
3000 nmol/g tissue in their fetuses. In DAHP group, BH4 levels were 590 nmol/g tissue in
the mothers and 860 nmol/g tissue in their fetuses. While in DAHP+BH4 group, BH4 levels
were 790 nmol/g tissue in the mothers and 1500 nmol/g tissue in their fetuses. Basal values

274
of dopamine, norepinephrine, and epinephrine were 0.4 !lg/g tissue, 3.5 llglg tissue and
53.2!lg/g tissue in the mothers, and 1.9!lg/g tissue, 17.9!lg/g tissue and 61.0 !lg/g tissue in
their fetuses, respectively. In DAHP group, dopamine, norepinephrine, and epinephrine
were 0.3 11g!g tissue, 2.4 !lg/g tissue and 19.5 !lg/g tissue in the mothers, and 0.4 !lg/g
tissue, 0.4!lg/g tissue and 16.6!lg/g tissue in their fetuses, respectively. While in DAHP+BH4
group dopamine, norepinephrine, and epinephrine were 0.4!lg/g tissue, 3.7 llglg tissue and
50.7 !lg/g tissue in the mothers, and 1.3!lg/g tissue, 7.2!lg/g tissue and 47.6!lg/g tissue in
their fetuses, respectively. (Table 3.)

Table 3. Biopterin, dopamine, norepinephrine, and epinephrine levels in

adrenal glands. (mean±SD)

Group Biopterin Dopamine Norepinephrine Epinephrine

pmol/g tissue ).t gig tissue ).tg/g tissue ).t g/g tissue
Mother Control 610 ± 300 0.4 ± 0.3 3.5 ± 0.7 53.2 ± 12.9
DAHP 590 ± 97 0.3 ± 0.1 2.4 ± 1.3 19.5 ± 4.4
DAHP+BH4 790 ± 390 0.4 ± 0.2 3.7 ± 1.8 50.7 ± 17.8
Fetus Control 3000 ± 1300 1.9 ± 0.5 17.9 ± 6.5 61.0 ± 33.4
DAHP 860 ± 140 0.4 ± 0.2 0.4 ± 0.3 16.6 ± 12.0
DAHP+BH4 1500 ± 790 1.3 ± 0.6 7.2 ± 3.0 47.6 ± 9.8

Basal values of striatum BH4 in the brain were 510 pmol/g tissue in the mothers and
440 pmol/g tissue in their fetuses. In DAHP group, BH4 levels were 400 pmol/g tissue in
the mothers and 220 pmol/g tissue in their fetuses. While in DAHP+BH4 group, BH4 levels
were 470 pmol/g tissue in the mothers and 480 pmol/g tissue in their fetuses. Basal values
of striatum dopamine were 1.3 11g/g tissue in the mothers, and 0.8 !lg/g tissue in their
fetuses, respectively. In DAHP group, dopamine levels were 1.0 !lg/g tissue in the mothers
and 0.5!lg/g tissue in their fetuses. While in DAHP+BH4 group, dopamine levels were 1.2
!lg/g tissue and 0.9 !lg/g tissue in their fetuses. (Table 4.)

Table 4. Biopterin and dopamine levels in striatum. (mean±SD)

Group Biopterin Dopamine

pmol/g tissue J.t g/g tissue
Mother Control 510 ± 120 1.3 ± 0.4
DAHP 400 ± 58 1.0 ± 0.5
DAHP+BH4 470 ± 120 1.2 ± 0.9
Fetus Control 440 ± 110 0.8 ± 0.5
DAHP 220 ± 26 0.5 ± 0.1
DAHP+BH4 480 ± 170 0.9 ± 0.2

These results suggest that the R-BH4 administered to the mothers passed through
the placenta and increased the BH4levels in their fetuses.

DISCUSSION
For the experimental research on a fetal treatment of BH4 deficiency, we made a
model of fetal guinea-pigs with BH4 deficiency. We have reported previously a model of
fetal guinea-pigs with BH4 deficiency by an oral administration of DAHP to their mothers 4•5•
In this study we performed an intravenous loading of DAHP with a small amount of BH4
continuously by a syringe pump, and successfully made a model of fetal BH4 deficiency,
which keep mother's biopterin levels normal.
In the DAHP group, total biopterin levels in fetal plasma, liver, adrenal and striatum
decreased approximately to 7, 7, 29 and 49% of the control group, respectively. While

275
plasma and liver phenylalanine levels elevated to 3.5, 2.5 times, adrenal and striatum
dopamine levels decreased to 21, 57%, and adrenal norepinephrine and epinephrine levels
decreased to 2, 27% of the control group, respectively. In the DAHP+BH4 group, these
changes mentioned above were almost reduced to the control group levels.
In this study BH4 administered to the mothers passed through their placenta and
stimulated the aromatic amino acid hydroxylases in their fetuses' organs. These results
indicate that the R-BH4 administration to the mother should be effective to the fetal treatment
for BH4 deficiency.

REFERENCES
1. I. Smith, J.L. Dhondt, Birth weight in patients with defective biopterin metabolism, Lancet, i:818(1985).
2. Y. Hase, Y. Okano, Y. Sawada, et al., Early treatment of inborn errors of biopterin metabolism, Acta
Paediatri. Jpn., 30:390(1988).
3. Y. Tani, T. Ishihara, Simultaneous measurement of tetrahydrobiopterin (THBP) and biogenic amines by
liquid chromatography with electrochemical detection, Life Sci., 46:373(1990).
4. M. Fujioka, H. Shintaku, G. Isshiki, eta!., Fetal, in:"Chemistry and Biology of Pteridines," H-CH. Curti us,
eta!., ed., Walter de Gruyter, Berlin (1990).
5. T. Nakajima, H. Shintaku, Y. Sawada, et al., Fetal guinea-pig model of tetrahydrobiopterin deficiency,
Pteridines, 3:35(1992).

276
EXPERIMENTAL RESEARCH ON A NEW TREATMENT FOR MATERNAL
PHENYLKETONURIA(PKU)

Takuji Imamura1, Haruo Shintaku1, Tatsuo Nakajima\ Yoshitomo Sawada2,

Gen Isshiki1 and Toshiaki Oura3

1Dept. of Pediatrics, Osaka City University Medical School, Osaka 545, Japan
2Dept. of Pediatrics, Juso Citizens' Hospital, Osaka 532, Japan
30saka Municipal Rehabilitation Center for the Disabled, Osaka 547, Japan

INTRODUCTION

More girls with phenylketonuria (PKU) enter childbearing ages, and most such women
are mentally normal, having been born since newborn screening was initiated in the 1970s
and treated from early infancy with a low phenylalanine (Phe) diet. Women with PKU not
treated prior to conception can have a pregnancy that results in serious fetal damage 1•
Maternal PKU as a cause of mental retardation and birth defects is a new phenomenon.
There will be an increased need for specific therapies in maternal PKU. Low Phe diet is
essential for the treatment of maternal PKU. It should be started before pregnancy and it
is necessary to maintain their plasma Phe levels around 5 mg/dl throughout their
pregnancy2• However they are usually controlled around 10 mg/dl because of the difficulty
of the diet therapy. We made an animal model of maternal PKU by the intravenous
injection of Phe to pregnant guinea-pigs, and examined plasma, liver and brain Phe levels
in their fetuses after an intravenous administration of 6R-5,6,7,8-tetrahydrobiopterin (R-
BH4) to the mothers.

MATERIALS AND METHODS

Guinea-pigs (Hertley strain) in the third trimester of pregnancy were anesthetized with
20mg/kg of 5% nembutal. A silicon catheter (inner diameter 0.5mm) was placed in the
carotid artery for a blood collection and another one was in the carotid vein for infusion
of Phe. The intravenous loading of Phe (80mg/kg/hour) were performed in one group (Phe
group) of the animals for 18 hours and in an another group (Phe+BH4 group) for 22 hours
continuously by the injection pump. The Phe+BH4 group were loaded with both Phe and
R-BH4 (2mg/kg/hour) simultaneously for the last 4 hours. The blood samples of the
mothers were obtained from arterial catheter and fetal blood was obtained by the umbilical
cord puncture or cardiopuncture. Plasma, liver and brain levels of BH4, Phe and Tyr were
measured by a high-performance liquid chromatography.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 277
RESULTS
Basal values of plasma BH4 were 53 nM in the mothers and 80 nM in their fetuses.
In Phe group, plasma BH4 levels were 120 nM in the mothers and 120 nM in their fetuses.
While in Phe+BH4 group, BH4 levels were 5600nM in the mothers and 2500nM in their
fetuses. Basal values of plasma Phe and Tyr were 0.6 mg/dl and 0.6 mg/dl in the mothers,
and 1.4 mg/dl and 1.9 mgldl in their fetuses, respectively. In Phe group, Phe and Tyr levels
were 10.2 mg/dl and 9.0 mgldl in the mothers, and 25.8 mg/dl and 20.4 mg/dl in their
fetuses, respectively. While in Phe+BH4 group Phe and Tyr levels were 7.0 mgldl and 18.1
mg/dl in mothers, and 9.7 mg/dl and 29.3 mg/dl in their fetuses, respectively.(Fig 1.)

40 10000

Mother • Fetus
•
~ • Phe

..
30 1000 ~
.§. 0 Tyr .s
"'
o; o;
>
~ Q)
...J
~ .!:
20 100 Q;
"""c:::
"0 a0
"' iii
"
.<=
0.. "'
E
"'
E
iQ 10 10 £
c::

0
Control Phe Phe+BH4 Control Phe Phe+BH4
group group group group group group

Fig 1. Plasma concentrations of BH4, Phe and Tyr in mothers and fetuses after administration of Phe or
Phe+BH4•

400 20
•
., Mother Fetus
.,
~"' 300
• Phe "'
$
!!!
CJ Tyr
• Blopterln
·~
g
0
E
.,>
Q)
.s
...J
200 10 "'>
o;
~ .3
"0
c::: ...
"'
Q)
.<=
:X:
IXl
0.. Q;
>
Q; 100 ::;
>
::;

0 0
Control Phe Phe+BH4 Control Phe Phe+BH4
group group group group group group

Fig 2. Liver concentrations of BH4 , Phe and Tyr in mothers and fetuses after administration of Phe or
Phe+BH4•

278
 Basal values of liver BH4 were 2.3 nmol/g tissue in the mothers and 0.8 nmol/g tissue
in their fetuses. In Phe group, BH4 levels were 6.0 nmol/g tissue in the mothers and 1.0
nmol/g tissue in their fetuses. While in Phe+BH4 group, BH4 levels were 19.5 nmol/g tissue
in the mothers and 1.3 nmol/g tissue in their fetuses. Basal values of liver Phe and Tyr
were 8.0 pg/g tissue 10.5 pg/g tissue in the mothers, and 17.9 pg/g tissue and 35.0 pg/g
tissue in their fetuses, respectively.ln Phe group, Phe and Tyr levels were 45.0 pg/g tissue
and 60.0 pg/g tissue in the mothers, and 150 pg/g tissue and 107 pg/g tissue in their
fetuses, respectively. While in Phe+BH4 group Phe and Tyr levels were 33.2 pg/g tissue
and 120 pg/g tissue in mothers, and 74.4 pg/g tissue and 337 pg/g tissue in their fetuses,
respectively.(Fig 2.)
Basal values of brain BH4 were 190 pmol/g tissue in the mothers and 205 pmol/g
tissue in their fetuses. In Phe group, BH4 levels were 223 pmol/g tissue in the mothers and
353 pmol/g tissue in their fetuses. While in Phe+BH4 group, BH4 levels were 410 pmol/g
tissue in the mothers and 411 pmol/g tissue in their fetuses. Basal values of brain Phe and
Tyr were 5.0 pg/g tissue and 7.0 pg/g tissue in the mothers, and 19 pg/g tissue and 37 pg/g
tissue in their fetuses, respectively. In Phe group, Phe and Tyr levels were 49 pg/g tissue
and 59 pg/g tissue in the mothers, and 120 pg/g tissue and 85 pg/g tissue in their fetuses,
respectively. While in Phe+BH4 group, Phe and Tyr levels were 35 pg/g tissue and 120 pg/g
tissue in mothers, and 63 pg/g tissue and 220 pg/g tissue in their fetuses, respectively.(Fig
3.)

3 0 0 r - - -- - - - - - . -- - - - - - - - 1000

Mother Fetus
• • •
Qj

.ii!". • • • Qj

J.. 200 100 g.:!!"

o;
>
Q)
.j
• Phe
Cl Tyr l
~
• Biopterin
~
..
'0
c
.,
.c
10
:t
<ll
c
a. '16
c
·e .n
<ll

Control Phe Phe+ BH4 Control Phe Phe+BH4

group group group group group group

Fig 3. Brain concentrations of BH4, Phe and Tyr in mothers and fetuses after administration of Phe or
Phe+BH4 •

This results suggests that the R-BH4 administered to the mothers passed through the
placenta and increased the BH4 levels in their fetuses. Phe levels were lower and Tyr levels
were higher in Phe+BH4 group than in Phe group of plasma, liver and brain . This results
suggests that the BH4 passed through the placenta stimulated Phenylalanine hydroxylase
activity in their fetal liver so that Phe decreased and Tyr increased in the fetal plasma, liver
and brain.

279
 DISCUSSION
Low Phe diet is essential for the treatment of maternal PKU. It should be started
before pregnancy and it is necessary to maintain their plasma Phe levels around 5 mg/dl
throughout their pregnancy. However they are usually controlled around 10 mg/dl because
of the difficulty of the diet therapy. We made an animal model of maternal PKU by the
intravenous injection of Phe to pregnant guinea-pigs, and examined plasma Phe levels in
their fetuses after an intravenous administration of BH4 to the mothers. In the pregnant
guinea-pigs with hyperphenylalaninemia their fetuses had very high levels of plasma Phe,
but after an administration of BH4 to the mothers with hyperphenylalaninemia their fetuses
had much lower plasma Phe levels than without a BH4 administration. These results
suggests that BH4 administered to the mothers passed through their placenta and stimulated
the phenylalanine hydroxylase in their fetuses' live~. It had been reported that human liver
phenylalanine hydroxylase activity in vitro are the same in fetuses after 8 gestational weeks
as in adults4• Therefore we think that the BH4 administration is effective for the therapy for
maternal PKU even in human if it combines with a mild low Phe diet.

REFERENCES
1. E.Drogari, !.Smith, M.Beaseley, and J.K.Lloyd, Timing of strict diet in relation to fetal damage in
maternal phenylketonuria, Lancet ii: 927-930 (1987)
2. W.B.Hanley, J.T.R. Clarke, and W. Schoonheyt, Maternal phenylketonuria(PKU)-a review. Clin.
Biochem. 20: 149-156 (1987)
3. P.A. Friedman, and S. Kaufman, A study of the development of phenylalanine hydroxylase in
fetuses of several mammalian species. Arch. Biochem. 146: 321-326 (1971)
4. N.C.R. Riiihil, Phenylalanine hydroxylase in human liver during development. Pediat. Res. 7: 1-4
(1973)

280
NITRIC OXIDE SYNTHASE: FUNCTION AND MECHANISM

Michael A. Marietta

Interdepartmental Program in Medicinal Chemistry, College of Pharmacy

Department of Biological Chemistry, Medical School
University of Michigan, Ann Arbor, MI 48109-1065 USA

INTRODUCTION

The discovery of nitric oxide (•NO) as a mammalian metabolic intermediate has

developed into a swiftly moving area of research. The intense interest and rapid progress
have developed primarily because •NO controls and influences a number of critical
physiological processes. The formation of •NO from L-arginine in mammalian cells is
catalyzed by the enzyme •NO synthase (NOS; EC 1.14.23) (Figure 1). The best
characterized examples of biological reactions controlled by •NO include vasodilation and
regulation of normal vascular tone, inhibition of platelet aggregation, neuronal
transmission and cytostasis [for reviews, see1-5]. In addition to these activities it appears
that abnormally high levels of •NO are also involved in the hyfotension associated with
endotoxic shock6, inflammatory response-induced tissue injury , mutagenesis8•9, and the
formation of carcinogenic N-nitrosamines 10. In tissues where •NO controls normal
vascular tone, low levels can also be deleterious such as in the case of pulmonary
hypertension in infants and adults. •NO has very recently been found to be useful in the
treatment of this type of hypertension.

L-arginine NG-hydroxy-L-arginine citrulline nitric oxide

Figure 1: The reaction catalyzed by nitric oxide synthase showing ~-hydroxy-L-arginine as an

intermediate.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling eta!., Plenum Press, New York, 1993 281
GENERAL CHARACTERIZATION OF NITRIC OXIDE SYNTHASE

The family of NOS isoforms thus far characterized generally fall into two categories:
(i) a constitutive form regulated by Ca2+ and calmodulin, and ~ii) a cytokine-inducible form
that is not known to be regulated post-transcriptionally 1 . All of the NOSs require
NADPH and 02 as co-substrates in the reaction yielding citrulline as the amino acid
product of the reaction along with •NO. Cytosolic and membrane-bound NOSs have been
isolated. Constitutive NOSs isolated from rat 12•13 and porcine cerebellum14 are cytosolic
=
proteins (Mr 150 - 160 kDa). The inducible NOS purified from LPSIIFN-y treated
murine macrophages is also a cytosolic protein that has a Mr = 130 kDa and is a dimer
under native conditions 15 ·16 . However, a constitutive endothelial NOS isoform purified
from bovine aortic endothelial cells is membrane bound with a Mr = 135 kDa17 . A number
of additional NOSs have been described, however, this short review will focus only on
those best characterized. The mechanistic results described have, for the most part, been
obtained from studies with the inducible macrophage NOS.
The amino acid sequences derived from the isolated cDNAs for the constitutive
NOSs from rat cerebellum, and bovine aortic endothelial cells have now been re~orted 18 -20
as well as the corresponding data for the murine macrophage inducible NOS 1-23 . The
derived sequence from rat brain showed significant homology to NADPH cytochrome P-
450 reductase 18 . The sequences associated with NADPH, FAD, and FMN were all highly
conserved. The cloni¥§ and functional expression of a human endothelial NOS has also
recently been reported . It shows 52% amino acid identity when compared to the rat brain
NOS and a predicted molecular weight of 144 kDa The sequence of the particulate bovine
aortic endothelial cell NOS contains a myristoylation consensus sequence at the N-
terminus which is absent in the macrophage and rat cerebellar NOS isoforms 19 . All the
constitutive NOSs show, as expected, a consensus sequence for calmodulin recof3nition.
Surprisingly, the macrophage eDNA also has a calmodulin recognition sequence21 - .

COFACTORS AND PROSTHETIC GROUPS

The NOSs are complicated proteins. The constitutive forms show an absolute
requirement for Ca2+ and calmodulin. Although a requirement was not observed, the
inducible macrophage isoform has been shown to have a tightly bound calmodulin that is
apparently activated by very low levels of Ca2+25 , suggesting a novel type of regulation.
All NOSs require NADPH for activity 5. As predicted by the eDNA sequence, the
macrophage NOS has been shown to contain 1 equivalent each of FAD and FMN per
subunit15 . Subsequent reports have also verified that the other NOSs contain these same
flavins, however, the reported stoichiometries differ from that of the macrophage26-28 . The
eDNA sequences predict that all NOSs should contain 1 equivalent each ofFAD and FMN.
Differences from this stoichiometry probably reflect loss of flavin during the purification.
The macrophage NOS has been shown to contain a cytochrome P-450 type iron-
protopo~hyrin IX (Fe-PPIX) prosthetic group that functions in the turnover of L-
arginine . CO inhibition of the reaction supports a catalytic role for this heme29 . Similar
results were subsequently reported for the cerebellar NOS isoform 30•31 . Participation of
the cofactor (6R)-tetrahydro-L-biopterin (H4B) was reported, first in the macrophage32•33 ,
and subsequently in the constitutive NOS isoform from porcine brain26. Investigations
from a number of laboratories have continued to support a role for the reduced pterin in the
reaction, however a clear function has yet to emerge from the studies carried out thus far.

MECHANISM OF THE REACTION

NG-h~droxy-L-arginine (NHA) has been shown to be an intermediate in the

reaction 34·3 . A key mechanistic result is that CO inhibits the formation of •NO from
NHA as well as from L-arginine (Pufahl and Marietta, unpublished results). This result

282
implicates the heme moiety in overall conversion. A scheme consistent with all the results
reported to date involves a P-450-type hydroxylation of L-arginine to yield the
intermediate NHA followed by oxidation of NHA by the ferrous-oxy heme complex. This
NHA radical is then nucleophilically attacked at the guanido carbon by the ferric peroxy
anion. Subsequent decomposition of this complex leads to •NO and citrulline5.

FUNCTION OF THE REDUCED PTERIN

The first report of the involvement of I4B preceded the finding of Fe-herne cofactor
and hence it was reasonable to propose that this pterin was involved in the hydroxylation
chemist~ that was known to take place. The results described above plus additional
studies3 •37 suggest a nontraditional role for this reduced pterin. Reported elsewhere in
this volume (Hevel and Marietta) are studies that show that NOS isolated in a pterin-
deficient state is relatively uncoupled with respect to NADPH utilization and product
formed. The enzyme can be made more coupled by the addition of exogenous I4B to a
pterin-deficient preparation. The is due specifically to the reduced pterin as other reducing
agents such as DTT and ascorbate acid do not effectively substitute. This effect of l4B is
observed at very low concentrations. The mechanism of this coupling effect is currently
under study.

ACKNOWLEDGMENTS

The author is grateful to Dr. Joanie Hevel, Robert Pufahl, and Kimberly White for the
studies summarized in this report. In addition, generous support from the Nlli (CA50414),
and the Burroughs Wellcome Fund is gratefully acknowledged.

REFERENCES

1. J. Garthwaite, Trends Neurosci. 14:60 (1991).

2. M.A. Marietta, M.A. Tayeh andJ.M. Hevei, BioFactors. 2:219 (1990).
3. L.J. Ignarro, Ann. Rev. of Pharmacal. Toxicol. 30:535 (1990).
4. S. Moncada, R.M.J. Pahner and E.A. Higgs, Pharmacal. Rev. 43:109 (1991).
5. M.A. Marietta, 1 Biol. Chem. in press (1993).
6. R.G. Kilbourn, S.S. Gross, A. Jubran, J. Adams, O.W. Griffith, R. Levi and R.F. Lodato, Proc. Nat!. Acad.
Sci. USA. 87:3629 (1990).
7. M.S. Mulligan, J.M. Revel, M.A. Marietta and P.A. Ward, Proc. Natl. Acad. Sci. USA. 88:6338 (1991).
8. T. Nguyen, D. Brunson, C.L. Crespi, B.W. Penman, J.S. Wishuok and S.R. Tannenbaum, Proc. Natl.
Acad. Sci. USA. 89:3030 (1992).
9. D.A. Wink, K.S. Kasprzak, C.M. Maragos, R.K. Eiespurn, M. Misra, T.M. Dunams, T.A. Cebula, W.H.
Koch, A.W. Andrews, J.S. Allen and L.K. Keefer, Science. 254:1001 (1991).
10. M. Miwa, D.J. Stuehr, M.A. Marietta, J.S. Wishnok and S.R. Tannenbaum, Carcinogenesis. 8:955
(1987).
11. U. Forstermaun, H.H.W. Schmidt, J.S. Pollock, H. Sheng, J.A. Mitchell, T.D. Warner, M. Nakane and F.
Murad, Biochem. Pharmacal. 42:1849 (1991).
12. D.S. Bredt and S.H. Snyder, Proc. Natl. Acad. Sci. USA. 87:682 (1990).
13. H.H.H.W. Schmidt, J .S. Pollack, M. Nakane, L.D. Gorsky, U. Forstermann and F. Murad, Proc. Natl.
Acad. Sci. USA. 88:365 (1991).
14. B. Mayer, M. John and E. Bohme, FEBS. 277:215 (1990).
15. J.M. Hevel, K.A. White and M.A. Marietta, J. Biol. Chem. 266:22789 (1991).
16. D.J. Stuehr, H.J. Cho, N.S. Kwon, M.F. Weise and C.F. Nathan, Proc. Natl. Acad. Sci. USA. 88:7773
(1991).
17. J.S. Pollock, U. Forstermaun, J.A. Mitchell, T.D. Warner, H.H.W. Schmidt, M. Nakane and F. Murad,
Proc. Natl. Acad. Sci. USA. 88:10480 (1991).
18. D.S. Bredt, P.M. Hwang, C.E. Glatt, C. Lowenstein, R.R. Reed and S.H. Snyder, Nature (London).
351:714 (1991).
19. S. Lamas, P.A. Marsden, G.K. Li, P. Tempst and T. Michel, Proc. Natl. Acad. Sci. USA. 89:6348 (1992).
20. K. Nishida, D.G. Harrison, J.P. Navas, A.A. Fisher, S.P. Dockery, M. Uematsu, R.M. Nerem, R.W.
Alexander and T.J. Murphy, J. Clin. Invest. 90:2092 (1992).

283
21. C.R. Lyons, G.J. Orloff and J.M. Cunningham, J. Biol. Chem. 267:6370 (1992).
22. C.J. Lowenstein, C.S. Glatt, D.S. Bredt and S.H. Snyder, Proc. Natl. Acad. Sci. USA. 89:6711 (1992).
23. Q.-W. Xie, H.J. Cho, J. Calaycay, R.A. Mumford, K.M. Swiderek, T.D. Lee, A. Ding, T. Troso and C.
Nathan, Science. 256:225 (1992).
24. S.P. Janssens, A. Shimouchi, T. Quertennous, D.B. Bloch and K.D. Bloch, J. Biol. Chem. 267:14519
(1992).
25. H.J. Cho, Q.-W. Xie, J. Calaycay, R.A. Mumford, K.M. Swiderek, T.D. Lee and C. Nathan, J. Exp. Med.
176:599 (1992).
26. B. Mayer, M. John, B. Heinzel, E.R. Werner, H. Wachter, G. Schultz and E. Bohme, FEBS Lett. 288:187
(1991).
27. H.H.H.W. Schmidt, R.M. Smith, M. Nakane and F. Murad, Biochemistry. 31:3243 (1992).
28. D.S. Bredt, C.D. Ferris and S.H. Snyder, J. Biol. Chem. 267:10976 (1992).
29. K.A. White and M.A. Marietta, Biochem. 31:6627 (1992).
30. D.J. Stuehr and M. Ikeda-Saito, J. Biol. Chem. 267:20547 (1992).
31. K. McMillan, D.S. Bredt, D.J. Hirsch, S.H. Snyder, J.E. Clark and B.S.S. Masters, Proc. Natl. Acad. Sci.
USA. 89:11141 (1992).
32. M.A. Tayeh and M.A. Marietta, J. Biol. Chem. 264:19654 (1989).
33. N.S. Kwon, C.F. Nathan and D.J. Stuehr, J. Biol. Chem. 264:20496 (1989).
34. D.J. Stuehr, N.S. Kwon, C.F. Nathan, O.W. Griffith, P.L. Feldman and J. Wiseman, J. Biol. Chem.
266:6259 (1991).
35. R.A. Pufahl, P.G. Nanjappan, R.W. Woodard and M.A. Marietta, Biochemistry. 31:1992 (1992).
36. J.M. Hevel and M.A. Marietta, Biochem. 31:7160 (1992).
37. J. Giovanelli, K.L. Campos and S. Kaufman, Proc. Natl. Acad. Sci. USA. 88:7091 (1991).

284
MACROPHAGE NITRIC OXIDE SYNTHASE: TETRAHYDROBIOPTERIN
DECREASES THE NADPH STOICHIOMETRY

Joan M. Hevell and Michael A. Marletta1,2

lCollege of Pharmacy
2Department of Biological Chemistry, School of Medicine
University of Michigan
Ann Arbor, MI USA 48109-1065

INTRODUCTION

Nitric oxide synthase (NOS; EC 1.14.23) is a P4so-type hemoprotein that catalyzes

the oxidation of L-arginine to citrulline and •NO. The involvement of •NO in the
maintenance of vascular tone, neuronal transmission, and cytostasis [for reviews see 1•2], as
well as the adverse effects of altered •NO synthesis which include hypotension 3, penile
erection dysfunction 4 and diabetes mellitus5 illustrate the physiological importance of •NO
and the need to understand the molecular mechanism of •NO synthesis. Cloning studies6-8
have identified three different genes which encode for the NOS's which are found in the
endothelium surrounding blood vessels, the brain, and activated macrophages. Although
the three NOS's vary slightly in size (130-155 kDa subunits), the overall sequence
homology and cofactor requirements suggest that the conversion of L-arginine to citrulline
and •NO occurs by the same enzymatic mechanism. The complex reaction carried out by
the NOS's involves an interelay of protoporphyrin IX Fe-herne, FAD, FMN and
(6R)-tetrahydrobiopterin (H4B) . Our work with the macrophage form of the NOS has
demonstrated that maximum activity occurs when these cofactors I prosthetic ~roups are
found bound to the enzyme in a 1:1 stoichiometry with the 130 kDa subunit10 -1 . Loss of
pterin during the purification of the NOS can occur, yielding pterin-deficient NOS which
possesses a specific activit1 that can be directly correlated to the amount of sub-saturating
bound pterin that is present 2.
The exact function of H4B in the NOS reaction is just beginning to unfold.
Studies with the brain 13•14 and macrophage 12 forms of the enzyme have indicated that ~B
is important for enzyme stability and is also able to increase the initial rate of citrulline I
•NO formation in a concentration-dependent manner. In addition, experiments with the
macrophage NOS and the redox stable deaza analog of (6R,S)-methyltetrahydropterin,
(6R,S)-methyl-5-deazatetrahydropterin, have suggested that the redox properties of ~B are
important for its function 12. However, unlike the aromatic amino acid hydroxylases, NOS
does not appear to utilize reducing equivalents from ~B to hydroxylate L-arginine. In the
present studies we have investigated the effect that H4B has on the NADPH stoichiometry
of the NOS reaction. The results of these experiments indicate that the presence of H4B
decreases the number of reducing equivalents required to synthesize citrulline and •NO.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 285
METHODS

Purification of NOS

Cell culture procedures, induction of murine macrophage NOS activity and the
preparation of lOO,OOOg supernatant were carried out as described previously 15 .
Purification of NOS was carried out as described previously12 using 2'5'-ADP Sepharose
4B and DEAE Bio-Gel A columns and a concentration step in an Amicon uXtrafiltration
system: A) inclusion of 5 j..tM I4B in only the purification buffers for the affinity column
resulted in pterin-deficient NOS. Previous studies12 have demonstrated that NOS purified
in this manner contains sub-stoichiometric quantites of pterin (approximately 0.2-0.5 mols
pterin : subunit), and demonstrates activity that is dependent upon the addition of H4B for
maximum velocity. B) On the other hand, inclusion of 5j..tM I4B in all buffers during the
purification and the concentration step resulted in NOS that contained approximately
1 mole pterin per subunit (as per Bevel & Marietta 12).

NADPH Stoichiometry and Peroxide Analysis

End-point analysis was employed to determine the NADPH stoichiometry with NOS
that had been purified in the presence and absence of pterin using varying concentrations of
limiting NADPH (typically 0-20 j..tM) and allowing the reaction to proceed until all the
NADPH was consumed. Product formation was followed using the [14C]arginine assay as
previously described 12. Experiments were done at 37° C and were set up under conditions
in which the NADPH was consumed within 5-7 minutes to avoid complications that could
arise due to NOS instability. Control experiments demonstrated that the rate of citrulline
formation was linear over this time period. Plots of citrulline formed vs. NADPH
concentration were made where the inverse of the slope of the line equaled the NADPH
stoichiometry (moles NADPH required to form one mole citrulline). The NADPH
stoichiometry was also determined under initial rate conditions by following NADPH
consumption at 339 nm on a Cary 3E UV-VIS spectrophotometer equipped with a Neslab
RTE-100 temperature controller set at 37° C. After approximately 4-5 minutes the
reactions were terminated with TCA and placed on ice. The reaction mixture was
neutralized and chromatographed later to quantitate citrulline formation 12.
Peroxide formation was measured using the iron I KSCN assay as described
previously 16. Briefly, 25 j..tL of 6N TCA was added to 475 j..tL of sample, followed by
75 j..tL of 130 mM ferrous ammonium sulfate and 75 j..tL of 2.1 M KSCN. The samples
were allowed to stand for at least 5 minutes before reading the absorbance at 474 nm.

RESULTS

NADPH Stoichiometry

Pterin-Deficient NOS. When pterin-deficient NOS (demonstrated approximately

30-50% maximum activity in the absence of exogenous H4B) was used and no additional
pterin was included in the assays, the NADPH stoichiometry was > 2. However, the
stoichiometry was consistently lower when 50 j..tM I4B was added to the reaction mixture.
For example, typical NADPH stoichiometries measured in the absence of pterin varied
between preparations of NOS and ranged from 2.22 to 2.55 (mean = 2.39 ± 0.12, n=7). On
the other hand, when H4B was added to the assays, the stoichiometry decreased in each
independent experiment occupying a range between 1.95 and 2.33 (mean= 2.17 ± 0.14,
n=7). The stoichiometry could also be decreased with (6R,S)-methyl tetrahydropterin, but it
was not as effective as H4B (data not shown). Since it was possible that the decrease in
NADPH stoichiometry afforded by H4B was due to the non-specific reducing agent present

286
in the H4B solution (DTT), both ascorbate and DTT were examined for their ability to
mimic the effect of H4B on the stoichiometry. At concentrations equivalent to that in the
H4B solution, ascorbate did not effect the NADPH stoichiometry (data not shown). When
the same experiment was done using DTI as the non-specific reducing agent, the results
varied. In some instances DTT had no effect on the stoichiometry (data not shown). In
other experiments the stoichiometry was decreased by DTI; e.g., from 2.22 to 2.14. In this
particular example, the NADPH stoichiometry was decreased even further (1.95) in the
presence of 50 11M H4B. Subsequent studies demonstrated that as little as 1 j..tM H4B was
able to substantially decrease the NADPH stoichiometry (from 2.48 to 2.11).

Pterin-Saturated NOS. It is known from previous studies that NOS purified in the
absence of H4B contains a sub-stoichiometric concentration of bound pterin, the amount of
which can be correlated to enzyme activity 12 . The amount of bound pterin can be
increased by incubating the NOS with a large excess of H4B, followed by gel filtration.
However, a stoichiometry of 1 mol pterin : 1 mol subunit can not be reached by this
reconstitution method. Therefore, although the NADPH stoichiometry had been
determined in the presence of H4B with pterin-deficient NOS, it was necessary to also
determine the stoichiometry with NOS that was fully active; that is, enzyme containing a
1 : 1 pterin: subunit stoichiometry. Pterin-saturated NOS can be prepared as described in
the Methods section. Under these conditions the NADPH stoichiometry was 2.00 ± 0.07
(n = 9). This value was not affected by changing the buffer concentration, by increasing
the concentration of L-arginine from 100 JlM to 200 JlM, or by varying the concentration of
NOS used in the assay (data not shown). In addition, NOS that had been applied to a gel
filtration column to remove DTT and unbound H4B demonstrated NADPH stoichiometry
values of 1.91, 1.97, and 2.09. Finally, as an independent measure of product formation,
NOz- I N03- levels were measured. In all cases the NADPH stoichiometry was 2.
The NADPH stoichiometry of the NOS reaction was also examined under initial rate
conditions. NADPH consumption was followed at 339 nm using NOS that had been
purified in the presence of H4B. After the reaction was terminated, the amount of NADPH
oxidized was calculated and the amount of [14C]citrulline formed was measured as
described in the Methods section. The rate of substrate-independent NADPH oxidation was
not subtracted from tl).e arginine-dependent rate. In the presence of L-arginine, the NADPH
stoichiometry was 1.91 ± 0.05 (n = 5).

Peroxide Formation

Initial rate experiments showed that in the absence of substrate, NOS oxidizes
NADPH at approximately 25% the rate of NADPH oxidation when L-arginine is present
(data not shown). Presumably the reducing equivalents are being used to reduce molecular
oxygen to produce HzOz (or Oz-, that will dismutate to HzOz) in the absence of substrate.
In the presence of substrate and fully active enzyme, we have assumed that all of the
NADPH consumption is coupled to citrulline formation. However, it is possible that some
uncoupled NADPH oxidation occurs during arginine turnover. In addition, uncoupled
NADPH oxidation may explain the need for more NADPH in the absence of H4B with
pterin-deficient NOS. The amount of peroxide formed during the oxidation of L-arginine
to •NO and citrulline was measured in assays employing end-point analysis. SOD was
included in these and subsequent assays to help prevent the reaction of Oz- with •N017 •
Formation of peroxide under these conditions was dependent upon the concentration of
NADPH (data not shown). When pterin-deficient NOS (demonstrated approximately 50-
60% maximum activity in the absence of exogenous H4B) was used in these assays, the
observed NADPH stoichiometry of 2.09 ± 0.02 (n=3) dropped to 1.62 ± 0.10 (n=3) when
corrected for uncoupled NADPH oxidation. When pterin-saturated NOS was used, the
observed stoichiometry of 1.93 ± 0.04 (n=3) also decreased to 1.63 ± 0.04 when uncoupled
NADPH oxidation was taken into account.

287
DISCUSSION

The NADPH stoichiometry is an important key to solving the mechanism of •NO

synthesis by NOS. The first report of NADPH concumption came from Stuehr and co-
workers18 using purified macrophage NOS. These studies demonstrated that 1.5
equivalents of NADPH were required for the synthesis of citrullline and •NO from
L-arginine. However, the possibility of uncoupled NADPH oxidation was not addressed
until Mayer and co-workers 19 demonstrated that the brain NOS produced peroxide at
suboptimal concentrations of L-arginine or f4B. Shortly after these reports the brain and
macrophage NOS's were identified as P45o-type hemoproteins 10•20 . In P45o-catalyzed
reactions uncoupled NADPH oxidation is a familiar event, which is influenced by many
factors including substrates, inhibitors, and components of the system.
Our results demonstrate that citrulline I •NO synthesis catalyzed by pterin-deficient
macrophage NOS is accompanied by a substanital amount of uncoupled NADPH oxidation
which can be decreased by adding H4B to the reaction mixtures. Interestingly, pterin-
saturated NOS also synthesized peroxide (or 02-, that will dismutate to H2~) during the
turnover of L-arginine; however, additional I4B did not influence the stoichiometry. The
ratio of NADPH oxidized : citrulline formed in both cases was -1.6 : 1 when the observed
stoichiometry was corrected for uncoupled NADPH oxidation. These results indicate that
one of the functions of H4B in the NOS reaction is to increase the efficien'if of electron
transfer from NADPH to the heme which is presumed to be at the active site 1 . Our efforts
are currently directed at determining the molecular mechanism by which I4B decreases
uncoupled NADPH oxidation.

ENDNOTES

This research was supported by NIH Grant CA50414.

REFERENCES

1. J. Garthwaite, Trends Neurosci. 14:60 (1991).

2. S. Moncada, R.M.J. Pahner and E.A. Higgs, Pharmacal. Rev. 43:109 (1991).
3. R.G. Kilbonrn, S.S. Gross, A. Jubran, J. Adams, O.W. Griffith, R. Levi and R.F. Lodato, Proc. Natl. Acad.
Sci. USA. 87:3629 (1990).
4. J. Rajfer, W.J. Aronson, P.A. Bush, F.J. Dorey and L.J. Ignarro, N. Eng. J. Med. 326:90 (1992).
5. H.H. Schmidt, T.D. Warner, K. Ishii, H. Sheng and F. Murad, Science. 255:721 (1992).
6. C.J. Lowenstein, C.S. Glatt, D.S. Bredt and S.H. Snyder, Proc. Nat/. Acad. Sci. USA. 89:6711 (1992).
7. C.R. Lyons, G.J. Orloff and J.M. Cunningham, J. Bioi. Chern. 267:6370 (1992).
8. Q.-W. Xie, H.J. Cho, J. Calaycay, R.A. Mumford, K.M. Swiderek, T.D. Lee, A. Ding, T. Troso and C.
Nathan, Science. 256:225 (1992).
9. M.A. Marietta, J. Biol. Chern. in press (1993).
10. K.A. White and M.A. Marietta, Biochern. 31:6627 (1992).
11. J.M. Revel, K.A. White and M.A. Marietta, J. Bioi. Chern. 266:22789 (1991).
12. J.M. Revel and M.A. Marietta, Biochern. 31:7160 (1992).
13. B. Mayer, M. John, B. Heinzel, E.R. Werner, H. Wachter, G. Schultz and E. Bohme, FEBS Lett. 288:187
(1991).
14. J. Giovanelli, K.L. Campos and S. Kaufman, Proc. Nat!. Acad. Sci. USA. 88:7091 (1991).
15. M.A. Tayeh and M.A. Marietta, J. Biol. Chern. 264:19654 (1989).
16. A. G. Hildebrandt, I. Roots, M. Tjoe and G. Heinemeyer, Methods Enzymol. 52:342 (1978).
17. J.S. Beckman, T.W. Beckman, J. Chen, P.A. Marshall and B.A. Freeman, Proc Nat/ Acad Sci US A.
87:1620 (1990).
18. D.J. Stoehr, N.S. Kwon, C.F. Nathan, O.W. Griffith, P.L. Feldman and J. Wiseman, J. Bioi. Chern.
266:6259 (1991).
19. B. Heinzel, M. John, P. Klatt, E. Bohme and B. Mayer, Biochern J. 627 (1992).
20. D.J. Stoehr and M. Ikeda-Saito, J. Bioi. Chern. 267:20547 (1992).

288
ROLE OF TETRAHYDROBIOPTERIN IN CYTOKINE-STIMULATED
METABOLISM OF TRYPTOPHAN AND HYDROXYLATION OF ARGININE

Sheldon Milstienl, Naoki Sakail, Seymour Kaufmanl,

Kuniaki Saito2, and Melvyn P. Heyes2

Laboratories of Neurochemistryl and Clinical Science2

National Institute of Mental Health
National Institutes of Health
Bethesda, MD 20892

INTRODUCTION

The mechanisms which lead to central nervous system pathology observed in

inflammatory neurological diseases, such as AIDS, meningitis, and multiple
sclerosis, are not understood. It is now clear, however, that many biochemical
systems are induced or activated by potent cytokines released by cells of the
immune system in these diseases and some potential candidates for mediators of
neuronal cell toxicity or death have been identified. It has long been known that
induction of pterin synthesis, as measured by increased levels of neopterin in
cerebrospinal fluid, serum and urine, is a useful surrogate marker for activation of
the immune system.l In 1975, we pointed out that elevated neopterin levels in
patients with HIV infection correlated with the stage of the disease.2 Although the
identification of a functional biological role of neopterin has remained elusive, the
discoveries by Werner and colleagues that cytokines stimulate a parallel increase in
tetrahydrobiopterin (BH4) synthesis and tryptophan degradation in human cells
suggested a possible functional biochemical link between these two pathways.3 It
has also now convincingly been demonstrated that stimulation of L-tryptophan
metabolism results in dramatic increases in serum and cerebrospinal fluid levels of
the neuroactive metabolites, kynurenine (KYN), kynurenic acid, and quinolinic
acid, arising as a result of cytokine activation of indoleamine 2,3-dioxygenase
activity.4 Furthermore, in a model system for HIV infection in humans, we
correlated tryptophan metabolite levels and neopterin in D-retrovirus infected
macaques. We found high correlations between quinolinic acid, neopterin, and
clinical and viral status of the macaques.S Therefore, since BH4 has been shown to
have cofactor activity with IDO in vitro (although it is not nearly as effective as
ascorbate),6 the rate limiting enzyme in the tryptophan metabolic pathway in most
tissues, we explored this putative biochemical relationship between pterins and
tryptophan metabolism (see Fig. 1) in several model systems in which pterin levels

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 289
could be manipulated independently. As discussed below, we found
overwhelming evidence against this hypothesis. However, our studies of another
BH4-dependent, cytokine-stimulated metabolic pathway6 - the oxidation of L-
arginine to nitric oxide - led us back to a possible connection between pterin
synthesis and tryptophan degradation.

,L-KYN

-
IDO _,-
L-TRP --~-KYN --> intermediates --> quinolinic acid

GTPCH
- -?-
--
GTP --> NH2TP --> intermediates --> BH4
- -

·······~NEO

Figure 1. Possible link between tryptophan metabolism and pterin synthesis. Cytokines
stimulate the rate-limiting enzymes in each pathway, 100 and GTPCH, and BH4 can
function as cofactor in the 100 reaction. The solid lines refer to extracellular transport of
the products which are determined as measures of activities of the respective pathways.

RESULTS AND DISCUSSION

Tryptophan Metabolism In Macrophages Is Independent Of Pterin Levels

It has been reported that macrophages are the primary source of the increased
neopterin levels found in serum of patients in which the immune system is
activated.8 In addition, Heyes et al have recently shown that cytokine-stimulated
human macrophages can metabolize L-tryptophan to quinolinic acid.9 As shown in
Fig. 2A, we found that interferon-gamma (IFN-y) induced a dose-dependent
stimulation of pterin synthesis and L-tryptophan metabolism, as measured by
neopterin and KYN production, respectively. However, when pterin synthesis was
inhibited by the addition of 2,4-diamino-6-hydroxypyrimidine (DAHP), a
competitive inhibitor of GTP cyclohydrolase (GTPCH), there was no significant
inhibition of KYN production (Fig. 2B). Since activated human macrophages
produce little (or no) BH4,8 it was not entirely surprising that inhibition of GTPCH
had no effect on KYN production. Furthermore, increasing intracellular BH4levels
by treatment with sepiapterin (SP), which is converted to BH4 by the salvage
pathway, also has no detectable effect on the rate of L-tryptophan metabolism.
These results strongly suggest that, at least in human macrophages, the metabolism
of L-tryptophan is independent of either pterin synthesis or intracellular neopterin
and biopterin levels.

The Effect Of Cytokines On L·Tryptophan Metabolism And Pterin Synthesis In

Other Human Cells And In C57BL6 Mice

In contrast to monocyte-derived macrophages, which cannot produce BH4,

THP-1 cells are a monocytic cell line which can synthesize BH4 as well as oxidize L-
tryptophan to KYN. However, although we found that cytokines stimulate both L-
tryptophan metabolism and pterin synthesis in these cells, the rate of production of

290
KYN was also unaffected by inhibition of pterin synthesis with DAHP.l 0 In
addition, the rate of production of KYN by primary human skin fibroblasts after
stimulation with IFN-y and TNF-a was the same in fibroblasts from Blf4-deficient
children as from normals. tO

A. 48 HOURS B. 24HOURS
8360
10 D Neo

B1o

KYN

10 100 1000 IFNrrNF IFNrrNF+DAHP IFNrrNF+SP

IFN-y (Units/ml)

Figure 2. Stimulation of tryptophan metabolism in human macrophages by IFN-J' is

independent of pterin synthesis. In A, macrophages were cultured for 48 h in the presence
of the indicated concentrations of IFN-y. In B, macrophages were incubated for 24 h with
250 U/ml IFN-y+ 100 U/ml TNF-a. in the absence or presence of 5 mM DAHP or 50 J!M
SP.

The kynurenine pathway of L-tryptophan metabolism in C57BL6 mice has been

shown to be particularly sensitive to treatment with cytokines or agents which
release cytokines.11 However, after chronic treatment of these mice with IFN-y,
under conditions which lead to dramatic increases in tissue 100 activity and
increased levels of L-tryptophan metabolites, we could not detect any
corresponding changes in tissue BH4 levels. tO
In sum, we have accumulated substantial negative evidence which strongly
argues against any physiological role for either neopterin or biopterin in L-
tryptophan metabolism. Thus, it is likely that the function, if any, of cytokine
stimulated increases in biopterin levels lies elsewhere.

Does BH4 Play A Role In Cytokine-Stimulated Production Of Nitric Oxide?

It is well established that oxidation of the guanidino group of L-arginine to

nitric oxide is a reaction catalyzed by a family of enzymes, called nitric oxide
synthases, which all require BH4 as a cofactor.12 While the exact role that BH4
plays in this reaction is still not understood, it has now been shown that inhibition
of pterin synthesis results in inhibition of nitric oxide formation by endothelial cells,
and smooth muscle cells.12
Although the requirement for BH4 was first demonstrated by Marietta and
coworkers in in vitro experiments on cytosol from macrophages,7 they did not show

291
that synthesis of BH4 by macrophages was absolutely essential for cytokine-
stimulated production of nitric oxide. In fact, as shown in Fig. 3, inhibition of
pterin synthesis by more than 95% by treatment of macrophages with DAHP only
has a small effect on nitric oxide production. This is likely due to the constitutive
synthesis of BH4 by these cells and the high affinity of nitric oxide synthase for BH4
since pretreatment of the cells with a combination of pterin synthesis inhibitors
(DAHP + N-acetylserotonin), reduced BH4 to nearly undetectable levels and
significantly inhibited nitric oxide synthesis. Furthermore, this effect was shown to
be specifically due to the loss of BH4, since repletion of BH4 levels by addition of
BH2 essentially normalized the nitric oxide production. Therefore, cytokine-
induced production of nitric oxide also appears to be absolutely dependent upon
BH4 synthesis. A more detailed study of BH4 and nitric oxide production by
murine macrophages has recently been published.13

A B 275+2.5
100
ill
';:J
...J
<
>
(f)
75
ll..
...J
........
;:-
~
......
50

>I..
0
zUlE-< 25
u~
Ul
ll..
0
DAHP DAHP+NAS DAHP+NAS+BH2

Figure 3. Effect of pterin depletion and resupplementation on nitric oxide production by

RAW264 cells. In A, cells were treated for 24 h with 100 U/ml IFN-y + 1.0 ng/ml LPS in the
absence or presence of 5 mM DAHP. In B, cells were pretreated for 12 h with 5 mM DAHP plus
5 mM N-acetyl serotonin (NAS) and then stimulated with IFN-y /LPS in the presence of 100 JlM
BH2 for 24 h. DAHP and SP were present during the entire period. The numbers above the
bars are the actual values. Both DAHP and SP inhibited GTPCH activity by >90%. The
numbers over the bars are the actual relative values.

Tetrahydrobiopterin, NMDA Receptor-Mediated Neuronal Cell Death and Nitric

Oxide

Glutamate neurotoxicity mediated through N-methyl-0-aspartate (NMDA) type

of L-glutamate receptors may play a role in neuronal cell death in ischemic brain
injury as well as in neurodegenerative disorders, such as Alzheimer and
Huntington diseases. Dawson et al., recently presented evidence that glutamate-

292
induced cytotoxicity in primary dissociated cortical cell cultures was mediated
through nitric oxide produced by the action of nitric oxide synthase on L-
arginine.14 Constitutive neuronal nitric oxide synthase is the likely source,
although recent studies have demonstrated that reactive nitrogen intermediates
produced from L-arginine by cytokine-activated microglia can mediate neuronal
cell injury and death.15 In addition, quinolinic acid is a potent NMDA receptor
agonist.16 Taken together, these facts and our data have led us to propose the
model below (Fig. 4) for the interaction of three metabolic pathways which are
activated in parallel by cytokines, pterin synthesis, L-tryptophan degradation, and
nitric oxide synthesis. This interaction could be responsible for some of the
pathological consequences of inflammatory neurological diseases. In this model,
we have shown a number of pathways for which there is some evidence, although
it is unlikely that they all occur within the same cell. Space limitations preclude a
detailed description (see references). Finally, this model highlights the different
potential pharmacological targets, including BH4 biosynthesis, which could be the
focus for the development of a multi-faceted approach for the treatment of some
devastating neurological diseases.

CYTOKINES

L-TRP
L·AAG f/00
~Nos..____ ....... L-~N ~ KYN

+ NO
interme.ates
QUIN

ARG .......

G~P
eve se
/
,
NO--
\
./
-
,-
--
1neurodegenerallon
1
NEURON

cGMP

Figure 4. Interactions between pterin synthesis, tryptophan degradation, and arginine

oxidation which can lead to neuronal cell injury and death.

293
ACKNOWLEDGMENTS

Dr. Naoki Sakai was supported by the NIH Intramural AIDS Targeted Antiviral
Program.

REFERENCES

1. H. Wachter, D. Fuchs, A. Hausen, G. Reibnegger, G. Weiss, E.R. Werner, and G. Wemer-

Felmayer. "Neopterin. Biochemistry, Methods, Clinical Application", W. de Gruyter, Berlin
(1992).
2. J.P. Abita, H. Cost, S. Milstien, S. Kaufman, and G. Salmot. Lancet 2:51 (1985).
3. E.R.Wemer, G. Wemer-Felmayer, D. Fuchs, A. Hausen, G.Reibnegger, and H. Wachter. Biochem. f.
262:861 (1989).
4. M.P. Heyes, et al. Brain 115: 1249 (1992).
5. M.P. Heyes, A. Lackner, S. Kaufman, and S. Milstien. AIDS 5:555 (1991).
6. Y. Ozaki, J.F. Reinhard, and C.A. Nichol. Biochem. Biophys. Res. Commun. 137:1106 (1986).
7. M.A. Tayeh and M.A. Marietta. J. Bioi. Chern. 264:19654 (1989).
8. C. Huber, J.R. Batchelor, D. Fuchs, A. Hausen, A. Lang, D. Niederwieser, G. Reibnegger, P.
Swetly, J. Troppmair, and H. Wachter. J. Exp. Med. 160:310 (1984).
9. M.P. Heyes, K. Saito, and S.P. Markey. Biochem. J. 283:633 (1992).
10. N. Sakai, K. Saito, M.P. Heyes, S. Kaufman, and S. Milstien. Biochem. J., submitted (1993).
11. K. Saito, A. Lackner, S.P. Markey, and M.P. Heyes. Neurosci. 51: 25 (1992).
12. R.G. Knowles and S. Moncada. TIBS 17:399 (1992).
13. N. Sakai, S. Kaufman, and S. Milstien. Malec. Pharmacal. 43:6 (1993).
14. V.L. Dawson, T.M. Dawson, E.D. London, D.S. Bredt, and S.H. Snyder. Proc. Natl. Acad. Sci.
U.S.A. 88:6368 (1991).
15. K.M. Boje and P.K. Arora. Brain Res. 587:250 (1992).
16. A.T. Malouf, R.L. Schnaar, and J.T. Coyle. J. Bioi. Chern. 259:12756 (1984).

294
 TETRAHYDROBIOPTERIN SYN1HESIS IS INDUCED BY LPS IN VASCULAR
SMOOm MUSCLE AND IS RATE-LIMITING FOR NITRIC OXIDE
PRODUCTION

StevenS. Gross*\ Roberto Levi*, Arelis Madera* Kwan Ha Park,

John Vane and Yoshiyuki Hattorit

*Department of Pharmacology
Cornell University Medical College
New York, NY 10021, USA
tThe William Harvey Research lnstitiute
St. Bartholomew's Medical College
London EC1M 6BQ, UK

INTRODUCTION

Cells produce tetrahydrobiopterin (BH4) by two distinct pathways; a de novo

synthetic pathway which uses GTP as a precursor and a salvage pathway which uses pre-
existing dihydropterins 1 (see Fig. 1). GTP cyclohydrolase I (GTPCH1; EC 3.5.4.16) is
the first and rate-limiting enzyme for the de novo BH4 synthetic pathway, leading to
synthesis of dihydroneopterin triphosphate (NH2 PPP). GTPCH1 has been shown to be
selectively inhibited by pterins, most potently by reduced pterins, including BH42'3 • Thus,
it is likely that de novo BH4 synthesis can be regulated by end-product inhibition.
Additionally, 2,4-diamino-6-hydroxypyrimadine (DAHP) selectively inhibits this enzyme
in vitro 4 and in vivo 5 . Subsequent metabolism of NH2PPP to BH4 is catalyzed by 6-
pyruvoyl tetrahydropterin synthase, producing 6-pyruvoyl tetrahydropterin (6-PT). 6-PT
is finally converted to BH4 by two successive NADPH-dependent hydroxylations
catalyzed by sepiapterin reductase. While the dihydropterins, BH2 and sepiapterin, are
not substrates for the de novo synthetic pathway, they are converted to BH4 via the pterin
salvage pathway which utilizes dihydrofolate reductase (DHFR; EC 1.5.1.3) for its final
step 1.
GTPCH1 is a constitutive enzyme in some tissues (e.g., adrenal medulla,
hypothalamus and liver6), however, it has been shown to be induced by cytokines (e.g.,
interferon-gamma, interleukin-2) in macrophages, lymphocytes and fibroblasts 7•9 . While
the role of GTPCH1 in tissues that contain constitutive GTPCH1 can be explained in most
cases by the presence of aromatic amino acid hydroxylases, BH4 induction by cytokines
occurs in cells which lack these, e.g., T lymphocytes (for review, see ref. 10). This has
fueled speculation that there may be yet undiscovered BH4-dependent enzymes 10 •
The prophesy of undiscovered BH4-dependent enzymes was fulfilled by the finding
that nitric oxide synthases (NOS) require BH4 for activity 11 ' 12 . NOS produces NO and
citrulline at the expense of L-arginine, NADPH and molecular oxygen (for review, see ref.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 295
 13). BH4 is a cofactor for each of the three isoforms of NOS identified to date 13 .
Although NOS certainly requires BH4 for function, the exact role of BH4 remains
controversial.
An intriguing possibility is that the major function of cytokine-induced BH4 is to
support activity of co-induced NOS. The validity of this hypothesis may depend on cell-
type. K won et al. 13 have shoWn. that macrophages in culture contain enough constitutive
BH4 to fully support cytokine-induced NOS activity. In contrast, fibroblasts 14 and
endothelial cells 15 in culture require de novo BH4 synthesis for optimal NO production
after cytokine activation. In the present report we characterize the role of BH4 for
cytokine-induced NO production in vascular smooth muscle (VSM).

cGMP/Gr
®

//' l GTP cycle hydrolase I

/ ' Dihydroneopterin

'f
/ triphosphate
// I 6-pyruvoyl tetrahydropterin
'
NO• +citrulline ARG ~~~
~ f Sepiapterin reductase

Q-BH2 BH4
~ 'Dihydrofolate reductase
Dihydropteridine BH2
reductase '
Sepiapterin
Figure 1. Enzymatic pathways for the synthesis of tetrahydrobiopterin (BH4) in eukaryotic cells and the
proposed relationship to nitric oxide synthase. GTP is indicated to play a dual role in the biology of NO;
it is both precursor to BH4 (a required cofactor of NOS) and precursor to cGMP (the mediator of vascular
actions of NO). The dashed arrow indicates that L-arginine(ARG)-derived NO activates soluble guanylyl
cyclase, thereby triggering the conversion of GTP to cGMP. [Reprinted with permission from ref. 16]

NO OVERPRODUCfiON BY VSM AND CYTOKINE-INDUCED CIRCULATORY

SHOCK

NO is a potent vasorelaxant and is the identity of the endothelial-derived relaxing

factor described in 1980 by Furchgott and Zawadski 17 • Although vascular smooth muscle
is the normal target of endothelial-derived NO, VSM itself can be induced by cytokines
and immunostimulants to produce copious quantites of N0 6•18-20 We have shown that
bacterial lipopolysaccharide (LPS) causes VSM to secrete nitrite, an accumulating stable
oxidation product of the unstable free radical N0 16 . LPS-induced nitrite production is
arginine-dependent, inhibited by N'"-substituted arginine analogs, abolished by inhibitors
of protein and mRNA synthesis and potentiated by interferon-y (IFN), indicating that
nitrite arises specifically via induction of the arginine:NO pathway. Thus, VSM can
become both source and target of NO; this results in profound vasodilatation.
Overproduction of NO has been implicated as the mechanistic basis of septic- and
cytokine-induced circulatory shock, conditions which are highly lethal 21 . It is significant
that the isoform(s) of NOS which is induced by cytyokines differs from the constitutive
isoforms of NOS in that the former lacks a requirement for calcium 13 . Thus, inducible
NOS can produce large quantites of NO in an unregulated fashion, limited only by the
availability of substrate.

296
INDUCI'ION BY LPS OF BH4 SYNTHESIS AND GTPCHl mRNA IN VSM

As shown in Fig. 2, BH4 is undetectable in the cytosol of untreated rat aortic VSM
in culture. However, BH4 appears after treatment with LPS. While IFN alone does not
induce BH4 synthesis, it markedly potentiates the action of LPS. This increase in
intracellular BH4 is abolished by the GTPCHl inhibitor DAHP, indicating that BH4
elevation arises from de novo synthesis via GTPCHI. Confirmation that GTPCHI is
induced in VSM is provided by our finding that GTPCHI mRNA is markedly enhanced
by LPSIIFN (Fig. 3). Reverse transcription-PeR was used to amplify a predicted 372 bp
nucleotide sequence corresponding to nucleotides 295-666 of the constitutive GTPCHI
from rat liver22. To confirm that the PCR fragment was indeed GTPCHI, it was
subcloned and sequenced. The nucleotide sequence was found to be 100% identical to
rat liver GTPCH 122, suggesting that constitutive and inducible GTPCHI are the same
protein. Although GTPCHI mRNA was clearly induced by LPSIIFN, low levels of
mRNA were also expressed constitutively, in control cells. The constitutive expression
of mRNA in the absence of detectable protein activity, suggests that additional factors
could be involved in regulation of GTPCHl protein translation and/or activity.
Interestingly, preliminary results suggest that DAHP may attenuate GTPCHl mRNA
induction by LPSIIFN. Further experiments will be required to confirm this possibiity.
In any event, our findings demonstrate that LPSIIFN, which we have shown to be a potent
inducer of NOS in VSM16, simultaneously induces GTPCHI mRNA and protein.

LPS ELICITS BIOPTERIN ACCUMULATION IN RASMC BY AN

ACTION WHICH IS POTENTIATED BY IFN AND BLOCKED BY DAHP·

BASA~ LPS LPS ~PS IFN

+ IFN + IFN
+ DAHP

Figure 2. Int1uence of inununostimulants on total biopterin content (oxidized plus reduced forms) of rat
aortic smooth muscle cells. Biopterin was assayed by HPLC after acidic oxidation6 in cytosol from cells
that were either untreated (basal), or treated for 12 h with LPS (30 !lg/m), IFN (50 ng/m1), the combination
of LPS and IFN, or the combination of LPS, IFN and DAHP (3 mM). Assays Bars indicate mean values
± SE of 3-4 replicate treatments, expressed as a function of protein concentration. "n.d." denotes no
detectable biopterin [reprinted with permission from ref. 16)

Figure 3. LPS induces GTPCHl mRNA in rat aortic smooth muscle cells. Total RNA was prepared by
guinidinium isothiocyanate extraction from intreated cells (lane I) or after a 4-hour treatment with a
combination ofLPS (30 !lg/ml) and IFN (50 ng/ml) alone (lane 2), or with DAHP (3 mM; Jane 3)). RNA
(l!lg) was amplified by RT -PCR using primers for GTPCHl (left panel) or the house-keeping gene,
glyceraldehyde-3-phosphate dehydrogenase (right panel). Both primer sets gave the predicted amplification
products.

SPECIFIC INHffiiDON OF GTPCHl ABOLISHES LPS-INDUCED NO SYNTHESIS

INVSM

Since we found that DAHP blocks LPSIIFN-induced BH4 synthesis in VSM, we

next investigated whether DAHP also inhibits LPSIIFN-induced NOS activity. As shown

297
 in Fig. 4, DAHP caused a concentration-dependent and complete inhibition of the
accumulation of nitrite in cell culture medium. This effect of DAHP was not associated
with a cytostatic effect ofDAHP; indeed, DAHP attenuated the inhibition of mitochondrial
respiration observed with LPS alone16 . Prevention of respiratory depression by DAHP is
consistent with the knowledge that large quantities of NO inhibit respiration by binding
to iron-sulfur centers in mitochondrial complexes I and II 13 . DAHP inhibits LPSIIFN-
induced NO synthesis by specific inhibition of GTPCHl and subsequent reduction of
intracellular BH4, since this effect is prevented by administration of the BH4 precursor
sepiapterin (Fig. 4). Importantly, the ability of sepiapterin to prevent NO synthesis
inhibition does not occur in the presence of 10 !JM methotrexate 16, a selective inhibitor
of DHFR which catalyzes the final step of sepiapterin conversion to BH4.
To further confirm that DAHP inhibits NO synthesis by selective inhibition of
GTPCHl, we investigated whether inhibition of NO synthesis could be reduced by
elevated intracellular GTP. Intracellular GTP was increased in VSM by administering
guanosine; guanosine is converted within the cells to GTP by the purine salvage
pathway 23 . As predicted, guanosine caused a rightward-shift in the concentration response
relationship for inhibition of NO synthesis by DAHP. In additional studies we found that
LPS-induced NOS activity could also be inhibited by N-acetylserotonin 16, an inhibitor of
the enzyme sepiapterin reductase which catalyzes the final 2 steps in de novo BH4
synthesis 24 . Taken together, our findings establish that induction of GTPCHl and de novo
synthesis of BH4 are necessary events for LPS-induced NO synthesis in VSM.

DAHP INHIBITS THE INDUCTION OF NO SYNTHESIS BY LPS/IFN INHIBITION OF LPS/IFN-INDUCED NO SYNTHESIS BY DAHP
IN RAT AORTIC SMOOTH MUSCLE CELLS BY A MECHANISM IN RAT AORTIC SMOOTH MUSCLE CELLS
WHICH IS PREVENTED BY SEPIAPTERIN IS COMPETITIVELY REDUCED BY GUANOSINE ADMINISTRATION

100
wc-
t:e
~§ 80
Zo
o-
tj ~ 60
::::>~
Oz
,::0 40
IF
zu
;;;.g
"-::::>

a. a::
20
...JO..

0
.01 .03 0.1 0.3 .01 .03 0.1 0.3 3
[DAHP] (mM) [DAHP] (mM)

Figure 4. Inhibition of LPSIIFN-induced NO synthesis in VSM: prevention by sepiapterin. Nitrite release

was quantified during a 24 h period following addition of LPS (30 f!g/ml) in combination with IFN (50
ng/rnl) and the indicated concentration ofDAHP, with or without sepiapterin (100 f!M}. Points are means
± SE of the nitrite concentration in 4 individual cell culture wells.

Figure 5. Inhibition of LPS/IFN-induced NO synthesis in VSM: protection by guanosine. Nitrite release

was quantified during a 24 h period following addition of LPSIIFN and the indicated concentration of
DAHP, with or without gunosine (100 f!M}. Points are means ± SE of the nitrite concentration in 4
individual cell culture wells.

It is noteworthy that providing excess BH4 to LPS/IFN treated VSM significantly

enhances NO production. Thus, administration of either sepiapterin or BH4 to VSM
causes a concentration-dependent increase in NO production, to levels approaching 200%
of that observed with LPSIIFN alone16 . Furthermore, in the presence of sepiapterin (1 00
!JM), the onset of LPSIIFN-induced NO synthesis occurs earlier16 . Although these
findings had been interpreted by us to indicate that BH4 levels are rate-limiting for
cytokine-induced NOS, this conclusion must be considered as tentative since we have
subsequently discovered that sepiapterin and BH4 also enhance cytokine-induced NOS
protein mass (Gross et al., manuscript submitted).

298
EFFECf OF GTPCHl INHffiiDON ON LPS-INDUCED NO SYNTHESIS IN VIVO

Our studies of VSM in culture raise the possibility that BH4 synthesis inhibitors
could have utility in vivo for treatment of conditions arising from vascular NO
overproduction, e.g., septic- and cytokine-induced shock. To test this possibility we have
investigated whether BH4 synthesis inhibitors diminish LPS-induced NO synthesis and
NO-mediated vascular dysfunctions in the rat. LPS (15 mg/kg, i.p., 6 h pretreatment)
caused an 80% increase in total plasma biopterin (BH4 and more oxidized species); this
efffect was abolished by prior treatment of animals with DAHP and MTX (I g/kg, i.p. and
1 mg/kg, i.v., 1 h prior to LPS). Under these conditions, DAHP/MTX diminished by
>60%, the 20-fold elevation in arginine-derived plasma nitrate obtained when LPS was
administered alone. This finding suggests that NO synthesis in vivo can be attenuated by
GTPCHI inhibition.
One important pathophysiological manifestation of NO overproduction within the
blood vessel is a blunted responsivity to vasoconstrictors. Indeed, diminished sensitivity
to pressor agents may be the major impediment to therapy of septic patients. A primary
role of NO in this phenomenon is indicated by the finding that the LPS-induced
impairment of vasoconstrictor responses can be overcome by the selective NOS inhibitor
N"'-methyl-L-arginine 25 '26 . Therefore it is significant that DAHP/MTX pretreatment also
causes a near-complete prevention of the LPS-induced hyporesponsivity to the
vasonstrictory effect of phenylephrine in rat aorta, ex vivo (Gross et al., manuscript in
preparation).

SUMMARY AND CONCLUSIONS

GTPCHI mRNA and BH4 synthesis is increased by LPS in vascular smooth

muscle. Our data suggest that induction of GTPCHI and NOS represent two arms of a
common pathway required for immunostimulant-evoked NO synthesis. This conclusion
is consistent with the view that the major function of immunostimulant-evoked BH4 is to
support NOS. Moreover, GTPCHI and other enzymes of the de novo BH4 synthetic
pathway may prove to be important targets for therapy of clinical conditions arising from
NO overproduction. As we begin to reveal the molecular events governing the induction
and expression of GTPCHl and NOS, additional therapeutic approaches for treating NO
overproduction are certain to be revealed.

Acknowledgements
The work was funded by NIH grants HL46403 (S.S.G.), HL34215 (R.L.) and a
grant from Strohtech.

REFERENCES

1. Nichol, C., Smith G. and Duch, D. (1985) Biosynthesis and metabolism of tetrahydropterin and
molybdopterin. Ann Rev Biochem 54: 729-764.
2. Ballahene, Z., Dhondt, J.-L. and Farriaux (1984) Guanosine triphosphate cyclohydrolase activity in
rat tissues. Biochem J. 217:59-65.
3. Shen, R.-S., Alam, A. and Zhang, Y.X. (1988) Inhibition of GTP cyclohydrolase I by pterins.
Biochem Biophys Acta 965: 9-15.
4. Gal., E.M., Nelson, J.M., and Sherman, A.D. (1978) Biopterin III. Purification and characterization
of enzymes involved in the cerebral synthesis of 7,8-dihydroneopterin. Neurochem. Res. 3: 69-88.

299
5 Kabasbi, T Hasegawa, H, Kaneko, E and Isbiyama, A (1991) Gastromtestmal serotorun depletiOn
due to tetrahydrobwptenn defiCiency J Pharmacal Exp Therap 256: 773-779
6 Fukushima T and Nixon, I C (1980) Analysis of reduced forms of bwptenn m bwlogiCa! tissues
and flmds Anal Bzochern 102: 176-188
7 Werner, E, Werner-Felmeyer, G , Fuchs D, Hausen, A, Ziebnegger, G, and Wachter, H (1989)
Parallel mductwn of tetrahydrobwptenn bwsynthesis and mdoleamme 2,3-dwxygenase activity m
human cells and cell hnes by mterferon-y Bzochern J 262 861-866
8 Ziegler I , Schott, K , Lubbert, M , Schwulera, U, and Bacher, A (1990) Control of
tetrahydrobwptenn synthesis m T lymphocytes by synergistic actwn of mterferon-y and
mterlukm-2 J Bzol Chern 265: 17026-17030
9 Werner, E R, Werner-Felmeyer, G, Fuchs, D, Hausen, A, Reibnegger, G, Yrm, J J, Pfleiderer, W,
and Wachter, H (1990) Tetrahydrobwptenn bwsynthetJc activitles m human macrophages,
fibroblasts, THP-1, and T 24 cellls J Bzol Chern 25: 3189-3192
10 Zeigler, I (1990) ProductiOn of ptendmes dunng hematopoiesis and T-lymphocyte prohferatwn
partial partiCipation m the control of cytokme signal transmissiOn Med Res Rev 10: 95-114
11 Kwon, N, C Nathan, and Stuehr, D (1989) Reduced bwptenn as a cofactor m the generatwn of
mtrogen oxides by munne macrophages J Bwl Chern 264 20496-20501
12 Tayeh, M A and M A Marietta (1989) Macrophage oxidatiOn of L-argmme to mtric oxide, mtnte
and mtrate Tetrahydrobwptenn IS reqmred as a cofactor J Bwl Chern 264 19654-19658
13 Nathan, C (1992) Nitric oxide as a secretory product ofmammahan cells FASEB J 6: 3051-
3064
14 Werner-Felmayer, G, Werner, E R, Fuch>, D, Hausen, A, Reibnegger, G and Wachter, H (1990)
Tetrahydrobwpterm-dependent formatwn of mtnte and mtrate m murme fibroblasts J Exp M ed
172 1599-1607
15 Gross, S , Stuehr, D , Aisaka, K , Jaffe, E A , Levi, R and Gnffith, 0 (1991) Cytokme-activated
endothehal cells express an Isoform of mtnc ox1de synthase wh1ch IS tetrahydrobwptenn-
dependent, calmodulm-mdependent, and resembles the macrophage Isoform m Its profile of
mhibition by W-substituted argmme analogs Bwchern Bwphys Res Comrnun 178 823--829
16 Gross, S S, and Lev1, R (1992) Tetrahydrobwptenn synthe>Is An absolute reqmrement for
cytokme-mduced mtnc ox1de generation by vascular smooth muscle J Bzol Chern , 267: 25722-
25729
17 Furchgott, R F and Zawadski, J V (1980) The obihgatory role of endothehal cells m the relaxatwn
of artenal smooth muscle by acetylcholme Nature 288:373-376
18 BusseR and Mulsch, A (1990) Inductwn ofmtnc oxide synthase by cytokmes m vascular ~mooth
muscle cells FEBS Lett 275: 87-90
19 Beasley, D, Schwartz, JH, and Brenner, B M (1991) Interlukm-1 mduces prolonged L-argmme
dependent cychc guanosme monophosphate and mtrite productwn m rat vascular smooth muscle
cells J Clm Invest 87 602-608
20 Schim, VB, Junquero, DC, Scott-Burden, T and Vanhoutte, PM (1991) Interleukm-1~ mduces
the productiOn of an L-argmme-denved relaxmg factor from cultured smooth muscle cells from rat
aorta Bwchern Bwphys Res Cornmun 176: 114-121
21 Parnllo, J E (1989) Textbook of CntJcal Care, 2"d edition (Shoemaker, W C , Ayers, S, Grenvik et
a!, eds) W B Saunders Pub!, Philadephia, p1006
22 Hatakeyama, K, Inoue, Y, Harada, T, and Kagamiyama, H (1991) Clonmg and sequencmg of
eDNA encodmg rat GTP cyclohydrolase I The firSt enzyme of the tetrahydrobwptenn bwsynthetJc
pathway J Bwl Chern 266 65-769
23 Hatakeyama, K , Harada, T and Kagam1yama, H (1992) IMP dehydrogenase Inhibitors reduce
mtracellular tetrahydrobwptenn levels through reductiOn of mtracellular GTP J Bwl Chern 267
20743-20739
24 Gal , EM , Nelson, J M, and Sherman, AD (1978) Bwptenn III Punficatwn and charactenzatwn
of enzymes mvolved m the cerebral synthesis of 7,8-dihydroneoptenn Neurochem Res 3: 69-88
25 Gross, S S , Madera, AM , Gr1ffith, 0 W , and Levi, R (1991) Endotoxm Impaus vasoconstnctwn
by mducmg m aortic smooth muscle cells a mtric oxide synthase Isoform d1stmct from that m
endothehal cells FASEB J 5: Al728
26 Jolou-Schaeffer, G, Gray, G A, Schott, C, Paratt, JR and Stoclet, J -C (1990) Loss of cellular
responsiveness mduced by endotoxm mvolves L-argmme pathway Am J Physwl 259 Hl038-
Hl043

300
6R-[3H]TETRAHYDROBIOPTERIN BINDING ACTIVITIES IN RAT
BRAIN

Yumiko Watanabe1,2, Hiroshi Morii1, Yasuo Nemoto1, Bernd Mayer3,

Ernst R. Werner4, Soichi Miwa5, and Yasuyoshi Watanabe1,2

1Dept. of Neuroscience, Osaka Bioscience Institute, Osaka 565,

2subfemtomole Biorecognition Project, Research Development Corporation
of Japan, Osaka 565, Japan, 3Institute ofPharmacol. and Toxicol., Graz
Univ., Graz, 4Institute of Medical Chemistry and Biochemistry, Innsbruck
Univ., Innsbruck, Austria, 5Dept. of Pharmacal., Fac. Med., Kyoto Univ.,
Kyoto 606, Japan

INTRODUCTION
6R-L-Erythro-5,6,7 ,8-Tetrahydrobiopterin (R-THBP) is a common cofactor for
phenylalanine, tyrosine, and tryptophan hydroxylases, the latter two of which are the rate-
limiting enzymes of catecholamines and serotonin biosyntheses, respectively. In an attempt
to answer the puzzle from the clinical evaluation that R-THBP and L-DOPA or 5-HTP
possess the additive effect on the treatment of atypical phenylketonuria, Parkinsonian
disease, and infantile autism, we recently found a novel role of R-THBP in the central
nervous system; R-THBP functions as a release-promotor of dopamine, serotonin, and
norepinephrine from the striatal and cortical nerve terminals 1 ,2. Amino acid
neurotransmitters in the extracellular space also increased by R-THBP treatment, but in this
case, the destruction of catecholaminergic components by 6-hydroxydopamine pretreatment
resulted in a complete blockade of amino acid release evoked by R-THBP2. Namely, the
effect of R-THBP on amino acid neurotransmitters is via monoaminergic stimulation. R-
THBP also evokes acetylcholine release possibly through serotonergic stimulation3. On the
contrary, when we reduce R-THBP content in the tissues including the brain by use of a
potent inhibitor of R-THBP biosynthesis, the tissue content of dopamine and its metabolites
very slightly decreased at around 10%, but such a treatment lowering R-THBP level resulted
in a severe decrease of dopamine release; the dopamine output from the striatum was reduced
to less than 50% of the control level. This result and other experimental data indicate that the
endogenous level of R-THBP can regulate dopamine release. The molecular mechanisms
underlying the R-THBP-induced neurotransmitter amine release have been studied in terms
of second messenger system and modification of autoreceptor system for presynaptic
dopamine terminals4. In the course of the study, the high-affinity binding protein specific
for [3H]R-THBP has been found in the crude membrane fraction of rat brain. However, the
binding activity was also found in the cytosol fraction. Here, we describe several lines of
evidence showing that the binding protein in the cytosol is almost entirely due to nitric oxide
synthase.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 301
EXPERIMENTAL PROCEDURES

Materials.
[3H]6RS-L-Erythro-5,6,7,8-tetrahydrobiopterin was prepared by hydrating 7,8-
dihydrobiopterin with tritiated sodium borohydride. Then, [3H]6R-tetrahydrobiopterin (R-
THBP) was purified using HPLC (Whatman Partisil10 SCX, 4.6 x 250 mm, mobile phase:
30 mM ammonium acetate, pH 3.5/0.1 mM ascorbic acid/ 0.1 mM cysteine). Ascorbic acid
and cysteine were included for the protection of product from oxidation. The sample fraction
coincides with the retention time of authentic R-THBP, clearly distinguishable from the 6S-
form. The radiochemical purity is better than 98%. Purified [3H]R-THBP was stored in 0.1
N HCl containing 1 mM ascorbic acid and 3 mM cysteine at -20°C until use. Unlabeled
pteridines were generous gifts from Suntory Ltd.
Preparations.
Male Wistar rats weighing 200-250 g (Nihon SLC) were anesthetized with
diethylether and decapitated. Brain tissues were homogenized using a Potter-Elvehjem glass
homogenizer with a Teflon pestle in 10 volumes of 0.32 M sucrose containing 5 mM Tris-
HCl (pH 7.4), 1 mM DTT and 1 mM EDTA. After centrifugation at 1,000 x g for 12 min,
the supernatant was further centrifuged at 17,000 x g for 30 min. The pellet was suspended
in a volume of 50 mM Tris-HCl (pH 7.4) equal to the original homogenizing buffer, washed
by recentrifugation at 17,000 g for 10 min, and the suspension was referred to as the P2
fraction. The 17,000 x g-supernatant from was further centrifuged at 103,000 x g for 60
min, and the resulting supernatant was taken and referred to as the cytosol fraction.

Binding Assay.
[3H]R-THBP binding was measured as described by Yumoto et a1.5. The standard
reaction mixture contained 50 mM Tris/HCl (pH7.4), 100 mM NaCl, 10 mM MgCl2, 2 mM
ascorbic acid, and 6 mM cysteine in a total volume of 0.4 ml. Incubation was performed at
37°C for 10 min.
Nitric Oxide Synthase Activity.
Nitric oxide synthase (NOS) activity was measured by monitoring the conversion of
[3H]arginine to [3H]citrulline as described6.

RESULTS AND DISCUSSION

Subcellular fractionation and regional distribution oUlHlR-THBP binding in the rat brain
In the rat cerebellum, [3H]R-THBP binding activity was measured after subcellular
fractionation. The binding activity was mostly located in the cytosol fraction (83% of total
activity in the homogenate), and less activity was detected in the P2 fraction (22%) and
washed P2 fraction (still 11% ). When NOS activity was compared using the same samples,
most (86%) of the activity existed in the cytosol fraction and 22% appeared in the P2
fraction.
Then, the regional distribution of [3H]R-THBP binding activity and NOS activity in
the cytosol fraction was studied. Both activities showed a similar pattern (Table 1); highest
in the cerebellum followed by midbrain and olfactory bulb, then both activities showed rather
diffuse pattern in the cerebral cortex, brain stem, hypothalamus/thalamus, striatum and
hippocampus. Such a pattern was similar to that reported for NOS and to that of [3H]R-
THBP binding activity observed in the P2 fraction of rat brain regions.
Kinetic rroperties ofR-THBP binding in cytosol fraction
By Scatchard plot analysis of [3H]R-THBP binding activity in the cytosol fraction of
rat cerebellum, Kd value was calculated to be 39 nM and Bmax value is 2645 fmoVmg
protein. The binding is highly specific for 6R-form; 6S-form has 3-orders of magnitude less
affinity. The specificity among the biopterin analogues was similar to that obtained in the P2
fraction of rat brain.

302
 Table 1. Distribution of [3H]R-THBP binding activity and nitric oxide synthase
activity in various brain regions of rat

brain regions [3H]R-THBP binding activity NOS activity

(fmoVmg protein) (pmoVmin/mg protein)

Mean SD Mean SD

cerebellum 1021 131 464 34

midbrain 590 29 272 14

olf. bulb 506 46 247 13

cerebral cortex 262 30 116 4

brain stem 239 29 92 3

hypothaVthal. 223 36 165 3
striatum 175 31 119 10

hippocampus 162 43 97 6

Each value was obtained from 4 rat brains with every three determinations for [3H]R-
THBP binding activity and duplicate determinations for NOS activity. SD represents the
standard deviation of the mean of the values from 4 animals.

Purification ofR-THBP binding activity from the cytosol fraction

[3H]R-THBP binding activity was purified from the cytosol fraction of rat brain by
using FPLC and conventional columns. By gel filtration column, [3H]R-THBP binding
activity showed a single peak with a molecular weight of ca. 340 kDa. Elution profile of
NOS activity was the same as this peak of [3H]R-THBP binding activity. Furthermore,
[3H]R-THBP binding activity was attached to 2',5'-ADP Sepharose column which is a good
affinity step for NOS. Therefore, we proceed to investigate [3H]R-THBP binding capacity
of purified NOS to apparent homogeneity.
R-THBP binding to NO synthase purified from porcine brain
[3H]R-THBP binding activity to the NOS purified from porcine brain7 has the
properties similar to that of cytosol fraction. Namely, the specificity, saturability, and time
course were similar. From the Scatchard plot analysis, the Kd and Bmax values were
calculated to be 38nM and 633 fmol/mg protein. A single entity of binding was observed. A
simple calculation from this result, if we assume the molecular weight of NOS being 340
kDa as a dimeric form, leads to the finding that 0.22 mol of [3H]R-THBP was bound to one
mol of the dimer. However, after determination of NOS activity in this purified enzyme
preparation, the enzyme activity was somehow inactivated at around 10% of its original
activity. Therefore, one mol of the dim eric form of the purified enzyme, if it maintains the
original active sites, may bind 2 mol of R-THBP.
Conclusion
Although the exact nature of the 6R-BH4 binding activity in the P2 fraction is still to
be characterized, [3H]R-THBP binding activity in the cytosol fraction showed similar

303
properties to those of NOS. These results taken together indicate that most of high affmity
R-THBP binding sites in the rat brain may be due to NOS and that a certain action ofR-
THBP may be evoked initially by NOS activation.

References
1. K. Koshimura, S. Miwa, K. Lee, M. Fujiwara, andY. Watanabe, J. Neurochem.,
54: 1391-1397' 1990.
2. N. Mataga, K. Imamura, andY. Watanabe, Brain Res., 551:64-71, 1991.
3. T. Ohue, K. Koshimura, Y. Akiyama, Y.Watanabe, and S. Miwa, Brain Res., 570:
173-179, 1992.
4. Yu. Watanabe, N. Mataga, T. Hayashi, T. Ishihara, T. Kanai, T. Noguchi, S. Miwa,
andY. Watanabe, Pteridines, 3:63-64, 1992.
5. N. Yumoto, Y. Watanabe, K. Watanabe, Yu. Watanabe, and 0. Hayaishi,
J. Neurochem., 46:125-132, 1986.
6. D.S. Bredt, and S.H. Snyder, Proc. Natl. Acad. Sci. USA, 87:682-685, 1990.
7. B. Mayer, M. John, B. Heinzel, E.R. Werner, H. Wachter, G. Schultz, and E. Bohme,
FEBS Lett., 288:187-191, 1991.

304
INDUCIBLE NITRIC OXIDE SYNTHASE ACTMTY IN HEPATOCYTES IS
DEPENDENT ON THE COINDUCTION OF TETRAHYDROBIOPTERIN
SYNTHESIS

M. Di Silvio, D.A. Geller, S.S. Gross, A. Nussler, P. Freeswick,

R.L. Simmons, T.R. Billiar

University of Pittsburgh
Department of Surgery
Pittsburgh, PA 15261
The William Harvey Institute
London, UK

Nitric oxide (NO) is a short-lived radical derived from the oxidation of one
of the two chemically equivalent quanido nitrogens of L-arginine 1• Three isoforms
of the enzyme which produces NO, NO synthase (NOS), have been identified thus
far. Two NOS isoforms, endothelial and neuronal are expressed constitutively
(eNOS) and release relatively small amounts of NO immediately upon stimulation.
A third isoform has been termed inducible NOS (iNOS) because it is not present in
resting cells but is expressed if cells are exposed to inflammatory stimuli, such as
bacterial lipopolysaccharide (LPS) and/or cytokines. Upon purification all three
isoforms are known to be dependent on tetrahydrobiopterin (BH4) for maximal
activity2-5• This observation adds to the list of four enzymes previously known to be
dependent on BH4 • Although the precise role of BH4 in the five-electron oxidation
ofL-arginine to NO and citrulline remains uncertain, recent reports using intact cells
in culture indicate that both endothelial cNOS6 and smooth muscle celf isoforms are
dependent on BH4 availability.
Tissues which express eNOS continuously (e.g. brain, endothelium) also
possess the BH4 synthesizing pathway. In contrast, most cells which express iNOS
upon stimulation, such as murine macrophages, fibroblasts, and vascular smooth
muscle cells probably do not produce BH4, or at least adequate amounts of BH4 in
the resting state. Upon stimulation these cells can be induced to produce BH4• For
example, interferon-g (IFN-g) is known to stimulate BH4 synthesis in both
macrophages 8 and fibroblast9 while LPS-induced BH4 synthesis in smooth muscle cells

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 305
increased by IFN-g7• We have shown the GTP-cyclohydrolase I mRNA is absent in
resting rat pulmonary artery smooth muscle cells but can be induced by the cytokines
interleuk:in-1 (IL-1) tumor necrosis factor (TNF), and IFN-g. Gross and Leve have
recently shown that cultured smooth muscle iNOS activity was 60-90% dependent on
de novo BH4 synthesis and 10-40% dependent on the conversion of BH2 to BH4 via
the salvage pathway.
We have previously shown that rat10•11 and human12 hepatocytes (HC) express
an iNOS when exposed to LPS and the cytokines TNF, IL-1, and IFN-g. In
unstimulated cells iNOS mRNA is absent but appears at two hours following
stimulation and then becomes maximal at eight hours 13 • Strong synergy is seen
between the three cytokines while LPS exhibits mild synergy when added with all
three cytokines. Dexamethasone partially suppresses the induction of iNOS in HC.
By cloning the iNOS eDNA from cytokine and LPS-stimulated human HC, we have
verified that human liver can express iNOS 14 and that a human iNOS truly exists. In
vivo studies have suggested that NO plays a protective role in the liver in
inflammation15•16• NO also has antimicrobial actions in the liver and can prevent the
development of the hepatic stage of malaria17• At the cellular level NO may play a
regulatory role with demonstrated effects on total protein synthesis 18 •19, soluble
quanylate cyclase20, mitochondrial aconitase21, and glyceralde-3-phosphate
dehydrogenase22 in liver cells.
Compared to the other cells which express iNOS, HC are unique because
enzymes besides iNOS which require B~ (e.g. phenylalanine hydroxylase) are
present constitutively in HC. We have examined cultured HC to determine if NO
synthesis is dependent on de novo BH4 synthesis or recycling via the salvage pathway
and if BH4 is increased in HC induced to express iNOS. Rat HC were placed in
culture and exposed to a combination of LPS (lO~tg/ml), TNF (500 U/ml), IL-1
(SU/ml), and IFN-g (100U/ml). N0-2 + N0-3 was measured as the stable end
product of NO formation. As we have shown before, the addition of these cytokines
+ LPS (cytokine mix: CM) results in a marked increased in NO synthesis measured
at 24 hours (Table I). The addition of 7mM 2.4-diamino-6-hydroxypryrimidine
(DAHP), an inhibitor of GTP cyclohydrolase I significantly suppressed NO synthesis.
Up to 80% inhibition was seen at 20 mM DAHP (not shown). This inhibition was
overcome by the simultaneous addition of BH4 • The addition of 100 ~tM
methotrexate (MTX) did not reduce NO synthesis. However, the addition DAHP
+ MTX almost completely inhibited NO synthesis. These data show that CM-treated
HC utilize BH4-derived primarily from de novo synthesis for NO production and that
NO synthesis may be limited by B~ availability.
To determine if BH4 synthesis was also induced or increased when HC were
stimulated to express iNOS, two approaches were taken. First, biopterin levels in
cultured HC were measured. Secondly, mRNA levels for GTP cyclohydrolase I, the
rate limiting enzyme for BH4 synthesis were measured in the stimulated ceHs. Total
cellular biopterin was measured after acidic oxidation of reduced forms of biopterin
using iodinine and followed by reverse phase HPLC (Table I). Higher biopterin
levels were found in CM-treated HC. Blocking NO with the NOS inhibitor, N°
monomethyl-L-arginine (NMA) decreased N0-2 + N0-3 levels but had no effect on
biopterin levels. DAHP reduced total biopterin levels to control levels while the
combination of DAHP and MTX markedly suppressed biopterin levels. These data
suggest that the salvage pathway may assist in maintaining total biopterin levels and

306
TABLE I. Supernatant N0-2 + N0-3 levels and cellular total biopterin levels in
hepatocytes 24 hours after exposure to the cytokine mix (CM: LPS,
TNF, IL-l, IFN-g).
No-2 + No-3 Biopterin ng/mg
nmoles/106 cells protein
Control 10 ± 1 0.45 ± .04
CM 328 ± 56 0.70 ± .07
CM + NMA ±2
16 0.6± .05
CM + DAHP (7mM) 248 ± 43 0.36 ± .07
CM + MTX 396 ± 84 0.61 ± .11
CM + DAHP + MTX 28 ± 9 Not detected

that biopterin levels can be reduced to undetectable levels if both de novo and
salvage pathways are blocked. Using a eDNA probe generated by RT polymerase
chain reaction (PCR) for GTP-cyclohydrolase I Northern blots were performed on
HC 8 hours after CM addition. A 2-3 fold increase in GTP-cyclohydrase I mRNA
was seen simultaneous to the known peak for iNOS expression. iNOS mRNA was
not detectable in control HC but was strongly induced by the 8 hour time point in
CM-treated HC. Taken together these data indicate that under CM stimulation BH4
synthesis is increased in HC. It is likely that this increase takes place to support
induced NO synthesis in HC. Whether constitutive levels of BH4 are adequate to
support lower levels of NO synthesis remain to be determined. In other experiments,
we have shown that the addition of BH4 to HC stimulated to express iNOS increases
total NO synthesis (not shown). It is possible that NO synthesis is limited by BH4
availability. How BH4 synthesis is regulated in cells expressing iNOS and how iNOS
expression influences other enzymes which utilize B~ is unknown.

TABLE II. mRNA levels for GTP-cyclohydrolase I and inducible nitric oxide
synthase (iNOS) in hepatocytes exposed to cytokine mix (CM).

Densitometry Units

GTP-cyclohydrolase I iNOS

Control 1888 Not detectable

CM (8 hrs) 5006 2298

307
 REFERENCES

1. S. Moncada, RM.J. Palmer, B.A. Higgs, J. Pharmacal Rev 43:109 (1991).

2. M.A. Tayeh, M.A. Marietta, J Biol Chern 264: 19654 (1989).
3. N. Kwon, C.F. Nathan, D. Stuehr, J Biol Chern, 264:20496 (1989).
4. B. Mayer, M. John, E. Bohme, FEBS Lett 277:215 (1990).
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F. Murad, Proc Natl Acad Sci USA 88:365 (1991).
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Schmidt, G. Weiss, H. Wachter, J Biol Chern 268:1842 (1993).
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8. E.RWerner, G. Werner-Felmayer, D. Fuchs, A. Hausen, G. Reibnegger, H.
Wachter, Biochem J 262:861 (1989).
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Wang, RL. Simmons, T.R Billiar, Proc Natl Acad Sci USA 90:522 (1993).
14. D.A. Geller, C.J. Lowenstein, RA. Shapiro, A.K. Nussler, M. Di Silvio, S.C.
Wang, D.K. Nakayama, R.L. Simmons, S.H. Snyder, T.R.Billiar, lProc Natl
Acad Sci USA (In Press).
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Simmons, J Leuk Biol48:565 (1990).
16. B.G. Harbrecht, T.RBilliar, J.Stadler, A.J. Demetris, J. Ochoa, RD. Curran,
RL. Simmons, J Leuk Biol52:390 (1992).
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18. T.R. Billiar, R.D. Curran, D.J. Stuehr, M.A.West, B.G. Bentz, R.L. Simmons,
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19. R.D. Curran, F.K. Ferrari, P.H. Kispert, J. Stadler, D.J. Stuehr, R.L. Simmons,
T.RBilliar, FASEB J 5:2085 (1991).
20. T.R. Billiar, R.D. Curran, B.G. Harbrecht, J. Stadler, D.L. Williams, J.B.
Ochoa, M. Di Silvio, RL. Simmons, S.A. Murray, Am J Physiol 262:C1077
(1992).
21. J. Stadler, T.R Billiar, RD. Curran, D.J. Stuehr, J.B. Ochoa, R.L. Simmons,
Am J Physiol 260:C910 (1991).
22. L. Molina y Vedia, B. Mcdonald, B. Reep, B. Brune, M. Di Silvio, T.RBilliar,
E.G. Lapetina, J Biol Chern 267:24929 (1992).

308
MODULATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN INTACT CELLS
BY INTRACELLULAR TETRAHYDROBIOPTERIN LEVELS

Gabriele Werner-Felmayer, Ernst R. Werner, Gunter Weiss, and Helmut Wachter

Institute for Medical Chemistry and Biochemistry, University of Innsbruck,

A-6020, Innsbruck, Austria

INTRODUCTION

An up to 100-fold increase of GTP cyclohydrolase I activity has been observed upon

treatment with interferon-gamma and tumour necrosis factor-alpha in a variety of human
and murine cell types in vitro, including macrophages and dermal fibroblasts. As a result,
intracellular tetrahydrobiopterin levels are highly increased in cytokine-treated cells as
compared to untreated controls (review1•2).
Besides being an essential cofactor for the hydroxylation of aromatic amino acids and
the cleavage of glyceryl ethers3, it has been demonstrated that tetrahydrobiopterin is
required for full activity of nitric oxide (NO) synthase. NO synthase, which occurs in
constitutive and cytokine-inducible isoforms, catalyzes the oxygenation of L-arginine to
L-citrulline and NO. NO formation depends on NADPH, flavins and Ca2+/calmodulin in
case of the constitutive enzyme (review4). The cytokine-inducible Ca2+-independent NO
synthase from murine macrophages has been demonstrated to contain calmodulin as an
integral part of the protein5•
The role of tetrahydrobiopterin in the NO synthase reaction is not yet clear. However,
stimulation of the NO synthase reaction by tetrahydrobiopterin has been observed for the
cytokine-inducible enzyme from activated macrophages and from liver of endotoxin-treated
rats, as well as for constitutive NO synthase purified from cerebellum, endothelial cells and
from neutrophils (review4). Further it was shown that tetrahydrobiopterin remains tightly
bound to NO synthase purified from cerebellum6· 8 and from cytokine-treated murine
macrophages9•

EFFECT OF INTRACELLULAR TETRAHYDROBIOPTERIN LEVELS

Cytokine-inducible NO synthase. Applying the strategy outlined in Fig. 1, we could

show that cytokine-induced NO synthase of murine fibroblasts depends on intracellular
tetrahydrobiopterin levels 10 • Murine fibroblasts form tetrahydrobiopterin 11 and
nitrite/nitrate10 when treated with interferon-gamma in combination with a second stimulus

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 309
such as tumour necrosis factor-alpha, interleukin-1 or lipopolysaccharide (LPS). Inhibition
of de novo tetrahydrobiopterin biosynthesis by 2,4-diarnino-6-hydroxypyrimidine (DAHP),
an inhibitor of GTP cyclohydrolase I 12, decreased intracellular tetrahydrobiopterin levels
and formation of nitrite/nitrate, indicating inhibition of NO synthase. Treating cells with
sepiapterin, which is converted to tetrahydrobiopterin via the salvage pathway13 led to
strongly increased intracellular tetrahydrobiopterin levels and increased formation of
nitrite/nitrate, suggesting that cytokine-induced NO synthase was not saturated with the
cofactor. Concurrent treatment of cells with sepiapterin counteracted NO synthase inhibition
by DAHP. Neither pterin 6-carboxylic acid nor isoxanthopterin, which are no substrates for
the salvage pathway, could overcome the effect of DAHP, thus indicating specificity for the
effect of tetrahydrobiopterin. Methotrexate, an inhibitor of dihydrofolate reductase which
converts 7,8-dihydrobiopterin formed from sepiapterin to the tetrahydro-derivative13, did not
interfere with NO synthase or the effect of DAHP. However, when applied in presence of
sepiapterin, formation of nitrite/nitrate was strongly inhibited. Maximal inhibition of NO
synthase was achieved by concomrnitant treatment of cells with DAHP, methotrexate and
sepiapterin due to inhibition of de novo and salvage pathway. In this case more than 80 %
of the residual biopterin occurred in its dihydro-form.
We could thus demonstrate that intracellular tetrahydrobiopterin levels modulate
cytokine-inducible NO synthase, that the enzyme is not saturated with tetrahydrobiopterin
and that intact cells use the tetrahydro- rather than the dihydro-derivative of biopterin in the
NO synthase reaction10 • Further, we could demonstrate that the cytotoxic effect of
interferon-gamma plus tumour necrosis factor-alpha on murine fibroblasts observed after
long-term treatment occurred due to formation of NO, since N'-monomethyl-L-arginine
(NMA), an inhibitor of NO synthase, could strongly reduce the cytotoxic effect of the
cytokines, which was overcome by excess of L-arginine but not by D-arginine10• The
cytotoxic effect was also abolished when stimulating cells in arginine-free culture medium.
When inhibiting tetrahydrobiopterin biosynthesis and NO synthesis with DAHP, the
cytotoxic effect of cytokines was significantly reduced. Cytotoxicity was restored by
concurrent addition of sepiapterin, indicating that not only NO synthase activity but also a
biological effect of the NO thus formed could be modulated by intracellular
tetrahydrobiopterin levels 10 •
Dependence of cytokine-inducible NO synthase on intracellular tetrahydrobiopterin
levels was also confirmed for murine endothelial cells14 and for rat vascular smooth muscle
cells 15 using the same strategy (Fig.l). In cytokine-treated murine macrophages, a
combination of DAHP with N-acetylserotonin, an inhibitor of sepiapterin reductase16,
strongly inhibited NO synthase17•

Constitutive NO synthase. Human umbilical vein endothelial cells (HUVEC) express

constitutive NO synthase which is stimulated by Ca2+ influx leading to increased
intracellular cGMP levels18• We could demonstrate that treatment of HUVEC with
sepiapterin (Fig. 1) leads to increased intracellular tetrahydrobiopterin levels and to a
concentration-dependent stimulation of NO synthase activity 19• Reducing intracellular
tetrahydrobiopterin levels by DAHP resulted in inhibition of NO synthase19• Inhibition of
constitutive NO synthase by inhibition of tetrahydrobiopterin biosynthesis with DAHP has
also been demonstrated for porcine aortic endothelial cells20• In HUVEC and in aortic
endothelial cells inhibition by DAHP could be reversed by addition of sepiapterin19•20•
Further, we showed that GTP cyclohydrolase I activity of HUVEC is stimulated up to 40-
fold by treatment with interferon-gamma, tumour necrosis factor-alpha and LPS 19 • The
thereby increased intracellular tetrahydrobiopterin levels led to a significant increase of
constitutive NO synthase activity19• As with a number of other human cells, cytokines did
not directly induce synthesis of a Ca2+-independent NO synthase 19• We obtained similar
results using the human cervix carcinoma cell line ME-18021 • These cells express a
constitutive brain-type NO synthase which is significantly increased by cytokines due to

310
increased intracellular tetrahydrobiopterin levels rather than to increased NO synthase
protein21 •

CONCLUSIONS

Taken together, our results indicate that cytokine-inducible as well as constitutive NO

synthase of intact cells depends on intracellular tetrahydrobiopterin levels. The effect of
methotrexate indicates that intact cells use tetrahydrobiopterin rather than dihydrobiopterin.
Furthermore, both types of enzyme are not saturated with this cofactor in intact cells, since
sepiapterin, which is converted to tetrahydrobiopterin, increased NO formation by cytokine-
inducible and constitutive NO synthase, respectively. Cytokines induce tetrahydrobiopterin
formation in murine fibroblasts in order to provide a cofactor for cytokine-induced NO
synthase. In human endothelial cells, which express only constitutive NO synthase, cytokine
treatment stimulates NO synthase by increasing intracellular tetrahydrobiopterin levels.
Patients suffering from septic complications leading to massive endogenous cytokine
production, show highly elevated neopterin excretion indicating activation of GTP
cyclohydrolase I22• It has been discussed whether inhibition of GTP cyclohydrolase I could
be used as a strategy to control cytokine-induced NO synthesis which plays an important
role in sepsis23"25• It was claimed that DAHP could be used for such a strategy since it had
no direct effect on constitutive NO synthase15 • However, this contrasts experimental
evidence19•20 : in line with observations on purified NO synthase from various sources
(review4), we find that DAHP treatment of cells is effective for both types of NO synthase
due to depletion of tetrahydrobiopterin10•19•20 •

cytokines

de novo j+ _
GTP-CH I - DAHP
+
/ I
neopterin
(in humans) 1 methotrexate
l -
tetrahydrobiopterin - DHFR - sepiapterin
~ salvage
inducible constitutive
NO synthase NO synthase
1 + 1 calcium
NO NO
1
nitrite + nitrate cGMP
long-term short-term
murine fibroblasts 10 HUVEC, ME-180 19 •21

FIGURE 1. Strategy for influencing NO synthase activity by modulating intracellular tetrahydrobiopterin

levels. DAHP, 2,4-diamino-6-hydroxy-pyrimidine; GTP-CH I, GTP cyclohydrolase I; DHFR, dihydrofolate
reductase.

REFERENCES
1. E. R. Werner, G. Werner-Felmayer, and H. Wachter, Tetrahydrobiopterin and Cytokines,
Proc.Soc.Exp.Biol.Med. 203, in press (1993).
2. E.R. Werner, G. Werner-Felmayer, G. Weiss, and H. Wachter, Stimulation of tetrahydro-biopterin
synthesis by cytokines in human and in murine cells, in: "Chemistry and Biology of Pteridines and
Folates", J.E. Ayling, M.G. Nair, and C.M. Baugh, eds., Plenum Press, New York, this volume (1993).

311
 3. S. Kaufman, The metabolic role of tetrahydrobiopterin, in:"Chemistry and Biology of Pteridines",
B.A. Cooper and V.M. Whitehead, eds., Walter De Gruyter, Berlin, New York (1986).
4. C. Nathan, Nitric oxide as a secretory product of mammalian cells, FASEB J. 6: 3051(1992).
5. H.J. Cho, Q. Xie, J. Calaycay, R.A. Mumford, K.M. Swiderek, T.D. Lee, and C. Nathan, Calmodulin
is a subunit of nitric oxide synthase from macrophages, J. Exp. Med.l76:599(1992).
6. B. Mayer, M. John, B. Heinzel, E.R. Werner, H. Wachter, G. Schultz, and E. Bohme, Brain nitric
oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase, FEBS Lett.
288:187(1991).
7. H.H. Schmidt, R.M. Smith, M. Nakane, and F. Murad, Ca2•/calmodulin-dependent NO synthase type
I: A bioptero-flavoprotein with Ca2•/calmodulin-independent diaphoraseandreductase activities,
Biochemistry 31:3243(1992).
8. P. Klatt, B. Heinzel, B. Mayer, E. Ambach, G. Werner-Felmayer, H. Wachter, and E.R. Werner,
Stimulation of human nitric oxide synthase by tetrahydrobiopterin and selective binding of the
cofactor, FEBS Lett. 305:160(1992).
9. J.M. Revel and M.A. Marietta, Macrophage nitric oxide synthase: relationship between enzyme-bound
tetrahydro-biopterin and synthase activity, Biochemistry 31:7160(1992).
10. G. Werner-Felmayer, E.R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, and H. Wachter,
Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts, J. Exp. Med.
172: 1599(1990).
11. E.R. Werner, G. Werner-Felmayer, D. Fuchs, A. Hausen, G. Reibnegger, J.J. Yim, and H. Wachter,
Impact of tumour necrosis factor-alpha and interferon-gamma on tetrahydrobiopterin synthesis in
murine fibroblasts and macrophages, Biochem. J. 280:709(1991).
12. E.M. GM, J.M. Nelson, and A.D. Sherman, Biopterin Ill. Purification and characterization of enzymes
involved in the cerebral synthesis of 7,8-dihydrobiopterin, Neurochem. Res. 3:69(1978).
13. C.A. Nichol, G.K. Smith, and D.S. Duch, Biosynthesis and metabolism of tetrahydrobiopterin and
molybdopterin, Annu. Rev. Biochem. 54:729(1985).
14. S.S. Gross, B.A. Jaffe, R. Levi, and R.G. Kilbourn, Cytokine-activated endothelial cells express an
isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and
inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages,
Biochem. Biophys. Res. Commun. 178:823(1991)
15. S.S. Gross and R. Levi, Tetrahydrobiopterin Synthesis, an absolute requirement for cytokine-induced
nitric oxide generation by vascular smooth muscle, J. Biol. Chem. 267:25722(1992).
16. S. Katoh, T. Sueoka, and S. Yamada. Direct inhibition of brain sepiapterin reductase by a
catecholamine and an indoleamine, Biochem. Biophys. Res. Commun. 105:75(1982).
17. N. Sakai, S. Kaufman, and S. Milstien, Tetrahydrobiopterin is required for cytokine-induced nitric
oxide production in a murine macrophage cell line (RAW 264), Mol. Pharmacal. 43:6(1993).
18. B. Mayer, K. Schmidt, P. Humbert, and E. Bohme, Biosynthesis of endothelium-derived relaxing
factor: a cytosolic enzyme in porcine aortic endothelial cells Ca2• -dependently converts L-arginine
into an activator of soluble guanylyl cyclase, Biochem. Biophys. Res. Commun. 164:678(1989).
19. G. Werner-Felmayer, E.R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, K. Schmidt, G. Weiss, and
H. Wachter, Pteridine biosynthesis in human endothelial cells- impact on nitric oxide-mediated
formation of cyclic GMP, J. Biol. Chem. 268:1842(1993).
20. K. Schmidt, E.R. Werner, B.Mayer, H. Wachter, and W.R. Kukovetz, Tetrahydro-biopterin-dependent
formation of endothelium-derived relaxing factor (nitric oxide) in aortic endothelial cells, Biochem. J.
281:297(1992).
21. G. Werner-Felmayer, E.R. Werner, D. Fuchs, A. Hausen, B. Mayer, G. Reibnegger, G. Weiss, and H.
Wachter, Ca2•/calmodulin-dependent nitric oxide synthase activity in the human cervix carcinoma cell
line ME-180, Biochem. J. 289: 357(1993).
22. W. Strohmaier, H. Redl, G. Schlag, and D. Inthorn, D-erythro-neopterin plasma levels in intensive
care patients with and without septic complications, Crit. Care Med. 15:757(1987).
23. R.G. Kilbourn and O.W. Griffith, Over-production of nitric oxide in cytokine-mediated and septic
shock, J. Natl. Cancer lnst. 84:827(1992).
24. G. Werner-Felmayer, E.R. Werner, G. Weiss, and H. Wachter, Tetrahydrobiopterin biosynthesis
inhibition for diminishing nitric oxide production in cytokine-mediated septic shock, J. Natl. Cancer
lnst. 84:1671(1992).
25. R.G. Kilbourn and O.W. Griffith, Inhibition of inducible nitric oxide synthase with inhibitors of
tetrahydrobiopterin biosynthesis, J. Natl. Cancer lnst. 84:1672(1992).

312
SR-L-erythro-5,6,7,8-TETRAHYDROBIOPTERIN: A REGULATOR OF
NEUROTRANSMITTER RELEASE

Kunia Koshimura, 1 Tetsuya Ohue, 1

Yasuyoshi Watanabe, 2 and Soichi Miwa 1

!Department of Pharmacology, Kyoto University

Faculty of Medicine, Kyoto 606, Japan
2Department of Neuroscience, Osaka Bioscience
Institute, 6-2-4 Furuedai, Suita, Osaka 565, Japan

INTRODUCTION
6R-L-erythro-Tetrahydrobiopterin (6R-BH 4 ) is a common
natural cofactor for hydroxylases of phenylalanine, tyrosine
and tryptophan, and is thought to play an important role in
the regulation of monoamine neurotransmitter biosynthesis 1 .
In fact, it is well known that in atypical phenylketonuria,
6R-BH 4 deficiency results in severe neurological problems as a
result of decreased biosynthesis of brain catecholamines and
5-hydroxytryptamine (5-HT) 2 • 3 Furthermore, it is reported
that the content of 6R-BH 4 is reduced in the brain and/or
cerebrospinal fluid of several neuropsychiatric diseases such
as Parkinson's disease 4 • 5 • 6 , Alzheimer's disease 7 , depression 8
and dystonia 9 . In some clinical trials, administration of
6R-BH 4 is reported to improve clinical signs and symptoms of
these diseases 3 • 6 • B-lO. These clinical findings imply that
the therapeutic efficacy of 6R-BH 4 are mainly due to enhance-
ment of neurotransmitter release. Therefore, we began to
examine the effects of 6R-BH 4 on release of neurotransmitters
such as dopamine (DA) and acetylcholine (ACh) in the brain
using in vivo brain microdialysis.

MATEIALS AND METHODS

In vivo brain microdialysis was performed under a
freely-moving condition as described elsewhere 11 . In brief,
under ether anesthesia, a U-shaped probe for brain microdialy-
sis was stereotactically implanted into the striatum and
hippocampus of male Wistar rats (200-250 g) for study of
release of DA and ACh, respectively, and fixed in place with
cranioplastic cement. After the rats had recovered from
anesthesia, the probe was perfused at a flow rate of 6.1
~1/min with Ringer solution (147 mM NaCl, 4 mM CaC1 2 and 2.3
mM KCl; pH 6 .1) and dialysa tes were collected every 20 min.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 313
DA recovered in stria tal dialysa tes was purified with an
alumina batch method, and analyzed using HPLC with electro-
chemical detection 11 . ACh was purified with a small column of
Sephadex G-10, and analyzed usinf HPLC with an enzyme reactor
and an electrochemical detector1 ,13.
6R-BH 4 and other pteridines were added to the perfusion
fluid at the indicated concentrations.
6R-BH 4 , 6S-BH 4 and L-erythro-biopterin were generous
gifts from Sun tory Institute of Biomedical Research, Osaka,
Japan.

2.0

c
·e 1.5
0
~
0
E
.s
G)
1.0
c
·e
CIS 0.5
c.
0
c
120 240 120 240
Time (min) Time (min)
Figure 1. Effects of infusion of 6R-BH 4 on the extracellular DA levels in
rat striatum monitored by in vivo brain microdialysis. A) 6R-BH4 was added
to the perfusion fluid (indicated by a bar) at the concentrations of 0.25
(.&), 0.5 (.). 1.0 (.)and 5.0 mM (~). Q; control. B) 6S-BH 4 (.)or
biopterin (.A) was added at 1 mM.

RESULTS AND DISCUSSION

Stimulation of Monoamine Neurotransmitter Release by 6R-BH4

Dopamine Release As shown in Fig. lA, DA levels in dialy-

sates of control rats were constant from the beginning of the
experiment up to 240 min. When various concentrations of 6R-
BH4 were added to the perfusion fluid, the DA levels increased
in a concentration-dependent manner, and reached a plateau at
1 mM with the EC 50 value of about 300 ~M. Following infusion
of 6S-BH 4 (a diastereoisomer of 6R-BH 4 ), the DA levels in-
creased but the increase was far smaller than that induced by
6R-BH 4 (Fig. lB and Fig. 2). In contrast, no increase was
observed following administration of biopterin, an oxidized
product of 6R-BH 4 (Fig. 1B). Notably, following administra-
tion of sepiapterin which is metabolized intracellularly to
6R-BH 4 via salvage pathway, the DA levels increased more
markedly than following administration of 6R-BH 4 (Fig. 2).
These results show that SR-form of BH 4 specifically possesses
DA releasing action.

314
 3.0

c
·e
Q
2.0
N
~
E
.s-
GI

·ec:as 1.0
a.
0
c

Control 6R 6S Sepia.

Figure 2. Effects of 6R-BH4 , 6S-BH4 and sepiapterin on DA levels in stri-

atal dialysates in the absence and presence of treatment with a-methyl-p-
tyrosine (a -MT, 250 mg/kg, ip).

The used concentrations of 6R-BH 4 seem to be high, but

when calculated using passage efficiency (called recovery; 3%)
of 6R-BH 4 through the dialysis membrane, the concentration of
6R-BH 4 in the extracellular fluid is estimated to be 30 /.L M
during infusion of 1 mM 6R-BH 4 .
The increase in DA levels in dialysates was abolished,
when 6R-BH 4 was administered after infusion of blockers of
voltage-dependent Na+ (tetrodotoxin; 40 J.LM) or ca 2 + (NKY-722;
0.5 mM) channels.

50

c 40
·e
Q
~
0 30
E
.s-
<
a. 20 1-1-
0 ::2::2
c 6 6

6R 6S Sepia.

Figure 3. Effects of 6R-BH 4 , 6S-BH 4 and sepiapterin on DOPA levels in

dialysates in the absence and presence of treatment with a -MT.

315
 These data taken together suggest that 6R-BH 4 stimulates
exocytotic DA release. However, since 6R-BH 4 is a cofactor
for tyrosine hydroxylase, there are two possibilities for
enhancement of DA release by 6R-BH 4 . One possibility is that
the enhancement is secondary to an increase in DA biosynthesis
resulting from increased availability of the cofactor. The
other possibility is that the enhancement is due to direct
stimulation of DA release, which is independent of cofactor
activity. To test these two possibilities, we examined the
effects of 6R-BH 4 and other pteridines after inhibition of DA
biosynthesis using an inhibitor of tyrosine hydroxylase, a-
methyl-p-tyrosine (a-MT).
After intraperitoneal injection of 250 mg/kg of a -MT,
the basal DA levels in dialysates markedly decreased (Fig. 2),
suggesting that DA biosynthesis was inhibited. When 6R-BH 4
was administered after a -MT, it can still stimulate DA re-
lease (more than 70% of the release persisted). In contrast,
the increase in DA release induced by 6S-BH 4 and sepiapterin
was completely abolished by pretreatment with a-MT (Fig. 2).
As shown in Fig. 3, the effects of 6R-BH 4 and other
pter idines on tyrosine hydroxylase activity in vivo were
determined using the same microdialysis system. That is, the
enzyme activity was indexed by extracellular DOPA levels
during continuous infusion of a DOPA decarboxylase inhibitor
(NSD 1015, 0.5 mM) as described previously 14 . The DOPA level
as an index of in vivo tyrosine hydroxylase activity was
increased (from the control value of 6.4 + 1.2 pmol/20 min) to
21 ~ 2.8 pmol/20 min following infusion of 6R-BH 4 (1 mM).
Following administration of 6S-BH 4 and sepiapterin, the DOPA
levels increased to 13.9 + 1. 8 pmol/20 min and 45.1 + 7. 2
pmol/20 min, respectively.-
When rats were pretreated with an intraperitoneal injec-
tion of a-MT (250 mg/kg), the DOPA level in a control group
gradually decreased to 1.2 ~ 0.2 pmol/20 min. After pretreat-
ment with a-MT, neither 6R-BH 4 nor other pteridines could
induce an increase in the DOPA levels. These results show
that tyrosine hydroxylase activity in vivo is completely
inhibited by a-MT, before or even after administration of 6R-
BH4 or other pteridines.
These results taken together strongly indicate that most
of the 6R-BH 4 -induced DA release is independent of DA biosyn-
thesis. In contrast, the increase in DA release induced by
6S-BH 4 or sepiapterin is secondary to an increase in DA bio-
synthesis. Thus, we conclude that 6R-BH 4 has a direct DA
releasing action, which is independent of its cofactor activi-
ty, whereas 6S-BH 4 or sepiapterin has only cofactor activity.

Other Monoamine Neurotrasmitter Release Mataga et al. 15

showed that 5-HT release in rat striatum, determined by in
vivo brain microdialysis, was enhanced by 6R-BH 4 . However, it
is at present unknown whether the enhancement is independent
of 5-HT biosynthesis or not.

Stimulation of Nonmonoamine Neurotransmitter Release by 6R-BH4

Acetylcholine Release To test whether this neurotransmit-

ter releasing action of 6R-BH 4 is specific for monoaminergic
neurons, we investigated the effects of 6R-BH 4 on ACh release
in the hippocampus, using the same brain microdialysis system.

316
 30
.......
s::
.E
0
~
0
E
sIll
Qj
>
~
..r:::
0
<C

Time (min) Time (min)

Fig. 4. Effects of infusion of 6R-BH 4 on ACh levels in dialysates of the
hippocampus in the absence and presence of treatment with reserpine.

When various concentrations of 6R-BH 4 were administered

through the dialysis probe, ACh levels in dialysates increased
concentration-dependently (Fig. 4A), but choline levels were
unchanged (data not shown).
As we have already shown, 6R-BH 4 stimulates release of
monoamine neurotransmitters. Conversely_, ACh release is
reported to be modulated by monoamines 16 ·I7 . Thus, there is
the possibility that 6R-BH 4 increases ACh release via monoami-
nergic system. Therefore, to test this possibility, we
examined the effects of 6R-BH 4 , after depletion of monoamines
using reserpine.
After pretreatment with reserpine (i.p. injection of 5
mg/kg and 3 mg/kg, 48 hand 24 h before experiments), the
content of DA, noradrenaline and 5-HT in the hippocampus
decreased to 0.1 + 0.1%, 1.2 + 1.1% and 28.2 + 4.5%, respec-
tively, of the control value (DA, 0.162 ~ 0.014-nmol/g tissue;
noradrenaline, 3.50 ~ 0.35 nmol/g; 5-HT, 0.987 ~ 0.066 nmol/g;
n=8).
When 6R-BH 4 was administered after pretreatment with
reserperine, it could no longer induce an increase in ACh
release (Fig. 4B). These results suggest that 6R-BH 4 indi-
rectly enhances ACh release via monoaminergic system.
To determine which monoamine (catecholamines or 5-HT) is
involved in the enhancement of ACh release, the effects of
6R-BH 4 on ACh release were investigated following selective
depletion of either catecholamines or 5-HT. After pretreat-
ment with a -MT (an inhibitor of tyrosine hydroxylase), the
6R-BH 4 -induced enhancement of ACh persisted, but after pre-
treatment with p-chlorophenylalanine (PCPA; an inhibitor of
tryptophan hydroxylase), the major portion of the increase was
abolished 18 . These results suggest that the 6R-BH 4 -induced
enhancement of ACh is mediated mainly by the 5-HTergic system.
Furthermore, the concentration of 5-hydroxyindoleacetic
acid (a major metabolite of 5-HT) in dialysates was elevated
following infusion of 6R-BH 4 18 . Administration of exogenous
5-HT or its precursor 5-hydroxytryptophan markedly enhanced
ACh release, but administration of DA, noradrenaline and their
common precursor L-DOPA had little effect on ACh release 18 .

317
These results taken together demonstrate that 6R-BH 4 indirect-
ly enhances ACh release mainly by activating the 5-HTergic
system.
Glutamate Release Mataga et a1. 15 showed that glutamate
release in rat striatum in vivo was enhanced by infusion of
6R-BH 4 through the dialysis probe. However, 6R-BH 4 had little
effect on glutamine levels in dialysates. Furthermore, the
enhancement of glutamate release was abolished after destruc-
tion of dopaminergic nerve terminals by continuous infusion of
a catecholamine neurotoxin, 6-hydroxydopamine. These results
show that enhancement of glutamate release is mediated by the
dopaminergic system.
Insights into Mechanism of Action

Since 6R-BH 4 is also known as a cofactor for nitric oxide

(NO) synthase and evidence is accumulating that NO is a sig-
naling molecule 19 , there is the possibility that the action of
6R-BH 4 is mediated by NO. However, inhibitors of NO synthase
such as NG-monomethyl-L-arginine and L-nitro-arginine (infused
through the dialysis probe) were without effect on 6R-BH 4 -
induced DA release in rat striatum (unpublished data). Thus
it is quite likely that the action of 6R-BH4 is not mediated
by NO.
To determine whether 6R-BH~ acts from the inside of the
cell or from the outside, the 1ntracellular concentration of
6R-BH 4 was selectively elevated by administration of sepiapt-
erin. Following infusion of sepiapterin through the dialysis
membrane, DA release monitored by brain microdialysis was
increased: the effect was more marked than that of 6R-BH 4 .
However, the increase was not observed, when sepiapterin was
administered after pretreatment with a -MT (Fig. 2). These
results suggest that an increase in the intracellular concen-
tration of 6R-BH 4 stimulates DA release secondarily to an
increase in biosynthesis of DA. These data taken together
strongly indicate that 6R-BH 4 acts from the outside of the
cell to stimulate DA release.
Furthermore, when 6S-BH4 or 6-methyl-tetrahydropterin (a
synthetic cofactor for tyrosine hydroxylase) was coadminis-
tered with 6R-BH 4 , they suppressed the increase in DA re-
lease induced by 6R-BH 4 alone (unpublished data). These data
indicate that 6S-BH 4 and 6-methyl-tetrahydropterin act as an
antagonist of 6R-BH4 at the same site as 6R-BH 4 . Thus it is
quite likely that the action of 6R-BH 4 is mediated by a spe-
cifc recognition site.
In a preliminary study using slice patch techniques, we
have found that 6R-BH 4 (at the concentration of ~M range)
inceases the frequency of firing of neurons in the brain and
activates several ion channels (unpublished data).
However, the information on cellular mechanism of action
of 6R-BH 4 is at present virtually absent and further intensive
study is needed.

REFERENCES
1. S. Miwa, Y. Watanabe, and 0. Hayaishi, Arch. Biochem. Biophys.
239:234 (1985).
2. S. Kaufman, S. Berlow, G.K. Summer, S. Milstien, J.D. Schulman,

318
 s. Orloff, S. Spielberg, and S. Pueschel, New Eng. J. Med.
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3. H.-c. Curtius, A. Niederwieser, M. Viscontini, A. Otten, J.S. Schaub,
S. Scheibenreiter, and H. Schmidt, Clin. Chim. Acta 93:251 (1979).
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and D.B.Calne, Science 204;624 (1979).
5. T. Nagatsu, T. Yamaguchi, T. Kato, T. Sugimoto, S. Matsuura, M. Akino,
I. Nagatsu, R. Iizuka, and H. Narabayashi, Clin. Chim. Acta 109:305
(1981).
6. T. Nagatsu, T. Yamaguchi, M.K. Rahman, J. Trocewicz, K. Oka,
Y. Hirata, I. Nagatsu, H. Narabayashi, T. Kondo, and R. Iizuka,
Adv. Neurol. 40:467 (1981).
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N.R. Cutler, and S. Rapoport, Arch. Neurol. 43:996 (1986).
8. H.-c. Curtius, A. Niederwieser, R.A. Levine, W. Lovenberg, B. Woggon,
and J. Angst, Lancet 1:657 (1983).
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A. Papavasiliou, A. Rayes, R. Eldridge, and R.S. Burns, Neurology
36:760 (1986).
10. S. Kaufman, G. Kapatos, R. Mcinnes, D. Schulman, and W. Rizzo,
Pediatrics 70:376 (1982).
11. K. Koshimura, S. Miwa, K. Lee, M. Fujiwara, and Y. Watanabe,
J. Neurochem. 54:1391 (1990).
12. K. Koshimura, S. Miwa, K. Lee, Y. Hayashi, H. Hasegawa, K. Hamahata,
M. Fujiwara, M. Kimura, andY. Itokawa, J. Neurochem. 54:533 (1990).
13. T. Ohue, K. Koshimura, Y. Akiyama, Y. Watanabe, and S. Miwa,
Brain Res. 570:173 (1992).
14. B.H.C. Westerink, J.B. De Vries, and R. Duram, J. Neurochem. 54:381
(1990).
15. N. Mataga, K. Imamura, andY. Watanabe, Brain Res. 551:64 (1991).
16. A. Ajima, Y. Yamaguchi, and T. Kato, Brain Res. 518:193 (1990).
17. D. Jackson, M.K. Stachowiak, J.P. Bruno, and M.J. Zigmond,
Brain Res. 457:259 (1988).
18. T. Ohue, K. Koshimura, Y. Takagi, Y. Watanabe, S. Miwa and T. Masaki,
Brain Res., in press.
19. J. Collier and P. Vallance, Trends Pharmacol. Sci. 10:427 (1989).

319
POSITRON EMISSION TOMOGRAPHY STUDIES ON SOME
NEUROTRANSMITTER RECEPTOR SYSTEMS WITH 6R-
TETRAHYDROBIOPTERIN PRETREATMENT

Yasuyoshi Watanabe1,2, Yoshihiro Tani3, Tadashi Kanai3,

Per Hartvig2,4, Osamu Inoue2,5, Jesper Andersson2.4,
Anders Lilja2,4, and Bengt LAngstrom2,4

lOsaka Bioscience Institute, Osaka 565, 2Subfemtomole

Biorecognition Project, Research Development
Corporation of Japan, Japan-Sweden, 3Suntory Institute
for Biomedical Research, Osaka 618, 4Uppsala
University PET Centre, UAS, S751 85 Uppsala, Sweden,
and 5National Institute of Radiological Sciences, Chiba
263, Japan

INTRODUCTION

Our recent microdialysis studies demonstrated the effect of

6R-L-erythro-5,6,7 ,8-tetrahydrobiopterin (6R-BH4, R-THBP) on the
promotion of release of dopamine, serotonin, and noradrenalinel,2.
In addition to this primary effect of R-THBP, glutamatergic,
GABAergic, and cholinergic systems were secondarily activated via
activation of such monoaminergic systems2-5. These results could
explain the mechanisms of clinical efficacy of R-THBP treatment on
some neuropsychiatric disorders such as infantile autism6,
depression and so on. Although our microdialysis studies have
been done using normal rats, the hypothesis that enhanced
neurotransmitters' release induced by R-THBP explains its
therapeutic effect could be tested in the patients and by peripheral

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 321
 administration. Therefore, we attempted to apply the peripheral
injection of R-THBP to evaluate its effect on neurotransmitter
dynamics by use of positron emission tomography (PET) in human
subjects and primates. Recently, we5 have demonstrated the
enhancement of DOPA turnover and dopamine release in vivo by
intravenous injection of R-THBP into the rhesus monkey by use of
PET and 3,4-dihydroxy-L-[B-11C]phenylalanine (DOPA). To extend
this result to dopamine and other neurotransmitter receptors, PET
studies using various 11 C-labelled receptor ligands have been
performed. Here, we summarize the effects of peripheral
administration of R-THBP on the dopaminergic, nicotinic and
muscarinic cholinergic receptor systems.

EXPERIMENTAL PROCEDURES

See details in the respective papers appears in this book.

Briefly, PET studies have been performed using ketamine-
anesthetized (in UUPC) or conscious (in NIRS) rhesus monkeys along
the guidelines from the Ethical Committee. Plasma concentration of
total biopterin was measured according to the method of
Fukushima and Nixon 7 with some modifications.

RESULTS AND DISCUSSION

Fig. 1 shows the concentration of plasma total biopterin at the

indicated times after intravenous injection of 10 mg/kg of R-THBP.
Total biopterin concentration decreased in a pseudo-first order
manner and still remained high (ca. 100-fold) from the control level
(0.02-0.03 nmol/ml plasma) even at 120 min after the injection (all
of PET scans were finished by this time). Essentially similar time
courses were obtained with 3 mg/kg and 30 mg/kg doses.
In tables I and II, the results of PET studies so far done are
summarized. The data in all brain regions are taken and calculated
but here we picked up the data in the striatum (subcortical region)
in Table I and in the frontal-temporal cortical region in Table II as
typical results. R-THBP pretreatment did not significantly affect
regional cerebral blood flow (rCBF) and regional glucose metabolic
rate (rCMRglc) nor dopamine Dt binding ([[11 C] S CH23 3 90).
However, R-THBP treatment can influence on dopamine D2 and D3
receptor bindings (N-[11C]methyl-spiperone and ( +)[11 C]UH23 2,
respectively) from the smaller dose and positively influence on
muscanmc (N-[11C]methyl-benztropine and N-[11C]methyl-
piperidylbenzilate) and nicotinic ((S)(- )[11 C]nicotine) cholinergic
receptors with somewhat larger doses (Table I). In the cortical

322
regions (Table II), the effect on N-[llC]methyl-piperidylbenzilate
binding was more marked than that in the striatum, while the
effects on N-[llC]methyl-benztropine and [llC]nicotine bindings are
somehow larger in the striatum than those in the cortical regions.
There are several subtypes of muscarinic cholinergic receptors in
the brain, which could explain the discrepancy of these data if both

100
·-.....""
c 0
•D.
APA204
QJ APA201

·--.5 ...
Q.
0 APA174
,Q
APA191

.....0 10 RA2=0.978
=
sc
fils
.!;;:;
Q.O
eo..S
oc

·-.........=
C'-'
0
1

c""QJ
Col
c
0
Col

.1 ~----._----~----._----~----~--~

0 100 200 300

Time after injection ofR-THBP lOmg/kg (min)
Fig.l. Change of plasma total biopterin concentration after
i.v.injection of R-THBP lOmg/kg in the rhesus monkeys.

ligands recognize different subtypes (usually both ligands are

considered for M1 and M2 subtypes). Another factor for this
discrepancy should be anesthesia; ketamine anesthesia in the case
of N-[llC]methyl-benztropine and no anesthesia in the case of N-
[llC]methyl-piperidylbenzilate.
In the near future, we will proceed to do PET study in the
patients in order to show the clinical efficacy of R-THBP and/or
related drugs. This study gives the bases for selection of the
tracers.

323
 Table 1. Pharmacological profile of R-THBP as evaluated
by PET in the striatum

Receptor R-THBP {i.v.)

Ligand (Function) 3mg/kg lOmg/kg 30mg/kg

H 2 15Q rCBF (-) (-) (-)

18FDG rCMRglc (-)

N -[IIC]methyl-benztropine
(S)(- )[ 11 C]nicotine
mACh
nACh
(-)
(-)
t l
(-)
~
[ 11 C}SCH23390 D1 (-) (-) (-)
N -[ uq methyl-spiperone
(+)(11C]UH232
Dz
D3 t (t) t
!

Receptor R-THBP (i.v.)

Ligand (Function) 2mg/kg 7mg/kg 20mg/kg

L-[IICJDOPA DA
turnover
(-)
t t
N -[llCJmethyl- mACh (-)
piperid ylbenzilate

All PET studies were perfomed in three rhesus monkeys except for
(llC}SCH23390.

(- ): None of the differences observed was statistically significant.

t: Statistically significant differences from the control baseline study
were observed (p<0.05).
l: Statistically diferences from the control baseline study were
observed (p<O.l ).

324
 Table 2. Pharmacological profile of R-THBP as evaluated
by PET in the cortex

Receptor R-THBP (i.v.)

Ligand (Function) 3mg/kg lOmg/kg 30mg/kg

H 215Q rCBF (-) (-) (-)

18FDG rCMRglc (-)

N- [! IC]methyl-benztropine
(S )(- )[llC) nicotine
mACh (-)
(-)
~
(-)
nACh

[ttC]SCH23390 Dt (-) (-) (-)

N -[IIC] methyl-spiperone 5-HT2 (-) (-) (-)
( + )[11C)UH232 (-)
DJ
~ ~

Receptor ---:-:---R....-_T_H-:-:B_P_,_(i_.v....:..)---::-:---::--
Ligand (Function) 2mg/kg 7mg/kg 20mg/kg

L-[ttC]DOPA DOPA (-)

turnover

N-(11C)methyl- mACh
piperidylbenzilate

All PET studies were perfomed in three rhesus monkeys except for
[ttC]SCH23390.

(- ): None of the differences observed was statistically significant.

~ : Statistically significant differences from the control baseline study
were observed (p<0.05).
~ : Statistically diferences from the control baseline study were
observed (p<O.l).

325
 REFERENCES

1. K. Koshimura, S. Miwa, K. Lee, M. Fujiwara, and Y. Watanabe, J.

Neurochem., 54:1391-1397, 1990.
2. N. Mataga, K. Imamura, and Y. Watanabe, Brain Res., 551:64-
71, 1991.
3. T. Ohue, K. Koshimura, Y. Akiyama, Y. Watanabe, and S. Miwa,
Brain Res., 570:173-179, 1992.
4. N. Mataga, K. Imamura, and Y. Watanabe, in: Frontiers and new
horizons in amino acid research, Amsterdam, Netherland, pp.
541-545, 1992.
5. Y. Watanabe, S. Miwa, N. Mataga, K. Imamura, Y. Tani, K.
Koshimura, T. Ohue, H. Onoe, Yu. Watanabe, T. Ishihara, P.
Hartvig, P. Bjurling, K.J. Lindner, B. Umgstrom, T. Noguchi, and
0. Hayaishi, Elsevier Science Publishers B. V., Amsterdam,
Netherland, pp. 317-331, 1992.
6. H. Naruse, T. Takesada, Y. Nakane, K. Yamazaki, T. Noguchi, Y.
Watanabe, and 0. Hayaishi, Proc. Japan Acad., 63B:231-233,
1987.
7. T. Fukushima and J.C. Nixon, Anal. Biochem., 102:176-188,
1980.

326
POSITRON EMISSION TOMOGRAPHY (PET) STUDY: THE EFFECTS OF 6R-L-
ERYTHR0-5,6,7,8-TETRAHYDROBIOPTERIN (R-THBP, SUN 0588) ON THE CENTRAL
DOPAMINE D1, D2 AND D3 RECEPTORS IN RHESUS MONKEY

Yoshihiro Tanil, Takafumi Ishihara I, Tadashi Kanail, Tomochika Ohnol,

Hirotaka Onoe2, Yasuyoshi Watanabe2, Jesper Andersson3, Anders Lil~ja3,
Goran Westerberg3, Per Hartvig3, and Bengt U.ngstrom3

1 Suntory Institute for Biomedical Research, Osaka 618, Japan

2 Osaka Bioscience Institute, Osaka 565, Japan
3 Upp~a1a University PET Centre, Uppsala S-751 85, Sweden

Introduction

Among the brain imaging techniques, X-ray computed tomography (CT) and magnetic
resonance imaging (MRI) give morphological images, but positron emission tomography
(PET), which gives functional images, allows monitoring non-invasively of energy metabolism,
local blood flow, some enzyme reactions and several types of receptor bindingsl. The
developments of PET technique and the selective radioligands has potentiated to improve our
knowledge of the physiology of neurotransmitter systems and to monitor drug action by directly
measuring changes in energy metabolism and receptor occupancy as results of altered storage
pool of neurotransmitter, neurotransmitter release, re-uptake, etc. in human and non human
primates2. Especially, the dopaminergic system has been well studied by PET, since the
dopaminergic terminals are highly concentrated in the striatum and the size of the striatum is
suitable for spatial resolution of PET. Within the components of dopaminergic system,
measurable postsynaptic components by PET are D1 and D2 receptors,3 and measurable
presynaptic components are turnover of dopamine4 and dopamine re-uptake site.5
Furthermore, in this paper, we attempt to analyse the dopamine D3 receptor by PET using the
selective ligand (+)[11C]UH232 (cis-(+ )-5-methoxy-1-methyl-2-(di-n-propylamino)tetralin),
which was characterized as an acting dopamine autoreceptor antagonist with the biochemical and
behavioral profile. 6 The aim of the present study is to clarify the effects of peripherally
administered R-THBP (SUN 0588, the chemically synthesized dihydrochlorides salt of R-
THBP, Suntory Limited) on the dopamine D1, D2 and D3 receptor systems in the living rhesus
monkey brain using PET technique.

Materials and Methods

Radiochemistry

llC-radionuclides was produced by 14N(p,a.)llC nuclear reactions using a cyclotron

(Scanditronix MC-17) at Uppsala University PET Centre (UUPC). [llCjSCH23390, N-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 327
[llC]methyl-spiperone and (+)[11C]UH232 were synthesized from the respective desmethyl
precursor and [llC]methyl iodide. Chemical and radiochemical purity were determined to be
>98% by high performance liquid chromatography (HPLC) and the solutions were passed
through a sterilized filter (pore size, 0.22Jlm). The specific activity of the ligands at the time of
injection (corresponding mass in Jlg) were as follows : [11C]SCH23390, 79-347 mCi/Jlmol
(3.0-14.7 Jlg); N-[llC]methyl-spiperone, 146-654 mCi/Jlmol (2.7-11.0 Jlg) and (+)[11C]-
UH232, 160-233 mCi/Jlmol (10.0-12.0 Jlg).

Procedure

Normal rhesus monkeys (Macaca mulatta) weighing 6.0-11.5 kg from the Primate
Laboratory, UUPC, were used after an overnight starvation. Anesthesia was induced with
ketamine (Ketalar® Parke-Davis, 100 mg i.m.) which was repeated as required. Catheters
were inserted into both sides of femoral vein, one for the injection of radioligand orR-THBP,
and the other for venous blood sampling. Venous blood samples (0.5 ml each) were collected
at 1, 2, 5, 10,30 and 60 min afterinjection ofradioligand and total radioactivity was counted in
well-counter. PET camera started immediately following intravenous administration of
radio ligand (radioactivity was 100-400 MBq) and the distribution of radioactivity was recorded.
The following frame sequence was used: 0-3 min, every 15 sec (12 frames); 3-15 min, every 3
min (4 frames); 15-60 min, every 5 min (9 frames), total25 frames. PET scan was performed
using PET cameras (GE PC4096-15WB and PC2048-15B which were equipped with 8 detector
rings giving 15 transaxial slices of the head interspaced 6 mm, a spatial resolution of 4-5 mm).
The position of the head of monkey was determined with reference to a map of horizontal
cryosections of rhesus monkey head (UUPC). Two consecutive PET scans were performed.
The first one was the baseline study and the second was R-THBP (SUN 0588) pretreatment
study. These studies were performed at least 2 hrs apart on the same day in the same rhesus
monkey. Animals recovered for at least 8 weeks prior to their second study according to the
Ethical Committees at the Medical Faculty of Uppsala University. After reconstruction of the
images, the regions of interest (ROis ; the striatum, hippocampus, thalamus, frontal, temporal,
occipital cortex, cerebellum and white matter) were delineated. The kinetics in different brain
regions were estimated after correction of the radioactivity for physical decay and being given a
dimensionless value by correcting for the injected dose per gram body weight. The
concentration of plasma total biopterin, pterin and neopterin were measured by HPLC with
fluorescence detector (FD) according to the methods of Fukushima & Nixon 7 with some
modifications. R-THBP (SUN 0588) was dissolved immediately before use in 0.1 M
phosphate buffer (pH 7 .15) containing 6 mM ascorbic acid and 3 mM L-cysteine and passed
through a filter (pore size, 0.22J.Lm). The intravenous injection of R-THBP was performed 1
hr before the radioligand injection, according to our previous results2.

Results and Discussion

Dopamine Dl : [llC]SCH23390

After injection of [11C]SCH23390, the uptake rapidly increased with time and followed by a
gradual decrease. The highest accumulation of radioactivity was found in the striatum. The
regional distribution of [11C]SCH23390 in the normal rhesus monkey was consistent with that
obtained in human brain3. Any dose (3, 10 and 30 mg/kg i.v., n=l each) of R-TIIBP showed
little effect on [11C]SCH23390 binding in comparison with control baseline study (n=2). By in
vitro experiments, it was also reported that R-THBP showed no effect on the [3H]SCH23390
binding to the rat striatal membrane fractionS. Accordingly, these results suggest that R-THBP
has little effect on the central dopamine Dl receptor.

Dopamine D2 : N-[IIC]methyl-spiperone

N-[llC]methyl-spiperone was taken up in the striatum rapidly and reached its maximum at
approximately 30 min and thereafter remained the same level, but in the cortex its radioactivity

328
reached the peak at around 10 min and gradually decreased. The three compartment model
could be applied for the calculation of receptor kinetics. In this case, the cerebellum with a
negligible amount of dopaminergic terminal was used as the reference brain region. A typical
result for calculation of k3 value in striatum and frontal cortex are shown in Fig.1.

sr---------------------~~ 3~-----------------------,
Striatum Frontal cortex
4

Q,l
,Q
y
3

~u 2 R-TIIBP: k3=0.0073

0 Baseline 0 Baseline
e R-THBP 10 mglkg e R- TIIHP 30 mg/kg
o~~-L~--~~~~~~~~ 0~~-L~--~~~~~~~~

0 20 40 60 80 100 0 w ~ w w ~
JCcbe(t)dt/Ccbe JCcbe(t)dt/Ccbe
Fig. 1 Example of k3 value determination and the effect of R-THBP pretreatment on k3 values in
the striatum and frontal cortex. The k3 values, the association rate x Bmax values, was calculated
by using three compartment model after reconstraction of the images and calculation of time-curve
of the decay-corrected radioactivites.

The k3 value showed a tendency to decrease with administration of a comparatively low dose
(3 mg/kg i.v.) of R-THBP in the striatum. The administration of R-THBP (10 mg/kg)
significantly decreased the k3 value in the striatium, whereas the k3 value in the frontal cortex
was not significantly altered even by large dose of R-THBP (up to 30 mg/kg i.v., n=3). N-
[IIC]methyl-spiperone has been evaluated as in vivo ligand for the dopamine D2 receptor in
receptor-rich brain region such as the striatum. However, it is well recognized that this ligand
has been characterized as binding to the serotonin 5-HT2 receptor approximately 90 % in the
frontal cortex9. Therefore, present PET results indicate that R-THBP exerts the action not on
the serotonin 5-HT2 receptor but on the dopamine D2 receptor.

Table 1. Effect of intravenous administration of R-TiffiP on the k3 value for

N-C 1CJmethyl-spiperone in the striatum and frontal cortex in the normal adult
rhesus monkeys.

k3 values
n Striatum Frontal cortex
Baseline 7 0.0232±0.0027 0.0098±0.0012
R-TiffiP 3 mg/kg 3 0.0145±0.0029 0.0085±0.0019
R-THBP 10 mg/kg 3 0.0057±0.0018* 0.0066±0.0008
R-TiffiP 30 mglkg 3 0.0133±0.0057 0.0070±o.0009
Each value represents the mean±SEM. Statistically significant difference
(Dunnet two-tailed test, Super ANOVATM) from control baseline study is
indicated:* p<0.05.

329
Dopamine D3: (+)[11C]UH232

(+)UH232 has been demonstrated as the most selective dopamine D3 autoreceptor antagonist
from the biochemical and behavioral studies6. The regional distribution of (+)[11C]UH232
uptake in the rhesus monkey brain showed a relatively high level in the striatum, thalamus and
frontal cortex, but low in cerebellum and white matter, whereas the striatal-cerebellar uptake
ratio of (+)[11C]UH232 was apparently lower than those of [11C]SCH23390 and N-
[11C]methyl-spiperone. These results suggest that the dopamine D3 autoreceptor is differently
distributed among the cerebral neurons, as reported by Socoloff et al.lO The pretreatment with
low dose (3 mg/kg i.v.) of R-Tl-IBP significantly decreased the binding potential (k3/k4
value,which represents Bmax/Kd value for dopamine D3 autoreceptor) in the frontal cortex, and
tended to decrease it in the striatum and hippocampus. However, R-Tl-IBP lOmg/kg i.v.
showed little effect on the k3/k4 values, and large dose (30 mg/kg i. v.) of R-Tl-IBP significantly
decreased k3/k4 value in the frontal cortex and thalamus. Although further experiments will be
required to explain these findings, these in vivo PET results indicate that systemically
administered R-THBP may influence on the dopamine presynaptic D3 autoreceptor.

REFERENCES
1. MJ.Phelps, H.Mazziotta and H.Schelbert, ed., in: Positron Emission Tomography and Autoradiography:
Principles and Applications for Brain and Heart, Raven Press, New York, (1986).
2. Y.Tani, T.Ishihara, T.Kanai,T.Ohno, Y.Watanabe, J.Andersson, A.Lilija, G. Westerberg, P.Hartvig and
B.Ungstrom• see another chapter in this book.
3.L.Farde, C.Halldin, S.Stone-Elander and G.Sedvall, Psychopharmacology, 92:278-284 (1987).
4.Y.Watanade, P.Hartvig, J.Tedroff, P.Bjurling, S.Miwa, T.Hayashi, T.Noguchi, and B.Ungstrom, in: Pterins
and Biogenic Amines in Neurology, Pediatrics and Immunology, N.Blau, H.-Ch.Curtius, R.A.Levine and
R.G.H.Cotton, eds., Lakeshore Publishing Company, Grosse Pointe, Michigan, pp.353-362 (1991).
5. J.Tedroff, S.-M.Aquilonins, A.Laihinen, U.Rinne, P.Hartvig, J.Andersson, H.Lundqvist, M.Haaparanta,
O.Solin, G.Antoni, A.D.Gee, J.Ulin and B.Ungstrom, Acta Neurol. Scand., 81:24-30 (1990).
6. K.Svensson, A.M.Johansson, T.Magnusson and A. Carlsson, Naunyn-Schmiedeberg's Arch. Pharmacal.,
334:234-245 (1986).
7. T.Fukushima and J.C.Nixon, Anal. Biochem., 102:176-188 (1980).
8. Y.Watanade, N.Mataga, T.Hayashi, T.Ishihara, T.Kanai, T.Noguchi, S.Miwa and Y.Watanabe, Pteridine, 3:63
(1992).
9. JJ.Frost, A.C.Smith, M.J.Kuhar, R.F.Dannals and H.N.Wagner Jr., Life Sci., 40:987-995 (1987).
10. P.Sokoloff, B.Giros, M.-P.Martres, M.-L.Bouthenet and J.-C.Schwartz, Nature, 347:146-151 (1990).

330
6R-L-ERYTHR0-5,6,7,8-TE1RAHYDROBIOP1ERIN (R-THBP, SUN 0588) ACTS ON THE
BRAIN MUSCARINIC AND NICOTINIC CHOLINERGIC RECEPTORS AS EVALUATED
BY POSITRON EMISSION TOMOGRAPHY (PET) IN RHESUS MONKEY

Yoshihiro Tani1, Takafumi Ishihara1, Tadashi Kanai1,Tomochika Ohno1,

Yasuyoshi Watanabe2, Jesper Andersson3, Anders Lilija3, Goran Westerberg3,
Per Hartvig3, and Bengt Ungstrom3

1 Suntory Institute for Biomedical Research, Osaka 618, Japan

2 Osaka Bioscience Institute, Osaka 565, Japan
3 Uppsala University PET Centre, Uppsala S-751 85, Sweden

Introduction

6R-L-erythro-5,6,7 ,8-tetrahydrobiopterin (R- THBP) is the natural cofactor for

phenylalanine, tyrosine and tryptophan monooxygenases, the latter two enzymes catalyses the
rate-limiting reactions in the synthesis of catecholamines and serotonin, respectively. Among
various types of pterins, R-THBP has attracted researcher's interest in recent years, since R-
THBP regulates not only the biosynthesis but also the release of biogenic amines such as
dopamine and serotoninl,2 and other neurotransmitters, glutamate2 and acetylcholine (ACh)3.
Ohue et al.3 reported that intracerebroventricular injection of R-THBP dose-dependently
increased extracellular ACh levels in the rat hippocampus as monitored by microdialysis
technique. Further, they demonstrated that the ACh releasing action of R-THBP was evoked
through excitation of monoaminergic system4. Therefore, experimental studies have been
planned on the cooperation of the monoaminergic with cholinergic functions to explain the
pharmacological role of R-THBP.
Positron emission tomography (PET) allows the monitoring non-invasively of energy
metabolism, local blood flow, some enzyme reactions and several types of receptor bindings
in vivoS by using various tracers labelled with positron emitting radionuclides llc, BN, 150
and 18p, By use of this imaging method, it is possible to study a variety of biochemical
processes quantitatively in a living animal and human. The aim of the present study is to
evaluate the effect of peripherally administered R-THBP (SUN 0588, the chemically
synthesized dihydrochlorides salt of R-THBP, Suntory Limited) on both the central
muscarinic cholinergic and nicotinic cholinergic receptor systems in anesthetized rhesus
monkeys using PET.

Materials and Methods

Radiochemistry

Radionuclides (llC, 150 and 18f) were produced by 14N(p, a)liC, 14N(d,n)150 and
20Ne(d, a)18p nuclear reactions, respectively, using a cyclotron (Scanditronix MC-17) at
Uppsala University PET Centre (UUPC). N-[llC]methyl-benztropine, (S)(-)[llC]nicotine,

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 331
were synthesized from the respective desmethyl precursor and [llC]methyl iodide. H215Q
and [18F]fluoro-deoxyglucose (18FDG) were prepared at UUPC by the routine procedures.
Chemical and radiochemical purities were determined to be >98% by high performance liquid
chromatography (HPLC) and then the solutions were passed through a sterilized filter (pore
size, 0.22 j.Lm). The specific activity of the ligands at the time of injection (corresponding
mass in j.Lg) were as follows: N-[llC]methyl-benztropine , 375-625 mCi/j.tmol (1.0-8.4 j.Lg) ;
(S)(-)[llC]nicotine, 113-999 mCi/j.tmol (1.2-9.7 j.Lg).

Procedure

Normal rhesus monkeys (Macaca mulatta) weighing 6.1-11.0 kg from the Primate
Laboratory, UUPC, were used after an overnight starvation. Anesthesia was induced with
ketamine (Ketalar® Parke-Davis, 100 mg i.m.) which was repeated as required. Catheters
were inserted into both sides of femoral vein, one for the injection of radio ligand orR-THBP,
and the other for venous blood sampling. Arterial catheterization was performed in the case
of the regional cerebral blood flow (rCBF) and the regional cerebral glucose metabolism
(rCMRglc) studies. Venous blood samples (0.5 ml each) were collected at 1, 2, 5, 10, 30
and 60 min after injection of radio ligand and total radioactivity was counted in well-counter.
PET camera started immediately following intravenous administration of radioligand
(radioactivity was 100-400 MBq) and the distribution of radioactivity was recorded. In
receptor studies,the following protocol for PET was used: 0-3 min, every 15 sec (12 frames);
3-15 min, every 3 min (4 frames); 15-60 min, every 5 min (9 frames), total25 frames. In
the case of rCBF study, continuous arterial blood sampling (3 ml/min) was performed by an
automated device for 145 sec. Images were recorded: 0-85 sec, every 5 sec (17 frames) and
85-125 sec, every 20 sec (2 frames), total 19 frames, after intravenous injection of sterilized
H2 15 0 (25-75 MBq/kg). In rCMRglc study,the following frame sequence was used: 0-5
min, every 60 sec (5 frames); 5-20 min, every 3 min (5 frames); 20-50 min, every 5 min (6
frames), total16 frames after intravenous injection of sterilized 18 FDG (8-20 MBq/kg). PET
scans were performed using PET cameras (GE PC4096-15WB and PC2048-15B which were
equipped with 8 detector rings giving 15 transaxial slices of the head interspaced 6 mm, a
spatial resolution of 4-5 mm). The position of the head of monkey was determined with
reference to a map of horizontal cryosections of rhesus monkey head (UUPC). Two
consecutive PET scan was performed in the [llC]ligand studies. The first one was the
baseline study and the second was R-THBP (SUN 0588) pretreatment study. These studies
were performed at least 2 hrs apart on the same day in the same rhesus monkey. Animals
recovered at least for 8 weeks prior to the next study according to the Ethical Committees at
the Medical Faculty of Uppsala University. After reconstruction of the images, the regions of
interest (ROis ; the striatum, hippocampus, thalamus, frontal, temporal, occipital cortex,
cerebellum and white matter) were delineated. The kinetics in the different regions were
estimated after correction of the radioactivity for physical decay and being given a
dimensionless value by correcting for the injected dose per gram body weight. In rCBF and
rCMRglc studies, the blood pC02 value, pH and plasma glucose concentration in the
beginning and end of each study were measured by blood gas system (ABL520,
©Radiometer, Copenhagen) and biochemistry analyzer (2700 select, YSI), respectively. The
concentration of plasma total biopterin, pterin and neopterin were measured by HPLC with
fluorescence detector (FD) according to the methods of Fukushima & Nixon6 with some
modifications. R-THBP (SUN 0588) was dissolved immediately before use in 0.1 M
phosphate buffer (pH 7.15) containing 6 mM ascorbic acid and 3 mM L-cysteine and then
passed through a filter (pore size, 0.22 j.Lm).

Results and Discussion

Muscarinic cholinergic system and the determination of pretreatment time of R-THBP

As Dewey et al.7 reported that N-[llC]methyl-benztropine is a suitable ligand for

investigations of the muscarinic cholinergic system by PET, we used this ligand to evaluate
the effect of R-THBP on the brain muscarinic cholinergic system. The uptake of N-

332
[llC]methyl-benztropine was observed in cerebral cortices (frontal, temporal and occipital
cortex), striatum and thalamus. In these brain regions, the uptake continued to increase until
60 min experimental period in rhesus monkey. First, we studied on pretreatment time of R-
THBP. When intravenous pretreatment with 10 mg/kg of R-THBP was performed 5, 20,
30, 60 and 180 min prior to the injection of N-[llC]methyl-benztropine, the most marked
decrease of the uptake ratio of N-[llC]methyl-benztropine into striatum, hippocampus,
thalamus and cerebral cortices against cerebellum or white matter was observed in the case of
60-min pretreatment. This result was consistent with our previous microdialysis study8 that
striatal dopamine output was significantly elevated 1-2 hr after intraperitoneal injection of R-
THBP (50 mg/kg). Therefore, the pretreatment time of R-THBP was decided to be 1 hr
before the radioligand injection.

Table 1. Effect of R-THBP on the uptake ratio of N-[11 C]methyl-benztropine into the
striatum, hippocampus, thalamus and cerebral cortices to that of cerebellum.

Brain region R-THBP

n 3 mglkg 10 mglkg 30 mgjkg

Striatum 3 -0.0637±0.0447 -0.1350±0.0450° -0.1203±0.0347°

Hippocampus 3 -0.0167±0.0254 -0.1653±0.0738 -0.1267±0.0364°

Thalamus 3 -0.0317±0.0266 -0.1450±0.0276* -0.1443±0.0378°

Frontal cortex 3 -0.0417±0.0626 -0.1787±0.1 064 -0.1720±0.0357*

Temporal cortex 3 -0.0403±0.0603 -0.1730±0.0870 -0.1693±0.0238*

Occipital cortex 3 -0.0420±0.0481 -0.1330±0.0502 -0.1927±0.0861

Two consecutive PET scans were performed on the same day in the same rhesus monkey.
The first one is the baseline study and the second is R-THBP pretreatment (1 hr before
injection of tracer) study. The uptake ratio is shown in the difference between the ba~eline
and R-THBP pretreatment study at 15- 60 min (9 frames). Each value represents the mean
±SEM of three normal adult rhesus monkey. Statistically significant differences (two-tailed
paired t-test) between baseline and R-THBP pretreatment study are indicated : 0 p<O.l, * p<0.05.

As shown in Table 1, the effect of R-THBP on muscarinic cholinergic system was dose-
dependent: while 3 mg/kg of R-THBP did not alter the uptake ratio from the baseline level, 10
and 30 mg/kg reduced the uptake ratio significantly in the thalamus, frontal and temporal
cortices and tended to decrease in the striatum and hippocampus. Although the mechanisms
underlying the fact that R-THBP reduced the uptake ratio of N -[llC]methyl-benztropine into
the various brain regions is not clear, this result may be interpreted to be due to ACh releasing
action of R-THBP, inasmuch as intracerebroventricular injection and infusion of R-THBP
produced dose-dependent increases in Ach level in the rat hippocampal dialysates3.4.

Nicotinic cholinergic system

(S)(-)[llC]nicotine uptake was highly distributed in the thalamus, striatum and cerebral
cortex, but low in the cerebellum and white matter. Intravenous injection of 10 mg/kg of R-
THBP significantly decreased (S)(-)[llC]nicotine uptake (about 10-20%) in the striatum, but
3 and 30 mg!kg exerted little effect. Although further experiments are needed to clarify the
effect of R-THBP on the nicotinic cholinergic system, these results suggest a possibility that
R-THBP may act also on the central nicotinic cholinergic receptor which may lead to new
avenues of investigation in regards to cognitive disorders9. The changes of plasma total
biopterin, pterin and neopterin concentrations in rhesus monkey were also measured. Both

333
plasma total biopterin and pterin levels were markedly increased with i.v. injection of R-
THBP and gradually decreased with time, while plasma total neopterin level did not change at
all.

rCBF and rCMRglc

The rCBF and rCMRglc were monitored by PET in normal rhesus monkey brain which
were assessed by use of Raichle's H215Q method and the graphical 18FDG method,
respectively. The values of rCBF and rCMRglc showed high levels in cortical and
subcortical regions, but relatively low in the cerebellum and white matter. Any dose of R-
THBP did not significantly affect rCBF and rCMRglc, without influencing the blood pC02
and pH values. Consequently, the changes in muscarinic and nicotinic cholinergic receptors
in vivo is not due to a change in rCBF.

REFERENCES
1. K.Koshimura, S.Miwa, K.Lee, M.Fujiwara and Y.Watanabe, J. Neurochem., 54:1391-1397 (1990).
2. N.Mataga, K.Imamura and Y.Watanabe, Brain Res., 551:64-71 (1991).
3. T.Ohue, K.Koshimura, K.Lee, Y.Watanabe and S.Miwa, Neurosci. Lett., 128:93-96 (1991).
4. T.Ohue, K.Koshimura, Y.Akiyama, Y.Watanabe and S.Miwa, Brain Res., 570:173-179 (1992).
5. M.J.Phelps, H.Mazziotta and H.Schelbert, ed., in: Positron Emission Tomography and Autoradiography:
Principles and Applications for Brain and Heart, Raven Press, New York, (1986).
6. T.Fukushima and J.C.Nixon, Anal. Biochem., 102:176-188 (1980).
7. S.L.Dewey, R.R.Macgregor, J.D.Brodie, B.Bendriem, P.T.King, N.D.Volkow, D.J.Schlyer, J.S.Fowler,
A.P.Wolf, S.J.Gatley and R.Hitzemann, Synapse, 5:213-223 (1990).
8 Y.Tani, N.Mataga, T.Ishihara, T.Kanai, Y.Watanabe and T.Noguchi, in: Neurobioloogy of Infantile
Autism, H.Naruse and E.M.Omitz, ed., Elsevier Science Publishers B.V., Amsterdam, pp.335-336. (1992).
9. E.D.Levin, Psychopharmacology, 108:417-431 (1992).

334
 EFFECT OF 6R-TETRAHYDROBIOPTERIN ON THE CENTRAL
MUSCARINIC CHOLINERGIC RECEPTOR AS EVALUATED BY
POSITRON EMISSION TOMOGRAPHY STUDIES USING
RHESUS MONKEY

Yasuyoshi Watanabel,2, Hirotaka Onoel,2, Masayasu

Tanaka3, Kaoru Kobayashi3, Kazutoshi Suziki2,3,
Yoshihiro Tani4, Soichi Miwa5, and Osamu Inoue2,3

lOsaka Bioscience Institute, Osaka 565, 2Subfemtomole

Biorecognition Project, Research Development
Corporation of Japan, Osaka 565, 3National Institute of
Radiological Sciences, Chiba 263, 4Suntory Institute for
Biomedical Research, Osaka 618, and 5Kyoto University
Faculty of Medicine, Kyoto 606-01, Japan

INTRODUCTION

Our recent microdialysis studies demonstrated the effect of

6R-L-erythro-5 ,6, 7 ,8-tetrahydrobiopterin (R- THBP) on the
acethylcholinergic system as well as dopaminergic, serotonergic,
and glutamatergic systems in the rat brain 1-3. Enhancement of
acetylcholine release by R-THBP occurs via activation of
serotonergic system3. More recently, by using positron emission
tomography (PET) with 3,4-dihydroxy-L-[B-llC]phenylalanine
(DOPA), we demonstrated the enhanc;ement of DOPA turnover in
vivo and maybe dopamine release in vivo by peripheral
administration of R-THBP4. Here, we intend to show the effect of
R-THBP on the muscarinic cholinergic receptor in unanesthetized
rhesus monkeys.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 335
 EXPERIMENTAL PROCEDURES

N- [11 C]Methyl-4-piperidylbenzilate ([11 C]NMPB ), an

antagonist for muscarinic M1 and Mz receptors, was synthesized
by N-methylation of 4-piperidylbenzilate with [11C]methyHodide.
The radiochemical purifies were better than 95%, and the specific
radioactivities ranges from 8 to 46 GBq/11mol5.
Three male rhesus monkeys (ages ranging 7-9 years old)
were used according to the guidelines ruled by the Ethical
Committee in National Institute of Radiological Sciences. They
received an intravenous injection of [11C]NMPB (185-333 MBq).
Prior to isotope injection, a transmission scan was performed for
attenuation correction. Serial dynamic scans were performed for
60 min immediately after the injection using a high-resolution
PET camera (Hamamatsu Photonics SHR2400). First, baseline run
for the control was performed with injection of [11 C]NMPB, and
then in the same position with the same animal, the second PET
study with [11C]NMPB was performed at 2-hr interval. Twenty
minutes before the second [11C]NMPB injection, the monkeys were
given 20 mg/kg of R-THBP dissolved in phosphate-buffered saline
within 5 min after preparation (40 mg/ml).
Irregular regions of interest (ROis) were defined on the
image of emission scan by computer-controlled delineation of
percentage isocontour with reference to MRI images of rhesus
monkey brain. The average values of left and right ROis were
used for calculation.

RESULTS AND DISCUSSION

The chemical structure of [11 C]NMPB is shown in Fig. 1 with

another muscarinic ligands so far developed for PET study. The
ratio of the radioactivities taken up into the cortices and striatum
to that into the cerebellum is comparably high in the case of
[11C]NMPB, which means the advantage for quantitative analysis5.
Saturation study has been done using unlabelled NMPB in the
rhesus monkey. Thirty micrograms per kilogram of NMPB
inhibited more than 80 % of the specific binding in the cortex,
while this dose did not significantly affect the accumulation of
radioactivity in the cerebellum. This result indicates that almost
all radioactivities accumulated in the cerebellum are not due to
the specific receptor binding and thus that we could use the

336
cerebellum as a reference. We therefore applied three
compartment model for quantitation of the receptor kinetics. In
this case, K3 value could be easily calculated by Patlak plot. The
results obtained from 3 rhesus monkeys were shown in Table 1.
Significant effect of R-THBP on the K3 value was seen in the part
of temporal and front-temporal cortices and thalamus. Not
statistically significant but considerable effect was observed in the
part of frontal cortical areas in PET slice numbers 5 and 6. These
areas may be related to dopaminergic and/or serotonergic
projection, and it might be possible that R-THBP is primarily
effective on such monoaminergic systems and then secondarily
affects on muscarinic cholinergic system as like our previous
results by microdialysis3. These results confirmed that R-THBP
could affect the mAChergic system and this implies that the
clinical efficacy of R-THBP could be mentioned even in the case of
demented patients with cholinergic defect as one of the clinical
features.

[ 11 C]Benztropine

eC]1RB
1

Fig. 1. Muscarinic Cholinergic Ligands for PET Study

337
 Table 1. Effects ofR-THBP (20 mglkg i.v.) on the k3 value of
in vivo muscarinic cholinergic receptor binding
in three rhesus monkeys as evaluated by PET

brain regions baseline R-THBP

mean S.E. mean S. E.
pons 0.0156 ± 0.0012 0.0152 ± 0.0011
striatum 0.0521 ± 0.0029 0.0518 ± 0.0029
thalamus 0.0388 ± 0.0023 0.0347 ± 0.0022 *
front. ex (S5) 0.0366 ± 0.0015 0.0346 ± 0.0016 0
front. ex (S6) 0.0404 ± 0.0019 0.0379 ± 0.0018 0
temp. ex 0.0427 ± 0.0019 0.0403 ± 0.0017*
front. temp. ex 0.0421 ± 0.0022 0.0388 ± 0.0017 *
occip. ex 0.0458 ± 0.0020 0.0451 ± 0.0022
hippocampus 0.0287 ± 0.0011 0.0282 ± 0.0011

Two-tailed paired t-test (n=6~8) * p<0.05, 0 p<O.l

REFERENCES

1. K. Koshimura, S. Miwa, K. Lee, M. Fujiwara, and Y. Watanabe,

J. Neurochem., 54:1391-1397, 1990.
2. N. Mataga, K. Imamura, and Y. Watanabe, Brain Res., 551:64-
71, 1991.
3. T. Ohue, K. Koshimura, Y. Akiyama, Y. Watanabe, and S.
Miwa, Brain Res., 570:173-179, 1992.
4. Y. Watanabe, S. Miwa, N. Mataga, K. Imamura, Y. Tani, K.
Koshimura, T. Ohue, H. Onoe, Yu. Watanabe, T. Ishihara, P.
Hartvig, P. Bjurling, K.J. Lindner, B. Langstrom, T. Noguchi,
and 0. Hayaishi, Elsevier Science Publishers B.V.,
Amsterdam, Nether land, pp. 317-331, 1992.
5. T. Suhara, 0. Inoue, K. Kobayashi K. Suzuki, and Y. Tateno,
Neurosci. Lett., 149:25-228, 1993.

338
INCREASE OF TETRAHYDROPTERINS IN CELL-FREE RETINAL EXTRACTS
IN RESPONSE TO LIGHT EXPOSURE

G. Cremer-Bartels, H. Gerding, K. Krause

Dept. of Ophthalmology, University of Munster, Germany

INTRODUCTION

The level of tetrahydrobiopterin (BH4) increases concomitant with exposure to light

in the mammalian retina under in-vitro and in-vivo conditions as recently reported 1•2•
Membrane bound visual pigments or other cell compartments may be suggested to be
involved in the phenomenon of photoreduction in the retina.
To get further information about photoreduction of pterins in the retina we examined
cell-free extracts of bovine retina by applying supernatants. We further on examined a
triamterene effect on retinal cell-free extracts since electroretinograms of rats are effected
by this substance3•

MATERIALS AND METHODS

Bovine eyes were prepared within two hours after slaughter. All animals were killed
in the early morning. Retina and pigment epithelium were homogenized (2 g in 1 ml
Ringer solution) and centrifuged with 45000 rpm for 30 minutes (Beckman L50). The
pooled supernatants were brought into 4 tubes. 100 - 500 ug triamterene (2,4,7-
triamino-6-phenyl-pteridine) per ml supernatant was added to 2 sets. Incubation at 37 C
was performed in Warburg apparatus with transparent walls for 30 minutes. Two sets were
exposed to about 40000 lx and two sets protected from light (0.01 lx). After incubation
dithioerythrol and perchloric acid was added as described by Brautigam et a1. 4• References
were obtained from Schircks, Jona Switzerland. The tetrahydropterins were determined after
HPLC separation by electrochemical detection: 0.4 V polarization voltage, detection
sensibility 0.5 rnA, methanol 5-10% of elution buffer. Light exposure varied seasonal:
winter approx. 25000 lx, summer approx. 50000 lx, fall approx. 35000 lx.

Chemistry and Biology of Pteridines and Folates, Edited by

I.E. Ayling et al., Plenum Press, New York, 1993 339
RESULTS

Tetrahydro-d-monapterin (MH4) appeared to be increased when retinal supernatants

were incubated in presence of triamterene. In the figures 1 and 2 examples of aligned
elution profiles in which triamterene was added are demonstrated.
The identification of the first peak (1) point to tetrahydromonapterin. Peak (1) see
figure 1,B was isolated, oxidized and monapterin confirmed by fluorescence identification.
The increase of MH4 in retinal cell-free extracts after incubation with triamterene is
demonstrated in figure 2.

Fig. 1. A standards 25 ng/ml MH4 (1) and 25 ng/ml BH4 (2); B retmal supernatant mcubated with tnamterene
m the dark; elution buffer 7.5 %methanol.

· ·~ ··· · : ····· · • · ··:·· · ··; .... .... : :.. ··:

I • : : 2 : :
I. I
~ /. II . . !i . .
_i~' ! ·i·· .. .. ........,li .... , .. .. ,
I I. B . . ' . .
.. ,....
.
.
. I
. . I
.
.
.
.

f\ : :. 1
I , I I I

p;·~~\
'- . :'1
. . t"'
. '····. \ \.:;:.~:-:-.::r.:-"

Fig. 2. A retmal supernatant mcubated wtth tnarnteren B control no tnarnterene added (1) MH4 (2} BH4
elution buffer 10% methanol.

340
 pmol BH4/g RETINA
120r-----~--------------------,

100
p<0.02
n•9

80

60

40

20

dark- light dark- light

+ triamterene
Fig. 3. BH4 increased significantly in retinal supernatants after incubation in light, see left side. This
phenomenon was not confirmed in presence of triamterene, see right side

Also tetrahydromonapterin (MH4) increased in response to light exposure when triamterene

was added before incubation. Under this experimental condition BH4 did not respond as
shown already in figure 3.

[%)
200r-----------------------------.

P<0.01
n•8
150

100

50

dark- light dark- light

BH4 MH4
Fig. 4. The samples incubated in the dark were set up to 100%. MH4 increased, see right side. BH4 was not
affected by exposure to light, see left side.

341
 pmol BH4/g RETINA
2sor-----~-----------------.

pc0.01
n-13
200

150 pc0.05
n•8
pc0.05
n•22
100

50
l
OLL~~LL~~LWU-~UU~~~

dark-light dark-light dark-light

WINTER SUMMER FALL
25000ix 500001x 350001x
Fig. 5. Light exposure of retinal homogenates resulted in a significant increase of B~ at all seasons, however
the highest content of B~ was found concomitant with high light intensities, see middle of figure 4.

The photohydration of BH4 of total retinal extracts seem to be dependent on illumination

(fig. 5)

DISCUSSION

The photohydration of BH4 and MH4 seems to be independent of membrane bound

visual pigments or other cell compartments. In the presence of triamterene however the
photohydration of B~ did not occur. This phenomenon may point to predominant binding
of triamterene to the reducing enzymes (DHPR or DHFR). We cannot explain why ~
increases in response to triamterene in the dark and in light respectively. A special
dihydromonapterin reducing enzyme may be speculated to occur in the retina which may
be involved in supply of MH4 as cofactor of neurotransmitter synthesis. The high
concentration of B~ in the retina in summer may sustain the idea of an illumination
dependent photohydration as an adaptive mechanism in the retina.

REFERENCES

I. Cremer-Bartels G, M Jektapour-Tabrizi, H Gerding, K Krause (1991). Photochem Photobio 53S l4S

2. Cremer-Bartels G, H Gerding, K Krause, M Jektapour-Tabrizi (1992). Pteridines 3, 75-76
3. Cremer-Bartels G (1993). This congress
4. Brautigam M., R Dreesen, H Herken (1982). Hoppe Seyler's Z. Physiol. Chern. 363, 341-343.

342
EFFECT OF TRIAMTERENE ON THE ELECTRORETINOGRAM OF LONG
EVANS RATS

G. Cremer-Bartels, H. Gerding, L. Hanneken, K. Krause

Dept. of Ophthalmology, University of Munster, Germany

INTRODUCTION

Tetrahydrobiopterin (BH4) is an important light-dependent cofactor in retinal

neurotransmitter synthesis 1•2 • BH4 resembles the rate-limiting coenzyme for tyrosine
hydroxylase in dopamine synthesis3• Hydroxylation is coupled with BH4 oxidation to
quinoid dihydrobiopterin (BH2), restoration of BH4 is being performed by dihydropteridine
reductase (DHPR) or dihydrofolate reductase (DHFR)4•5• Roberts and Hall6 reported an
induction of DHPR-activity by triamterene (2,4, 7-triamino-6-phenyl-pteridine). If this effect
exists in the retina an accelerated restoration of BH4 from BH2 could be postulated,
followed by an increase of dopamine metabolism and a possible effect on the
electroretinogram (ERG).

MATERIALS AND METHODS

Long Evans rats (n=15) received triamterene with tap water (approx. 5 mg
triamterene/day). Animals were kept under constant light conditions (LD 12h/12h; O.oi
lx/2.0 lx). After 8 hours of dark adaptation ERG were recorded under Ketanest anaesthesia
excluding diurnal variations of ERG amplitudes. A scotopic ERG recording was followed
by a 2 minute Ganzfeld illumination. ERG recordings were made every 2 minutes during
the subsequent readaptation phase using a Nicolet Compaq unit. The following parameter
were used: background illumination 17.0 cd/qm; flash illuminance 0.81 cd/qms.
Triamterene was dissolved in tap water with a final concentration of lg/1. Triamterene
uptake was controlled by urinary excretion of hydroxytriamterene by HPLC (fluorescence
detector). ERG recordings were done in three series. First before triamterene administration,
a second series after 11 days of triamterene uptake, a final third one 10 weeks after
cessation of triamterene.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 343
 a-Wave (JN)
300,------------------------------------.

1 4 7 10 13 16 19 22
Time (min)
b-Wave (JN)
700,------------------------------------,

1 4 7 10 13
Time (min)
Fig.!. a- and b-wave amplitudes in dependence of readaptation time:
* before triamterene administration (n=15);
+ after 11 days of triamterene uptake (n = 8);
"' 10 weeks after cessation of triamterene (n = 8).
Dark adapted rats were illuminated (17 cd/sqm) in the interval (1 min, 3 min). At t = 22
min rats under triamterene differed significantly (p = 0.025) from untreated animals.

344
RESULTS

After 11 days of triamterene administration a significant effect on a- and b-wave

amplitudes can be seen (Fig. 1). The overall electrophysiological retinal reaction in
response to light stimuli is reduced; the amplitudes recorded during dark adaptation in the
beginning of measurements as well as amplitudes after the 20-minute readaptation period
are reduced under triamterene. This reduction of about 2/3 is similar in a- and b-waves.
Readaptation dynamic does not change, peak amplitudes are reached after a 10-minute
readaptation time. This effect is completely reversible: 10 weeks after cessation of
triamterene all electrophysiological levels of the first series were measured again in the
third series.

DISCUSSION

Our results demonstrate an influence of orally administered triamterene in rats on the

retinal functional status. As shown in fig. 1, the synthetic pterin derivative triamterene
reduces a- and b-wave amplitudes of the ERG significantly in rats. This amplitude effect
may be explained by a triamterene mediated increase of retinal BH4 concentration6 and a
subsequent elevation of dopamine levels in the retina. This interpretation is consistent with
experiments of Starr7 and Dawis and Niemeye~, who found a reduction of b-wave
amplitudes in perfused retinae concomitant to increasing dopamine concentrations. Recent
experiments of Cremer-Bartels et al. 9 showed that additionally to BH4 another reduced
pterin, tetrahydromonapterin may influence ERG amplitudes by way of the discussed
mechanism. Our results sustain the hypothesis of a pterin mediated influence on
biochemical adaptation mechanism in the retina.

REFERENCES

1. PM Iuvone, CL Galli, CK Garrison-Gund, NF Neff (1978) Science 202, 901-902

2. PM Iuvone, JF Reinhard, MW Abou-Donia, CH Viveros, CA Nichol (1985) Brain Research 359:
392- 396
3. A Niederwieser, P Joller, R Seger, N Blau, A Prader, JD Bettex, R Luthy, B Hirschel, A Vetter
(1986) Klin Wochenschrift 64: 333 - 337
4. CA Nichol, GK Smith, DS Duch (1985) Ann Rev Biochem 54: 729 - 764
5. S Kaufman (1986) In: Cooper BA, VM Whitehead: Chemistry and Biology of Pteridines. de
Gruyter, Berlin New York
6. D Roberts, TC Hall (1967) Biochemical Pharmacology 17: 481 -484
7. MS Starr (1975) Exp Eye Res 21: 79 - 87
8. SM Dawis, G. Niemeyer (1986) Invest Ophthalmol Vis Sci 27: 330- 335
9. G Cremer-Bartels, H Gerding, K Krause (1993) lOth International Symposium Chemistry and
Biology of Pteridines and Folates, Alabama, USA.

345
IMMUNOENZYMATIC LABELING OF BIOPTERIN AND NEOPTERIN IN
THE PIGMENT EPITHELIUM OF BOVINE RETINA

H. Gerding', E. Vollmer, G. Cremer-Bartels',

H. Rokos 3, K. Krause' and H. Busse'

'Dept. of Ophthalmology
2Dept. of Pathology
University of Munster
Domagkstr. 15, W-4400 Munster, Germany
3Fa. Henning, 1000 Berlin, Germany

INTRODUCTION

The different chromatophores of the ectoderm can be classified according to the

chemical structure of pigments and their spectral properties (Table 1). Pterin-derivatives are
found as a major pigment in a subclass of chromatophores that was previously observed
only in the ectoderm of poikilotherms, crustaceae and cephalopods. It is characteristic of
these cells that pterins are bound to cytoplasmic particles (pterinosomes) of about 0.5 pm
in diameter (2). Equivalent organelles have never been confirmed in any ectodermal cells
of higher vertebrates.
Newer concepts (1,6) on the biology of chromatophores clearly demonstrate that the
classification of ectodermal chromatophoric cells is by far more variant than previously
suggested. These new concepts clearly establish mixed forms of chromatophores with
different pigment classes within one cell type. Besides that it seems that different pigments
or pigment derivatives are interactive in these cells. On the background of these
observations and our previous results concerning the analysis of biopterin in retinal pigment
epithelium (RPE) of bovine eyes (8) further biochemical and immunenzymatic studies were
performed in order to quantify and localize this pterin in RPE cells.

MATERIALS AND METHODS

Retinal pigment epithelium was isolated from bovine eyes within 1-2 hours after death
using the preparative methods as reported by Glocklin and Potts (10). Quantitative analysis
of pterins was performed according to our previous method (9) after acidic oxidation and

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 347
 Table 1. Classification of pigment cells

CHROMATOPHORE ORGANELLE PIGMENT COLOUR

melanophore melanosome melanin yellow
red
brown
black
iridophore reflecting guanine variable
platelets adenine
hypoxanthine
uric acid
xanthophore 1. pterinosome pterins yellow
2. carotinoid- carotinoids orange
vesicles red
erythrophore 1. pterinosome pterins yellow
2. carotinoid- orange
vesicles red

ion exchange chromatography (DOWEX H+, 50 WX 8). For quantitative analysis HPLC
with fluorescence detection was used. DNA-concentration was determined by the Burton
method (3). Labeling of biopterin and neopterin in bovine pigment epithelium was achieved
with the APAAP (Alkaline Phosphatase/Anti-Alkaline Phosphatase)-complex technique of
Cordell et al. (4). For primary labeling the recently available biopterin (sheep) antibody was
used (11). Further antibodies for the labeling procedure were: second GAS (goat-antisheep),
third RAG (rabbit-anti-goat). Tissue used for the labeling procedure was not subjected to
oxidative steps. For control staining hematoxylin was applied according to Mayer.

RESULTS AND DISCUSSION

Results of the quantitative analysis of bovine retinal pigment epithelium (RPE) are
listed in table 2. Biopterin in RPE revealed to be the major of analyzed pterin derivatives
at this location. The ratio between biopterin and neopterin was 1:36. The concentration of
monapterin in RPE was nearly by a factor of 7. 7 higher than neopterin. Compared to
neuroretinal concentrations (9) biopterin is nearly 12-fold higher in RPE. On the basis of
approximate comparison biopterin concentration in RPE is ranking in the upper range of
reported tissue analyses (5,7).
Immunenzymatic labeling of biopterin and neopterin in RPE-cells is demonstrated on
figure 1. Specific positivity occurs as a red/brown stain on the original histological
photographs. Generally staining of neopterin was less intensive when using similar antibody
dilutions (1: 10-1 00). Staining of neopterin and biopterin was mainly found in the cytoplasm
and seems to be bound to subcellular microsomal particles smaller than 1 pm.

348
Table 2. Quantitative results of pterin analysis in RPE of bovine eyes (ng/mg DNA).

Neopterin Monapterin Biopterin

mean 4.2 32.5 152.0

S.D. 1.8 16.0 56.0

Figure I. Positive biopterin label of bovine retinal pigment epithelium. Specific staining occurs originally as
a red/brown coloration of subcellular particles of the cytoplasm and demonstrates on the photographs as a
fine dark granulation. Additionally melanin granules can be seen on the photograph. Original magnification
was lOOOx.

The relative high concentration of biopterin in RPE is supporting the hypothesis of a

specific function at this location. The results of our immunenzymatic staining experiments
demonstrate that pterin positively seems to be bound to subcellular particles that resemble
those previously observed as pterinosomes only in the ectoderm of lower vertebrates.

REFERENCES
1. Bagnara, J.T., Matsumoto, Ferries, W., et al., 1979, Common origin of pigment cells. Science
203:410
2. Bagnara, J.T., and Hadley, M.E., 1973, "Chromatophores and Color Change". Prentice Hall,
Englewood Cliffs
3. Burton, K., 1955, A study on the conditions and mechanism of the diphenylamine reaction for the
colorimetric estimation of desoxyribonucleic acid. Biochemistry 62:315
4. Cordell, J.L., Falini, B., Erber et al., 1984, Immunenzymatic labeling of monoclonal antibodies using
immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP
complexes). J Histochem Cytochem 32:219

349
 5. Duch, D.S., Bowers, S., Woolf, J.H., and Nichol, C.A., 1984, Biopterin cofactor biosynthesis: GTP
cyclohydrolase, neopterin and biopterin in tissues and body fluids of mammalian species. Life
Sciences 35:1895
6. Frost, S.K., Borchert, M., Carson, M.K., 1989, Drug induced and genetic hypermelanism: effect on
pigment cell differentiation. Pig Cell Res 2:182
7. Fukushima & Nixon (1980) Analysis of reduced forms of biopterin in biological tissues and fluids.
Anal Biochem 102:176
8. Cremer-Bartels & Gerding, 1987, Pterins in bovine, rat, quail and human retina. In: Pfleiderer eta!.
(eds.) Biochemical and Clinical Aspects of Pteridines. De Gruyter, Berlin
9. Gerding, H., Krause, K., Cremer-Bartels, G. and Hanneken, L., 1990, Pterins in bovine, rat, quail
and human retina. In: Curtius eta!., (eds.) Chemistry and Biology of Pteridines 1989, De
Gruyter, Berlin
10. Glocklin & Potts (1962) The metabolism of the retinal pigment cell epithelium. Invest Ophthalmol
1:111
11. Rokos & Hey (1990) New immunoassays for determination of biopterin and neopterin in body
fluids. In: Curtius eta!. (eds.) Chemistry and Biology of Pteridines 1989, De Gruyter, Berlin

350
REDUCED PTERINS AS SCAVENGERS FOR REACTIVE OXYGEN SPECIES

Rong-sen Shen and Yixian Zhang'

Department of Human Biological Chemistry and Genetics

The University of Texas Medical Branch, Galveston
Texas 77555-0652, U. S. A.

INTRODUCTION

Pterins are widely distributed in nature in three forms: tetrahydro, dihydro, and
oxidized. The most studied pterin is L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), the
electron donor for mixed function oxidases, such as Phe hydroxylase during the
conversion of Phe to Tyr, and Tyr and Trp hydroxylases during biogenic amine
synthesis. 1 BH4 also serves as the cofactor for nitric oxide synthase which produces
nitric oxide from the vascular endothelium, platelets, neutrophils, and neurons. 2 Upon
oxidation to the quinonoid dihydrobiopterin (qBH2), BH4 is regenerated by
dihydropteridine reductase (NAD[P]H:6, 7-dihydropteridine oxidoreductase, EC
1.6.99.7;DHPR) using NADH as cofactor. 3 BH4 and DHPR are ubiquitous.4 Their
parallel distribution in some organs and blood cells which apparently do not synthesize
catecholamine neurotransmitters and/or nitric oxide, implies that BH4 and DHPR may
involve in other physiological functions.
Heales et al. 5 suggested that BH4 may serve as free radical scavenger. We were
the first to confirm this antioxidant role for BH4 by showing that BH4 inhibits
dopamine autoxidation. 6 We then proposed and tested the BH4 /DHPR antioxidant
system in rat pheochromocytoma (PC 12) cells, and found that it protected the host
against oxidative damage. 7 Recently, Kojima et al. 8 demonstrated that 5,6,7,8-
tetrahydroneopterin acts as a scavenger for superoxide radicals (Ot) generated in
mouse splenic cells by xanthine oxidase/xanthine (XO/X) reactions. These findings
strongly indicate that reduced pterins, tetrahydropterins in particular, play an important
role as a physiological antioxidant in mammalian cells.
The purpose of this study was to evaluate the antioxidant property of reduced
pterins in an in vitro 0 2·- and H 20 2-generating system, and in two mammalian cell
culture models.

*Present address:Department of Cell Biology, Baylor College of Medicine, Houston,

Texas 77030, U. S. A.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 351
MATERIALS AND METHODS

Chemicals And Enzymes. All pterins were purchased from Dr. B. Schircks Labs. (Jona,
Switzerland). Sheep liver DHPR, horseradish peroxidase (HPO), buttermilk XO,
bovine liver catalase, Dulbecco's phosphate buffered saline (DPBS), 5-amino-2,3-
dihydro-1,4-phthalazindione (luminol), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide (MTI) were obtained from Sigma (St. Louis, MO).

Cell Culture Conditions. All cells were grown in medium (RPMI 1640 for rat PC 12
cells; Dulbecco's modified Eagle for mouse macrophages J774A.1) supplemented with
5% fetal calf serum and 5% heat-inactivated horse serum (hereafter called the
complete medium), and were maintained at 36.5 °C in a humidified atmosphere of 5%
C02 in air? Murine J774A.1 cells were supplemented with L-glutamine (2.05 mM).
Before the experiment, cells were harvested and incubated in complete medium in 96-
well flat-bottomed plates (0.2 mL/well; 5 x 104 cells/mL for PC 12 and 2 x10S cells/mL
for J774A.1) for 24 h. The complete medium was then replaced with DPBS (9.6 g/L,
pH 7.4) or medium that was not supplemented with serum (hereafter called the serum-
free medium) but contained test substances. The BH4/DHPR antioxidant system
included 0.1 mM BH4, 3 U /mL HPO, 0.2 mM NADH, and 0.1 U /mL DHPR. The
cells were further incubated for a certain period and then assessed for injury by the
[ 3H]-thymidine incorporation, expressed in %incorporation, and MTI assay, expressed
in absorbance at 600 nm?

Chemiluminescence. Luminol-enhanced chemiluminescence, produced through the

action of Ot and Hz0 2 in basic solution, was measured in a Beckman LS 6800 liquid
scintillation system in the single-photon count mode. The reaction mixture (final
volume 2 mL) contained DPBS (4.9 g/L, pH 7.4), pterin, 3 JLM xanthine, 40 J.£0/mL
XO, 10 J.£M luminol, and 0.1 M boric acid solution (pH 10.5) in a 20-mL borosilicate
vial. Chemiluminescence intensity was measured in cpm at about 30 sec intervals for
5 min. Inhibition of chemiluminescence by pterins was expressed in % of control,
based on reading at 1 min after luminol addition. IC50 value was calculated from the
dose response curve.

RESULTS AND DISCUSSION

We have demonstrated that the BH4 /qBH2 redox-cycling couple, driven by

DHPR at the expense of NADH, serves as an antioxidant system to remove reactive
oxygen species (ROS) in PC 12 cells? As Hz0 2 is the central molecule in the ROS
cascade, its removal is an important means of reducing oxidative damage in vivo.
Figure 1A shows that the BH4/DHPR antioxidant system protects PC 12 cells against
oxidative damage caused by XO /X reactions. The antioxidant effect of the
BH4/DHPR system was equivalent to catalase, indicating removal of Hz0 2 by both
treatments. Ascorbic acid was as effective as the BH4/DHPR antioxidant system, but
became ineffective when the amount of XO increased from 50 to 100 J.£0/mL,
indicating the superiority of the enzymatic over the nonenzymatic antioxidants. Figure
1B shows that relative [3H]-thymidine incorporation in J774A.1 cells decreased
progressively with increasing H 20 2 concentrations, yielding an IC50 value of 0.2 mM.
The BH4 /DHPR antioxidant system as well as catalase and ascorbic acid (1 mM, data
not shown) reduced the Hz0 2 cytotoxicity by increasing the IC50 value to 0.35 mM.
However, HPO alone was ineffective, reinforcing our concept that the antioxidant
property of the BH4 /DHPR system does not derive from the activitiy of peroxidase. 7

352
 100 A B
Q)
c
:u ~ 80
1-
:5 5
..!-, :g 60
I ._
L-Je-0
I"')

~ 8 40
:g .5
Qi
a:: 20
'-

0 ~~ -~-.---.-.-.___:;ec___J
0 20 40 60 80 100 0.0 0.1 0.2 0.3 0.4 0.5
Xanthine oxidase,..uU/ml o
H2 2 concentration, mM
Figure 1. Use ofBH4/DHPR antioxidant system to reduce oxidative damage in two mammalian cell lines.
Panel A: Rat PC 12 cells stressed with XO/X reactions in serum-free medium for 2 h. Xanthine
concentration was held at 0.5 mM. Control(O); Ascorbic acid, 1 mM(•); Catalase, 1,250 U/mL(.o.); The
BH4/DHPR antioxidant system(•). Panel B: Murine J774A.1 cells exposed to H 20 2 for 2 h in DPBS.
Control(o); HPO, 3 UjmL(+); Catalase, 1,250 UjmL(.o.); The BH4/DHPR antioxidant system(•).

Figure 2 shows that luminol-enhanced chemiluminescence was inhibited by all

ten pterins tested. The inhibitory effect of these agents can be arranged in the
following decreasing order: (6R,S)-6-methyl-5,6,7,8-tetrahydropterin(v) > 7,8-dihydro-
D-neopterin (..-) = BRie) > (6R,S)-5,6,7,8-tetrahydroneopterin (•) = 6,7-dimethyl-
5,6,7,8-tetrahydropterin( o) > 5,6,7,8-tetrahydropterin( +) > 7,8-dihydro-L-biopterin(A)
> L-sepiapterin( •) > > D-neopterin( •) > L-biopterin ( o ). Mean IC50 values were
about 0.14 ~tM for five tetrahydropterins, 3.85 ~tM for three dihydropterins, and > 400
~tM for two oxidized pterins. The IC50 values of ascorbic acid and glutathione were 0.4
~tM and 0.9 ~tM, respectively (data not shown). Thus, the antioxidant property of
pterins is associated with the reduced forms only, and the efficiency of
tetrahydropterins is superior than ascorbic acid and glutathione.

120~--------------------------~----------------;
,.....
~c: 100
0
0
~
0

~
Q)
0
cQ)
0
~
.Ec:
:3
.E
Q)
..c:
(,)

0.010 0.100 1.000 10.000 100.000

Concentration, .uM
Figure 2. Inhibition of luminol-enhanced chemiluminescence by pterins.

353
 The MIT-oxidizable substances, which measure the number and mitochondrial
activity of living cells, in mouse macrophages were reduced to about half of the control
value when cells were exposed to UV light for 5 h, but recovered to about 75% of the
control when cells were pretreated with the BH4 /DHPR antioxidant system (Table 1).
NADH and BH4 alone was equally effective as compared to the BH4/DHPR
antioxidant system, whereas HPO and catalase were ineffective, indicating UV light
damage to the cells may not be just oxidative in nature.

Table 1. Reversal of UV light cytotoxicity in J774A.1 cells by the BH4/DHPR

antioxidant system. The cells in DPBS were plated in 96-well flat-bottomed plates,
pretreated with antioxidative agents, and then exposed to germicidal UV light for 5 h
in a Baker steril guard hood (VBN-600). Values represent mean ± SD of ten
determinations. Statistical analysis was performed by the Student's t-test.

Treatment Absorbance at 600 nm

Control at 0 h 0.262 ± 0.020*

BH4 /DHPR antioxidant system 0.200 ± 0.036*
NADH,0.1mM 0.191 ± 0.006*
BH4, 0.1 mM o.183 ± o.ow*
HPO, 3 U/ml 0.142 ± 0.013
DHPR, 0.1 Ujml 0.135 ± 0.015
Catalase, 625 U/ml 0.122 ± 0.020
Control at 5 h 0.136 ± 0.008

*p < 0.005 vs. control at 5 h.

The BH4/DHPR antioxidant system protects PC 12 and J774A.1 cells against

oxidative damage caused by Hz0 2 and XO/X reactions. The efficiency of this
antioxidant system against ROS is equivalent to or even better than ascorbic acid and
catalase. Moreover, this system reduces cell injury due to exposure to UV light.
Because of their low specificity for hydrogen donors, 9 mammalian peroxidases may
transfer electrons from BH4 to peroxides like HPO does. This implies that BH4,
peroxidases, NADH, and DHPR may constitute a physiological antioxidant system,
similar to that of glutathione, glutathione peroxidase, NADPH, and glutathione
reductase. This system reqires catalytic amount of BH4 and may be improved for
therapeutic purpose by replacing BH4 with synthetic tetrahydropterins.

REFERENCES

1. S. Kaufman and D.B. Fisher, in:"Molecular Mechanisms of Oxygen Activation," 0.

Hayaishi, ed., p. 285, Academic Press Inc., New York (1974).
2. J. Collier and P. Vallance, Brit. Med. J. 302:1289 (1991).
3. S. Kaufman, Pharmacal. Rev. 18:61 (1966).
4. H. Rembold and W.L. Gyure, Angew. Che. Int. Ed. 11:1061 (1972).
5. S.J.R. Heales, J.A. Blair, C. Meinschad and I. Ziegler, Cell Biochem. Function 6:191
(1988).
6. R.-S. Sherr, NeuroToxicol. 12:201 (1991).
7. R.-S. Shen and Y. Zhang, Chem.-Biol. Interactions 78:307 (1991).
8. S. Kojima, T, Icho, Y. Kajiwara and K. Kubota, FEBS Lett. 304:163 (1992).
9. J. Putter and R. Becker, in:"Methods of Enzymatic Analysis," Vol. 3, H.U.
Bergmeyer, ed., p. 286, Verlag Chemie, Weinheim (1983).

354
CHEMISTRY AND BIOLOGY OF THE
MOLYBDENUM COFACTORS

K. V. Rajagopalan, Jean L. Johnson, Margot M. Wuebbens,

Diana M. Pitterle, James C. Hilton, Teresa R. Zurick, and Robert M. Garrett

Department of Biochemistry
Duke University Medical Center
Durham, NC 27710

INTRODUCTION

All molybdenum-containing enzymes other than nitrogenase carry out either oxidative
hydroxylation or reductive dehydroxylation of their substrates as exemplified by the reactions
catalyzed by sulfite oxidase and nitrate reductase respectively, shown below. The results of
X-ray absorbance fine structure studies on sulfite oxidase, xanthine dehydrofenase and other
enzymes showed ligation of Mo to two to three thiolate ligands in all cases. In addition, the
ligand field of the metal contained either 2 Mo=O or 1 Mo=O and 1 Mo=S bonds. Because of
this, the term oxomolybdenum enzyme has been used to describe these proteins.

S03= + H20 + 2 Cytochrome c (III) ~ S04= + 2 Cytochrome c (II)+ 2 H+

Studies in our laboratory on the nature of association of Mo with the protein of sulfite
oxidase led to the identification and structural characterization of the molybdenum cofactor, a
complex of the metal with a unique organic molecule termed molybdopterin (MPT). 2 The
proposed structure of the cofactor (Figure 1) features a dithiolene group coordinated to Mo
through its sulfur atoms. The 4-carbon alkyl side chain also distinguishes molybdopterin
from other metabolically functional pterins. The tetrahydro state of the pterin ring was an
empirical assignment based on the susceptibility of the cofactor to oxidative loss of function.

Figure 1. Proposed structure of the molybdenum cofactor of liver sulfite oxidase.

355
 The cofactor itself has not yet been isolated due to its extreme lability when released
from its protective protein environment. Elucidation of the cofactor structure devolved on its
conversion to stable oxidized derivatives termed Form A and Form B3 and on the discovery
that urothione, a long known thienopterin, is the metabolic degradation product of the
cofactor. 4 Definitive proof of the structure of molybdopterin was derived by conversion to
the dicarboxamidomethyl derivative (camMPT) and structural characterization of the latter
(Figure 2).5

SULFITE OXIDASE

ANAEROBIC DENATURATION
IN THE PRESENCE OF IODOACETAMIDE

CHROMATOGRAPHY ON QAE-SEPHADEX
ELUTION WITH 10 mM HCI

AIR OXIDATION

HN~N'r-~=~-CHOH-CH,OPO, =
HN~NAN)
2
I
CH2 CH2
I

I I
H2NC CNH 2
II II
0 0
Figure 2. Formation of dicarboxamidomethyl molybdopterin from the molybdenum cofactor of sulfite
oxidase.

EVIDENCE FOR DITHIOLENE-Mo COORDINATION

Most of the well-characterized molybdoenzymes are complex proteins containing

one or more prosthetic groups in addition to the molybdenum cofactor, such as heme, flavin
and Se. The presence of these strong chromophores makes it very difficult to discern the
underlying weak spectroscopic properties of the molybdenum ligand field. The recent fmding
that DMSO reductase from Rhodobacter sphaeroides contains only molybdenum as its
prosthetic group 6 has made it possible to apply techniques such as resonance Raman and
magnetic circular dichroism spectroscopy to probe the molybdenum ligand field in that
enzyme.
The unique feature of the proposed structure of the cofactor is the dithiolene ring
consisting of the C=C bond, the dithiolene sulfurs and the Mo atom. In order to obtain in
situ evidence for the presence of this chelate complex at the molybdenum center of DMSO
reductase, we have carried out resonance Raman spectroscopy and magnetic circular
dichroism spectroscopy on the enzyme. The results of these studies are summarized below.

Resonance Raman spectroscopy

The molybdenum cofactor of DMSO reductase can exist with the metal in three different
valence states, Mo(VI), Mo(V) and Mo(IV). The absorption spectra of the Mo(VI) and
Mo(IV) forms of the enzyme contain bands that make them well suited for resonance Raman
spectroscopy.? In the C=C stretching region of the spectrum, bands at 1575 cm-1 for the
oxidized and at 1568 cm-1 for the reduced DMSO reductase have been assigned to C=C

356
stretching of the dithiolene. The shift in the position of the band upon reduction of the Mo
has been adduced as evidence for the proposed dithiolene-Mo coordination.
Data from the Mo-ligand stretching regions of the spectra are summarized in Table 1.
Mo-S stretching frequencies in the range of 350-390 cm-1 have been observed for several
dithiolene-Mo complexes. The two major bands seen at 350 cm-1 and 370 cm-1 in the
oxidized enzyme are shifted to 352 cm-1 and 383 cm-1 respectively in the reduced enzyme.
More importantly, all of these bands occur at lower frequencies in DMSO reductase isolated
from cells grown in the presence of [34S]sulfate in the medium. These data showing the
presence of C=C stretching and Mo-S stretching bands that are sensitive to the oxidation
state of Mo in R. sphaeroides DMSO reductase provide evidence for the existence of a
dithiolene chelate structure in the molybdenum cofactor of the enzyme. 7

Table 1. Mo-S stretching resonance Raman

bands in DMSO reductase.

Sample 32s

Oxidized enzyme 350 cm-1 341 cm- 1

370 cm- 1 367 cm- 1

Reduced enzyme 352 cm- 1 347 cm- 1

383 cm- 1 379 cm-1

Magnetic Circular Dichroism Spectroscopy

In the case of chromophores containing paramagnetic species, structural information

may be derived from the temperature-dependent MCD spectra. In the case of DMSO
reductase, the paramagnetic Mo(V) state can be generated by the anaerobic addition of one
electron equivalent of reduced methylviologen. In buffered aqueous solution, only a small
fraction of the enzyme is present in the Mo(V) state owing to equilibration among the
Mo(VI), Mo(V) and Mo(IV) states. However, when the reduction of the enzyme was carried
out in the presence of 50% glycerol, used to create a frozen glass, a tight complex of Mo-
glycerol was formed, with essentially all of the metal trapped in the Mo(V) state. This
happenstance facilitated detection of the optical properties of the Mo(V) center by variable
temperature MCD. As shown in Figure 3, the MCD spectrum in the 300 nm to 800 nm range
features three positive C-terms at 606, 442 and 358 nm and three negative C-terms at 701,
530 and 400 nm, all arising from an S = 1/2 ground state. The nearly constant energy
separations and the alternating signs of the six bands permit the assignment of the MCD
bands to dithiolene-n to Mo(V) charge transfer transitions. 8

MOL YBDOPTERIN DINUCLEOTIDES

In order to confirm the presence of molybdopterin in DMSO reductase, purified enzyme

was subjected to the procedure shown in Figure 2 for the preparation of camMPT. However,
the 10 mM HCl eluate from QAE-sephadex did not contain camMPT. Further experiments
showed that elution of the column with 50 mM HCl released a pterin displaying a 380 nm
absorption band resembling that of camMPT but showing additional features at shorter
wavelengths. 9 The HPLC elution behavior of the compound was not affected by treatment
with alkaline phosphatase, but treatment with nucleotide pyrophosphatase yielded camMPT
and a second product identical to guanosine monophosphate. 9 These data showed that the
molybdenum cofactor of DMSO reductase contains molybdopterin guanine dinucleotide

357
 (MOD). Soon thereafter the cofactor of another prokaryotic enzyme, CO dehydrogenase
from Pseudomonas carboxydoflava, was shown to contain molybdopterin cytosine
dinucleotide (MCD). 10 More recently dinucleotide variants containing adenine (MAD) and
hypoxanthine (MHO) have also been discovered. 11 The dinucleotide structure is shown in
Figure 4.

,.. 10 I
I
E 8
I
I

,..u
I
6 I
\..... - ...
' ' ..... ___
I
:i 4 ''

w
E
2 ------ -----
0

400

e
,.. 200
u
,.. 0~~~--~r---~~~
:a:
~ -200

-400

300 400 500 600 700 800

nm
Figure 3. Temperature sensitive MCD spectra of DMSO reductase. The
top panel shows the absorption spectra of the Mo(VI) form (dashed line)
and the glycerol-coordinated Mo(V) form (solid line) of the enzyme. The
lower panel displays the temperature-dependent MCD spectra of the
glycerol-coordinated Mo(V) form at a magnetic field of 4.5 T. The four
spectra, in the order of decreasing magnitude, were recorded at temperatures
of 1.61, 4.22, 9.6 and 27.3 K.

MOLYBDOPTERIN GUANINE DINUCLEOTIDE (MGD) =X

OH OH
X VARIANT
Cytosine MCD
Adenine MAD
Hypoxanthine MHD
Figure 4. The molybdopterin dinucleotides. The pterin ring is shown in the tetrahydro form.

358
 The distribution of the molybdopterin variants in a number of molybdoenzymes is
shown in Table 2. All of the eukaryotic enzymes analyzed so far contain only molybdopterin.
The dinucleotides have been found only in eubacteria and methanogens. It is noteworthy that
nitrate reductase and CO dehydrogenase from the same organism, P. carboxydoflava, contain
exclusively different dinucleotides. Among those listed in Table 2, xanthine dehydrogenase is
the only enzyme present in both eukaryotes and prokaryotes. Interestingly it is also the only
eubacterial enzyme not to have a dinucleotide in its cofactor.

Table 2. Distribution of molybdenum cofactor variant forms.

Enzyme Source Pterin Other cofactors

Sulfite oxidase animals MPT heme

xanthine dehydrogenase animals FAD, Fe/S
xanthine dehydrogenase Pseudomonas aeruginosa FAD, Fe/S
Xanthine dehydrogenase Pseudomonas putida
Nitrate reductase com heme, FAD
Nitrate reductase Chlorella vulgaris heme, FAD
Aldehyde ferredoxin oxidoreductase1 Pyrococcus furiosus Fe/S
Formaldehyde ferredoxin oxidoreductase1 Thermococcus litoralis Fe/S

DMSO reductase Rhodobacter sphaeroides MGD none

DMSO reductase Escherichia coli Fe/S
Nitrate reductase Rhodobacter sphaeroides none
Nitrate reductase Escherichia coli Fe/S, Se
Nitrate reductase Pseudomonas carboxydojlava _2
Formate dehydrogenase Escherichia coli Fe/S, Se
Formate dehydrogenase Methanobacterium formicicum Fe/S, Se
Formylmethanofuran dehydrogenase Methanobacterium barkeri
Formylmethanofuran dehydrogenase Methanobacterium thermoautotrophicum

CO dehydrogenase Pseudomonas carboxydojlava MCD FAD, Fe/S

Quinoline oxidoreductase Pseudomonas putida
Quinoline oxidoreductase Rhodococcus spec. Bl

Formylmethanofuran dehydrogenase M. thermoautotrophicum MAD

Formylmethanofuran dehydrogenase M. thermoautotrophicum MHD

I These enzymes contain W instead of Mo.
2Dash indicates not known.

Tungsten-containing Enzymes

Mukund and Adams have recently discovered and purified four tungsten-containing
enzymes from hyperthermophilic archaea. 12 These enzymes catalyze reactions similar to
those of molybdoenzymes from mesophilic organisms. Structural characterization of the
alkylated pterins from the tungsten cofactors of these enzymes showed that all four contain
molybdopterin rather than any of the molybdopterin dinucleotides. 13 These findings under-
score the fact that the molybdopterin structure has remained immutable throughout the course
of evolution and document the occurrence of molybdopterin in tungsten- as well as in
molybdenum-containing cofactors.

359
MOLYBDENUM COFACTOR BIOSYNTHESIS

A combination of genetic and biochemical approaches has enabled considerable headway

to be made in unraveling the complexity of the mode of biosynthesis of the cofactor. In
Escherichia coli, mutations in several genomic loci result in pleiotropic loss of all molybdo-
enzyme activities. The use of chl mutants has revealed that synthesis of the cofactor in E. coli
is a confluence of four distinct pathways: formation of the carbon skeleton; attachment of the
dithiolene sulfurs; uptake, processing and attachment of Mo; and generation of MGD from
MPT.

The Sulfur Pathway

The chlA locus in E. coli has been cloned and shown to encode five proteins. Earlier
studies showed that loss of function of chlA4 or chlA5 leads to accumulation of a precursor
that was initially visualized through its oxidation product, compound Z. Recent studies on
the structure of precursor Z have shown that it is identical to compound Z except that the
pterin ring in the precursor is in the dihydro state. 14 These structures are shown in Figure 5.

FigureS. The structures of compound Z (left) and precursor Z (right).

The conversion of precursor Z to molybdopterin can be effected in vitro in the presence

of a heterodimeric protein, termed convertint factor, composed of the peptides encoded by
chlA4 (8.5 kDa) and chA5 (16 kDa) genes.l5, 6 The smaller peptide has been found to be the
carrier of a labile sulfur serving as the immediate source of the dithiolene sulfur(s). 15 The
ultimate donor of the sulfur has yet to be identified, but the sulfur pathway includes the chlN
gene product, one of the two proteins encoded by the chlE locus. 17 The current state of
understanding of the sulfur incorporation pathway is summarized in Figure 6.

ChiN
"S" + ATP + ChiA4 ChiA4-S + ADP + Pi
ChiA4-S + ChiA5 Converting Factor-S
Converting Factor-s + Precursor Z Converting Factor-MPT
Figure 6. Formation of MPT from precursor Z. The Chi designation refers to the peptide encoded by the
corresponding chl gene. The exact oligomeric form of the converting factor has not been determined. The
MPT formed in the reaction remains bound to the converting factor.l 6

Molybdenum Insertion
Mutants in chiD and chlG display the pleiotropic phenotype when grown at low
concentrations of molybdate but contain active molybdoenzymes when grown on high
molybdate. These loci appear to be involved in the uptake and processing of molybdate, but
the proteins encoded by these loci have not yet been identified. 18

360
Biosynthesis of Precursor Z

Precursor Z is produced in large amounts by E. coli chlA5 and chiN mutants. The
oxidized product, compound Z, can easily be purified to homogeneity from whole cultures of
these mutants. To test the possibility that the carbon atoms of precursor Z are derived from
those of both the guanine and the ribose components of guanosine, chiN cells were grown in
the presence of uniformly labeled [14C]guanosine. The presence of 14C in compound Z
purified from the culture demonstrated that guanosine had indeed served as a precursor. To
examine the fractional distribution of label in the pterin ring vs. the side chain, a two step
procedure for the separation of the side chain carbons from the pterin ring was designed.
Alkaline permanganate treatment caused elimination of three side chain carbons and generated
pterin-6-carboxylate. The latter was purified by HPLC and its specific radioactivity was
measured. UV irradiation of the sample caused elimination of C02 and yielded unsubstituted
pterin. After HPLC purification, the specific radioactivity of the pterin could also be
measured. The fact that the specific radioactivity of the pterin compound decreased at each
step showed that at least two, and possibly all four, of the side chain carbons were derived
from the guanosine. These data suggested that an enzyme similar to GTP cyclohydrolase I
may be involved in the conversion of guanosine or one of its phosphorylated forms into
precursor Z (M. M. Wuebbens and K. V. Rajagopalan, unpublished).
In the reactions catalyzed by both cyclohydrolase I and cyclohydrolase II, the C-8 of the
guanine ring is eliminated as formate. Thus, neither biopterin in animals nor folates or
riboflavin in plants and microorganisms will be labeled when [8-14C]guanosine is used as the
precursor. To examine whether the same holds true for molybdopterin biosynthesis, chiN
cells were grown in the presence of [8-14C]guanosine. Unexpectedly significant radioactivity
was present in the compound Z. Degradation of compound Z to pterin-6-carboxylate and then
to pterin showed that all of the 14C was associated with the C-1' carbon of the side chain. In
contrast very little radioactivity was present in the bulk pterin-6-carboxylate obtained by
permanganate treatment of the whole cell culture. These findings showed that in the
conversion of guanosine to precursor Z, the C-8 of guanosine is retained to generate C-1' of
the side chain and suggested that the ten carbons of precursor Z could all be derived from the
10 carbons of guanosine. Since the label from the ribose of guanosine is incorporated into
some or all of the 2' to 4' carbons of the side chain, these data imply that the C-8 of the
guanine ring is inserted between C-2' and C-3' of the ribose in the process of generating
precursor Z. The mechanism of this intriguing reaction remains to be explored.

Formation of MGD from MPT

The chlB mutants of E. coli had been known for some time to produce MPT capable of
reconstituting nit-1 nitrate reductase, yet displayed the pleiotropic absence of molybdo-
enzymes characteristic of the entire class of chl mutants. 18 The nit-1 nitrate reductase
reconstitution was shown to be the result of cofactor transfer from the chlB extract to the apo
nitrate reductase and not due to activation of a nit-1 precursor. 19 Moreover, upon treatment
with 12/KI at pH 2.5 and 100 °C, chlB cells yielded much higher levels of the fluorescent
cofactor derivative Form A than did wild type cells. These results showed that chlB mutant
cells contain MPT, but did not indicate whether they also contain MGD since the conditions
used for generation of Form A lead to cleavage of the pyrophosphate bond. In order to
address this question, conditions were established for the conversion of MGD to a new
derivative, Form A-GMP. 19 Indeed, the results indicated that while significant levels of
MGD were present in wild type cells, extracts of chlB cells were devoid of this material.
Thus it was concluded that the product of the chlB gene locus is essential for the conversion
of MPT to MGD.

361
CLONING OF RAT LIVER SULFITE OXIDASE

The amino acid sequences of sulfite oxidase from rat liver and chicken liver have been
determined20,21 and found to contain two invariant cysteines which are also present in the
deduced amino acid sequences of several plant nitrate reductases. The eDNA of rat liver
sulfite oxidase has been cloned and expressed in E. coli to yield active enzyme (R. M. Garrett
and K. V. Rajagopalan, unpublished). Despite the presence of both MPT and MGD in the
host cells, the purified expressed protein was found to contain only MPT. The basis for this
selectivity remains to be explored. This expression system should facilitate detailed structure-
function studies on the enzyme, especially in relation to the interactions of the molybdenum
cofactor with the protein.

CONCLUSIONS

The severity of the pathology observed in molybdenum cofactor deficiency underscores

the essentiality of molybdenum and molybdopterin for normal human development. The
studies reported here have revealed numerous complexities associated with various aspects of
the chemistry and biology of the cofactor. These include (1) the role of the pterin ring in the
catalytic steps of molybdoenzyme reactions (2) the conformation of the cofactor at the
molybdenum centers of the enzymes (3) the sulfur mobilization pathway for the formation of
the dithiolene moiety (4) the molybdenum processing steps (5) the unusual reaction involved
in the formation of the pterin ring of molybdopterin and (6) storage, stabilization and transfer
of the cofactor. Studies directed at understanding these aspects of the cofactor are currently in
progress.

REFERENCES
1. C.D. Garner and S. Bristow, in: "Molybdenum Enzymes," T.G. Spiro, ed., Wiley-Interscience, New
York (1985).
2. K.V. Rajagopalan, in: "Advances in Enzymology and Related Areas of Molecular Biology," A. Meister,
ed., John Wiley & Sons, New York (1991).
3. J.L. Johnson, B.E. Hainline, K.V. Rajagopalan, and B.H. Arison, J. Biol. Chern. 259:5414 (1984).
4. J.L. Johnson and K.V. Rajagopalan, Proc. Natl. Acad. Sci. USA 79:6856 (1982).
5. S.P. Kramer, J.L. Johnson, A.A. Ribeiro, D.S. Millington, and K.V. Rajagopalan, J. Biol. Chern.
262:16357 (1987).
6. N.R. Bastian, C.J. Kay, M.J. Barber, and K.V. Rajagopalan, J. Biol. Chern. 266:45 (1991).
7. S. Gruber, L. Kilpatrick, N.R. Bastian, K.V. Rajagopalan, and T.G. Spiro, J. Am. Chern. Soc.
112:8179 (1990).
8. M.G. Finnegan, J. Hilton, K.V. Rajagopalan, and M.K. Johnson, J. Inorg. Chern. (1993), in press.
9. J.L. Johnson, N.R. Bastian, and K.V. Rajagopalan, Proc. Natl. Acad. Sci. USA 87:3190 (1990).
10. J.L. Johnson, K.V. Rajagopalan, and 0. Meyer, Arch. Biochem. Biophys. 283:542 (1990).
11. G. Borner, M. Karrasch, and R.K. Thauer, FEBS Lett. 290:31 (1991).
12. S. Mukund and M.W.W. Adams, J. Biol. Chern. 265:11508 (1990).
13. J.L. Johnson, K.V. Rajagopalan, S. Mukund, and M.W.W. Adams, J. Biol. Chern. 268:4848 (1993).
14. M.M. Wuebbens and K.V. Rajagopalan, J. Bioi. Chern. (1993), in press.
15. D.M. Pitterle and K.V. Rajagopalan, J. Bioi. Chern. (1993), in press.
16. D.M. Pitterle, J.L. Johnson, and K.V. Rajagopalan, J. Bioi. Chern. (1993), in press.
17. T. Nohno, Y. Kasai, and T. Saito, J. Bacterial. 170:4097 (1988).
18. V. Stewart, Microbial. Rev. 52:190 (1988).
19. J.L. Johnson, L.W. lndermaur, and K.V. Rajagopalan, J. Biol. Chern. 266:12140 (1991).
20. M.J. Barber and P.J. Neame, J. Bioi. Chern. 265:20912 (1990).
21. P.J. Neame and M.J. Barber, J. Biol. Chern. 264:20894 (1989).

362
STUDIES ON THE MOLYBDENUM COACTOR. SYNTHESIS OF
(±)-FORM B (DEPHOSPHO)

Edward C. Taylor and Ihab S. Darwish

Department of Chemistry, Princeton University, Princeton, NJ 08544

The molybdenum cofactor (Moco), which is found in all molybdenum-containing

enzymes with the single exception of nitrogenase, consists of a pterin (termed
molybdopterin) carrying a unique ene-dithiol functionality which is complexed with MoVI.
Several tungsten-containing enzymes from hyperthermophilic bacteria have also been shown
to contain molybdopterin (see Fig. 1). More complex molybdopterin dinucleotides
incorporating adenine, guanine, hypoxanthine and cytosine have been identified from
bacteria.1-4

In seminal studies on the structure of Moco, Rajagopalan and coworkers treated

Moco-containing molybdoenzymes with 0.01 M Tris-HCl at pH 2.5 at 100 °C in the presence
of potassium iodide and iodine and obtained a highly fluorescent oxidized pterin substituted
at position 6 with an acetylenic sidechain, but which had lost both of the sulfur atoms present
in molybdopterin itself.5 This degradation product, termed Form A, has been obtained from
all molybdopterin-containing enzymes examined thus far by Rajagopalan. The structure
proposed by Rajagopalan has been been rigorously confirmed by total synthesis, and its
absolute configuration established.6 When this controlled decomposition of Moco-
containing molybdoenzymes was carried out in the absence of iodine, a second degradation
product was obtained which contains only one of the two sulfur atoms present in
molybdopterin itself, for which the structure shown in Fig. 1 was proposed.5,7 The only
other known naturally-occurring thieno[2,3-.!i]pterin is urothione, the urinary metabolite of
Moco; its structure has been confirmed as a methylthio derivative of Form B, again by an
unequivocal total synthesis. 8 The present paper details the synthesis of (±)-Form B
(dephospho), which provides final confirmation of the structure of Form B as originally
proposed by Rajagopalan.

Initial attempts to prepare a thieno[2,3-.!i)pyrazine intermediate for a proposed

synthesis of Form B were unsuccessful. For example, a variety of palladium-catalyzed
carbon-carbon coupling reactions of acetylenic substrates with 2-amino-3-cyano-5-bromo(or
iodo)-6-chloropyrazine failed, despite the encouraging precedent described earlier by us of
the conversion of 2-amino-3-cyano-5-bromopyrazine to 6-substituted pterins by a similar
strategy.9 In earlier work, we had described an efficient entry to dihydrothieno[2,3-
.!i]pyrazines by way of a thiourea-mediated cyclization of a series of 2-amino-3-cyano-5-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 363
 0
\\It
s
Mo
o N~s·
·s
N .,... OP03-
H2N)!.N N OH
H
The Molybdenum Cofactor Form A
(from xanthine oxidase)
0

HN:.rN~X
H2N,h.N N~sYoPo3-
0H
Form B, X=H
Urothione, X=SMe

Figure 1

vinylpyrazines.lO In an adaptation of this methodology, we were able to convert 1 to trans-

2-amino-3-cyano-5-butenyl-6-chloropyrazine (2), which was cyclized with thiourea in
ethanol to the dihydrothieno[2,3-Q]pyrazine 5 (Fig. 2). However, attempts to aromatize this
model substrate were either completely unsuccessful, or went astray, as illustrated by the
oxidation of 5 with four equivalents of DDQ in aqueous dioxane to give a very low yield of
the corresponding 6-acetylthienopyrazine 6. An attempt to prepare a fused
dihydrothienopyrazine from the more complex potential intermediate olefin 4 (prepared from
the phosphonium salt 2 and 2,3-0-isopropylidene-D-glyceraldehyde either under Wittig or
Homer-Emmons conditions) was also unsuccessful, as was an attempt to unmask the latent
mercapto functionality in 7 in the hope of effecting intramolecular cyclization to the same
fused dihydrothienopyrazine.

The synthetic route which eventually proved successful is outlined in Figs. 3 and 4.
The chloromethyl substituent in the key pyrazine intermediate 1 was transformed into a
formyl group by the Krohnke method, which involves initial conversion to the pyridinium
salt 8, followed by condensation with 4-dimethylaminonitrosobenzene in the presence of
excess potassium carbonate to give a nitrone (9) which is then hydrolyzed to 10 at 0 °C with

364
NC'r!N~X
N''ll N~ Cl
+ RCHO
H2

1, X= Cl 3, R = CH 2 CH3

oxo
2, X = PPh3+ Cl" 4,R=-r'.

thiourea !
DDQ

NC)N:('C I
pyridine
Figure 2
NC
)
N
:c+N~ II
D
H2N N Cl (98%) H2N N'" Cl Cl-
1 8

K2 co, j ON-o-NM e 2

ONMe,
6 N HCI
I N~
NC)NXCH O NC
"
H 2NlN~ 6'
N
H2N N Cl 0 oc Cl

10 9

Figure 3

365
HCl. Condensation of this Q-chloroaldehyde with 1-acetoxy-3-mercapto-2-propanone in the
presence of triethylamine led directly to the fused thieno[2,3-.h]pyrazine 11, which now
contains the elements of the proposed Form B sidechain correctly positioned on the fused
(aromatic) thiophene ring. Conversion of this acetoxyketone to the glycol 12 was
accomplished with sodium borohydride in a mixture of ethanol and THF; the basic reaction
conditions proved sufficient for removal of the acetoxy grouping as well. Protection of this
diol as its acetonide 13 was then readily accomplished with 2,2-dimethoxypropane and p_-
toluenesulfonic acid in refluxing benzene.

NC~J(~
Et3N
10 + HslfoAc
I ~ I
0 H2N N S OAc
11 0

MeO OMe
NaBH 1 4

)(.
NC:(~
I ~ I NC)(~
. -: I
H2N N S 0 p-TsOH H2N N S OH
13
guanidine ! 0~ 4A
12
OH

H2N,4N
N:S:N~
I ~
N
~
S
I
0
1 N NaOH

I!.
JcN
H,NAN N~0
HN "'

14 0~ 15 0~

/ 20% HOAc, 1!.

(±)-Form B (dephospho)

Figure 4

The remainder of the synthesis was straightforward. Ring closure with guanidine in
methanol yielded 14 in excellent yield. The 4-amino group on the pyrimidine ring was
hydrolyzed with 1 N sodium hydroxide to give a homogeneous solution of the resulting
thienopterin 15 as its mono-sodium salt. Final removal of the acetonide protecting group
with aqueous acetic acid then provided (±)-Form B (dephospho), which was identical in
every respect except optical rotation with an authentic sample of Form B (dephospho)
obtained by degradation of Moco-containing molybdoenzymes. 1

366
References
1. K. V. Rajagopalan, Abstr. 204th National ACS Meeting, Washington, D.C. August
(1992), Inorg. Chern.# 173.
2. J. L. Johnson, K. V. Rajagopalan, and 0. Meyer, Arch. Biochem. Biophys. 283:542
(1990).
3. J. L. Johnson, N. R. Bastian, and K. V. Rajagopalan, Proc. Nat!. Acad. Sci. U.S A.
87:3190 (1990).
4. G. N. George, R. C. Prince, S. Mukund, and M. W. W. Adams, J. Am. Chern. Soc.
114:3521 (1992).
5. J. L. Johnson, B. E. Hainline, K. V. Rajagopalan, and B. H. Arison, J. Bioi. Chern.
259:5414 (1984).
6. E. C. Taylor, P. S. Ray, I. S. Darwish, J. L. Johnson, and K. V. Rajagopalan, J. Am.
Chern. Soc. 111:7664 (1989).
7. K. V. Rajagopalan, Biochem. Soc. Trans. 13:401 (1985).
8. E. C. Taylor and L.A. Reiter, J. Am. Chern. Soc. 111:285 (1989).
9. E. C. Taylor and P. S. Ray, J. Org. Chern. 52:3997 (1987).
10. E. C. Taylor and A. L. Sabb, J. Org. Chern. 53:5839 (1988).
11. A brief report of an alternate approach to Form B (dephospho) from 7-acetylthieno[3,2-
gJpterin has appeared: K. Ushio, M. Ishizuka, M. Kogushi, S. Fukui, and T. Toraya,
Biochem. Biophys. Res. Commun. 135:256 (1986). Desulfurization of urothione as a route to
Form B has also been reported, although this procedure is neither of synthetic significance,
nor an acceptable proof of structure; see Ref. 5 and M. Goto, A. Sakurai, K. Ohta, and H.
Yamakami, J. Biochem. (Tokyo) 65:611 (1969).

367
MOLYBDENUM-PTERIN COMPLEXES: A FUNCTIONAL AND STRUC-
TURAL MODEL FOR THE BINDING SITE IN THE ENZYME DIMETHYL
SULFOXIDE REDUCTASE
Berthold Fischer 1 , Helmut Schmalle1 , Erich Dubler 1 , Max Viscontini 2

1 Anorganisch-chemisches Institut der Universitat, Winterthurerstr. 190,

CH-8057 Zurich, Switzerland
2 0rganisch-chemisches Institut der Universitat, Winterthurerstr. 190,

CH-8057 Zurich, Switzerland

INTRODUCTION

In addition to the nitrogenases there is a large group of molybdenum containing

enzymes catalyzing oxidation and reduction of various substrates 1 .
Several bacterial species possess the ability to reduce dimethyl sulfoxide with the help
of the molybdenum dependant enzyme dimethyl sulfoxide reductase 2 • Although the dif-
ferent molybdenum-enzymes have molecular weights of at least 100 000, most of them
have a common cofactor with a molecular weight of about 1500. This cofactor contains
the active metal center coordinated by different ligands. One possible ligand found
in the vicinity of molybdenum is a pterin unit. Rajagopalan and coworkers proposed
a model for the cofactor consisting of a C(6)-substituted tetrahydropterin, where two
sulfur atoms of the sidechain, but not the pterin nucleus, are coordinating to the molyb-
denum(VI) atom 3 . The oxidation state of the molybdenum atom switches between+ VI
and +IV in the enzyme reactions.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 369
 With the first molybdenum-dihydropterin complex, trichloro(2' ,5-quinonoid-7 ,8-di-
hydro-(2'H+ ,6H)-6,B-L-biopterin)oxomolybdenum(IV) [MoOCh(H+ -q-BH 2 )] (1 ), it was
demonstrated that a chelating coordination of molybdenum by the the pterin nucleus
through 0(4) and N(5) is also possible (Figure 1) 4 . This complex displays an interesting
behaviour in solution, as could be followed by 13 C-NMR spectroscopy. Dissolving the
isolated solid in such solvents as methanol or dimethyl sulfoxide, a slow equilibrium
reaction takes place resulting in the educts tetrahydro-1-biopterin [BH 4] and a Mo(VI)-
complex in addit.un to the Mo0Cl 3 (H+ -q-BH 2 ) complex (1 ).

RESULTS

The new compound trichloro(2' ,5-quinonoid-7,8-dihydro-(2'H+ ,6H)-pterin)oxomo-

lybdenum(IV) [Mo0Ch(H+-q-PH 2 )] (2) was synthesized in the same manner as 1 from
the educts Mo0 2 Ch and tetrahydropterin · 2 HCl [PH 4 ]. The X-ray structure of the
new complex confirms the oxidation state of +IV for the molybdenum atom (Figure 2).

Figure 2. Structure of MoOCb(H+ -q-PH2) (2).

The first coordination sphere around the molybdenum atom in 1 is identical with
that in 2; only in the second coordination sphere of 1 a weak interaction in solution
between a Mo-coordinated chlorine atom and an OH-group from the BHTsidechain can
be discussed. But the properties of the two complexes are quite different.
The complex Mo0Ch(H+-q-PH 2 ) (2) is able to reduce the substrate dimethyl sulfoxide
[DMSO] to dimethyl sulfide [DMS] under very mild conditions, as can be shown by
13 C- NMR and mass spectrometry. In figure 3 a the spectrum displays the expected six
13 C-NMR signals of MoOCh(H+ -q-PH 2 ) (2) which are appearing in practically exact

the same range as in Mo0Cl 3 (H+-q-BH 2 ) (1). In contrast to 1, during seven hours there
is almost no change in the spectrum of 2 besides a very small amount of PH 4 formed.
Then the signals of deuterated DMS and of fully oxidized pterin begin to rise and after
about 20 h the spectrum in figure 3 b is obtained.
Control experiments with the pure substances PH 4 , q-PH 2 and Mo(IV)Cl 4 in DMSO in
no case yielded DMS. The deuterated DMS was additionally identified by mass spec-
trome_try. A quantitative determination of the produced DMS revealed a 1:2 ratio of 2 :
DMS. This means that not only the oxidation of the quinonoid dihydropterin to pterin

370
 f ..
uJ
C4 C 2

. .L

j I
C8a

..
C4a

.l •• ; . a
t '' I
.. ,
d
C6 I

DMS
b

200 180 100 1<0 121) 100 80 00 <0 201•pm

Figure 3. 13 C-NMR spectrum of MoOCh(H+ -q-PH 2 ) (2) in DMSO-d6 , room temperature,

argon: a after 3 h. b after 20 h. Arrows in b indicate signals of 2. The six new signals
correspond to oxidized pterin. DMS = Dimethyl Sulfide-d6 .

has caused the reduction of DMSO to DMS, but also the oxidation of Mo(IV) to
Mo(VI). In natural enzyme systems involving quinonoid dihydropterins, these will nor-
mally not be oxidized to pterins but reduced again to tetrahydropterins by the enzyme
dihydropteridine reductase. The effective electron transfer will be carried out by t he
Mo(IV)/Mo(VI) shuttle. On the basis of these two structures and supported by spec-
troscopical data, we propose a participation of the pterin nucleus in the enzyme reaction
of molybdenum dependent oxidoreductases and a new structure of t he Molybdenum Co-
factor in its reduced form (Figure 4).

~ ~o
-
/I
- Mo - - S
"c-CHOH
I! \
)C's
0 N
HN: x
I
CH2

H~N/ ~
2-
N OP0 3
H

X = H, CH 3 , PROTEIN
Figure 4 . Proposal of an extendend Molybdenum Cofactor in its reduced form.

The function of the tetrahydropterins could be that of a reducing agens to Mo(VI)

and the quinonoid dihydropterins may be useful as stabilizing ligands to the reactive
Mo(IV)-state. But this ligand does not coordinate too strongly like other standard
chelating ligands. Thus the reactivity toward substrates is not hindered and can be
tuned very finely as we could show with our simple models MoOCh(H+ -q-BH 2 ) (1) and
Mo0Cb(H+-q-PH 2 ) (2).

371
 In a further experiment we tried to "regenerate" the enzyme model, a crucial de-
mand for a functional model. In figure 5 are displayed in addition to the 13 C-NMR
spectra of PH 4 a and Mo0Cb(H+-q-PH 2 ) (2) b the spectrum of fully oxidized complex
2 after 14 days c. This spectrum exhibits only the six signals of the fully oxidized pterin
and presumably contains Mo(VI). After addition of PH 4 under argon, no PH4 signals
could be detected but only the "regenerated" complex 2 additionally to the still present
pterin signals d.

l
C 4a

Ill
PH~ a

jL_
,M
.oHL
MoOCb (W q- P1!2)

l!
C4a
C6
·~ I
• IIIII • • lllf

'
' '
2 week.~ later

200
rulditiou of PH,

180 160 140 120 100 110 60

L:_
•• 20ppm

Figure 5. 13 C-NMR spectra of PH 4 a, Mo0Cb(H+-q-PH 2 ) (2) b, same solution 2 weeks

later c and after new addition of PH 4 d. DMSO-d6, room temperature, argon.

The crystallographic and all other experimental data will be published in Helvetica
Chimica Acta.

REFERENCES

1. "Molybdenum Enzymes" , T.G . Spiro, ed., J . Wiley, New York, Met. Ions Bioi. 7 (1985).
2. N.R. Bastian, C.J. Kay, M.J. Barber, and K.V. Rajagopalan, J. Bioi. Chem. 266:45 (1991).
3. J.L. Johnson, B.E. Hainline, and K.V. Rajagopalan, J. Bioi. Chem. 255:1783 (1980) .
4. B. Fischer, J. Strahle, and M. Viscontini Helv. Chim. Acta 74:1544 (1991).

372
HUMAN MOLYBDENUM COFACTOR DEFICIENCY

Jean L. Johnson,1 K. V. Rajagopalan,1 and Sybe K. Wadman2

1Department of Biochemistry
Duke University Medical Center
Durham, NC 27710
2University Children's Hospital
Het Wilhelmina Kinderziekenhuis
Utrecht, The Netherlands

INTRODUCTION

Molybdenum cofactor deficiency is an inborn error of metabolism first identified in

1980. 1•2 Cofactor deficient patients exhibit combined deficiencies of three molybdoenzymes,
sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase, all of which depend on the
presence of a tightly bound molybdenum-molybdopterin complex for catalytic activity. The
study of molybdenum cofactor deficiency has been carried out in parallel with studies
defining the molecular structure of the molybdenum cofactor and those establishing the
pathway of its biosynthesis in microorganisms. Thus, when the first patient was identified,
very little information was available regarding even basic structural aspects of the cofactor.
However, as more patients have been characterized, more information has been obtained to
shed light on the structure of the pterin they lack. Many of the human studies have been
greatly facilitated by study of enzymes and biosynthetic intermediates in molybdopterin
mutants in microorganisms. Transfer of this knowledge to studies of the human patient
population is an ongoing process which may lead ultimately to the design of effective
therapeutic agents for correction or alleviation of the cofactor deficiency disease.

THE PATIENT POPULATION: OCCURRENCE OF THE DEFICIENCY

DISEASE AND CLINICAL SYMPTOMS

Molybdenum cofactor deficiency has been identified in 47 patients in 42 families.

Although some variability in severity of symptoms and age of onset has been observed, the
key clinical symptom is severe convulsions, not responding to therapy, presenting early after
birth. Those patients who survive beyond the first few days usually develop severe
neurologic abnormalities, dislocated ocular lenses, mental retardation and mild to severe
xanthinuria. Other common and less common clinical symptoms are summarized in Table 1.
Molybdenum cofactor deficiency has been identified in a variety of ethnic groups among the
populations of Central and Southern Europe, North America, Asia, and Northern Africa. The

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 373
disease is inherited as an autosomal recessive trait with obligate heterozygotes displaying no
symptoms. Pathology studies have been carried out in a number of cases and have shown a
severe encephalopathy with marked neuronal loss and demyelination in the white matter
accompanied by gliosis and diffuse spongiosis.

Table 1. Signs and symptoms of molybdenum cofactor deficiency.

Common (seen in more than 50% of patients) Seizures, tonic/clonic

Feeding difficulties
Hypotonia
Hypertonicity
Myoclonia
Pyramidal syndrome
Spastic tetraplegia
Dilated ventricles
Brain hypodensity
Brain atrophy
Bilateral ectopic lenses
Unresponsiveness to light
Psychomotor retardation

Less Common (seen in 50% of patients or less) Vomiting

Opisthotonus
Hydrocephalus
Enophthalmus
Nystagmus
Ring of Brushfield spots

ROLE OF THE MOLYBDENUM COFACTOR IN ANIMAL METABOLISM

The molybdenum cofactor is required in animals for the activities of sulfite oxidase,
xanthine dehydrogenase and aldehyde oxidase, and the metabolic pathways involving these
enzymes are disturbed in patients with molybdenum cofactor deficiency. Sulfite oxidase
functions in the degradative pathway of sulfur amino acids as indicated in Figure 1. In the
absence of sulfite oxidase activity, patients accumulate and excrete elevated levels of sulfite,
thiosulfate, S-sulfocysteine and taurine. The absence of xanthine dehydrogenase leads to an
increase in xanthine and hypoxanthine with decreased uric acid levels as in classic
xanthinuria. The consequences of the absence of aldehyde oxidase are less well-defined,
although the enzyme is known to catalyze the hydroxylation of a variety of heterocyclic
substrates and may function as a part of the body's general detoxification system.
Of the several metabolic blocks imposed by the absence of these molybdoenzyme
activities, the one responsible for the severe clinical ramifications appears to be that resulting
from a deficiency in sulfite oxidase. At least twelve cases of isolated deficiency of sulfite
oxidase have been identified, with clinical symptoms, metabolite patterns (other than
oxypurine profiles), and neuropathology all very similar to those observed in molybdenum
cofactor deficiency. 2 In addition, the recent identification of two classes of xanthinuric
patients, those with isolated xanthinuria and those with a combined deficiency of xanthine
dehydrogenase and aldehyde oxidase, has revealed that even a combined deficiency of the
latter two enzymes is a benign clinical syndrome with none of the devastating sequelae
associated with sulfite oxidase or molybdenum cofactor deficiencies. 3

374
 coo- coo-
COO- Aminotransferase + I + I
I .___ H3N-CH H3N-CH
O=C I I
CH 2 CH 2
I I
CH 2 I -
SH S203
I
SH

1
CYSTEINE S-8ULFOCYSTEINE

!
B-MERCAPTOPYRUVATE
02 Cysteine Dioxygenase
Transsulfurase

coo- coo-
l + I +
H2S + O=C H3N-CH C02 H N-CH H3N -CH 2
I I 3 I 2
I
I
CH 3 CH 2 ~ CH 2 - CH 2
I - I - I -
PYRUVATE so2 so2 so3
s2o3=
!
CYSTEINE SULFINATE HYPOTAURINE TAURINE
THIOSULFATE
Aminotransferase

coo-
l
O=C
I
CH 2
I -
so2
B-SULFINYL PYRUVATE

f'- PYRUVATE

!
so3 =
Sulfite Oxidase

so4=
Figure 1. Pathway of degradation of sulfur amino acids showing involvement of sulfite oxidase.
Metabolites that are elevated in molybdenum cofactor deficiency are indicated in bold type.

MOLYBDENUM COFACTOR BIOSYNTHESIS IN MAN:

CHARACTERIZATION OF THE MOLECULAR DEFECTS IN
MOLYBDENUM COFACTOR DEFICIENCY

Structural studies of the molybdenum cofactor and its stable degradation products have
shown that it consists of the metal ligated to a unique pterin species termed molybdopterin
(Figure 2). 4 Although the molybdenum cofactor bound to molybdoenzymes is extremely
stable within its protective environment, it decays rapidly upon release, first by metal
dissociation to molybdopterin, and then to numerous nonfunctional pterin degradation
products. The instability of the free cofactor precludes its use as a therapeutic agent for
molybdenum cofactor deficient patients and suggests that a rational approach to therapy can
evolve only after gaining a more thorough understanding of cofactor biosynthesis and the
specific defects in cofactor deficient patients.
The first indication that molybdopterin biosynthesis in man proceeds by a multistep
pathway and that cofactor deficient patients segregate into groups with defects at different
enzymatic steps came from fibroblast complementation studies. 5 Co-culture of binary
combinations of cells from various patients produced active molybdenum cofactor, as
indicated by the presence of sulfite oxidase activity. Complementation occurred with or

375
without heterokaryon formation, suggesting that a diffusible intermediate produced by one
cell line was being taken up and activated to molybdopterin by the complementing cell line.
Experiments using conditioned medium from which the donor cells had been physically
removed further established that cells from group B patients were the source of the diffusible
material which was utilized by cells from group A patients.
An evaluation of the levels of molybdoenzymes and molybdenum cofactor in cultured
fibroblasts indicated that characterization of the diffusible intermediate produced by group B
cells and of the processes in group A cells required to activate it to molybdopterin would be
very difficult to accomplish. An alternative and very successful approach was to turn to
related studies, carried out in this laboratory, of molybdopterin biosynthesis in micro-
organisms.4 The identification of a late precursor of molybdopterin, termed precursor Z
(Figure 2), and characterization of a protein that converts precursor Z to molybdopterin,
termed converting factor, in molybdopterin mutants of E. coli made it possible to assay for
their presence in group A and group B cofactor deficient patients. 5 As indicated in Figure 2,
these studies revealed that a molybdopterin precursor indistinguishable from the microbial
precursor Z does accumulate in group B patients. Precursor Z was identified in urine samples
from several patients using a functional assay that measured the generation of molybdopterin
in the presence of exogenously added E. coli converting factor and by quantitation of a
fluorescent degradation product of precursor Z produced by iodine oxidation. Also
summarized in Figure 2 is the observation that tissues from group A patients contain a
converting factor activity that recognizes exogenously added precursor Z, producing
molybdopterin. This activity was initially identified in fibroblasts from group A patients, and
has more recently been partially purified and characterized from liver tissue.

Defective in E. coli ch/A 1, ch/A2 and ch/A3 mutants

and in group A molybdenum cofactor deficient patients

PRECURSORZ

0
Converting Factor

H
l Defective in E. coli ch/A4 and ch/A5 mutants
and in group B molybdenum cofactor deficient patients

HNAyNrr=r-CHOH-CH20P03=

H2N
~ N. A)
N
SH SH
MOLYBDOPTERIN

0 l
HN~NT-t=\-CHOH
:

CH,OPO,=

H2 N~NAN) l., /s H Mo MOLYBDENUM COFACTOR

c/'a
Figure 2. Late steps in the biosynthesis of molybdopterin. The defects identified in group A aiid group B
molybdenum cofactor deficient patients are indicated.

376
DIAGNOSIS: QUANTITATION OF PTERIN METABOLITES

The diagnosis of molybdenum cofactor deficiency is based upon clinical symptoms,

metabolic excretion profiles, and when appropriate tissue samples are available, on direct
assay of sulfite oxidase and xanthine dehydrogenase activities. Because the molybdoenzymes
are present in high concentrations primarily in liver and kidney, verification of the deficiency
by enzymatic analysis is not always possible. Sulfite oxidase activity, but not xanthine
dehydrogenase activity, is expressed at low levels in cultured fibroblasts and can be
quantitated in this source material.6
When assessing altered metabolite levels, an oxypurine profile often serves as the first
and simplest test to demonstrate a deficiency in an enzyme dependent on the molybdenum
cofactor. It is important to quantitate xanthine and hypoxanthine levels as well as uric acid
concentations, since some cofactor deficient patients have been observed with uric acid levels
close to the normal range. Altered sulfur metabolites, including elevated sulfite, S-
sulfocysteine and thiosulfate are indicative of sulfite oxidase deficiency. A pterin metabolite
screening test has been developed recently and has proven to be extremely useful in
confirmation of molybdenum cofactor deficiency and evaluation of the complementation
group status of an identified patient. The metabolic degradation product of the molybdenum
cofactor is a methylated, dephospho thienopterin, urothione (Figure 3).7 Molybdenum
cofactor deficient patients excrete no urothione, whereas urinary concentrations in normal
individuals range from 50 to 600 ng/mg creatinine. The published method for quantitation of
urothione has been modified to include an iodine oxidation step which does not affect the
urothione analysis, but which oxidizes any precursor Z present in the sample to its
fluorescent degradation product, compound Z (Figure 3). 8 Compound Z is separated from
the uncharged urothione during chromatography on an anion exchange resin, and the two
pterins are quantitated by HPLC chromatography (Figure 4). The absence of urothione
confirms a diagnosis of molybdenum cofactor deficiency, while the absence or presence of
compound Z in the same urine sample defines the A or B complementation group,
respectively, obviating the need for lengthy and expensive cell co-culture experiments.

Figure 3. Structures of urothione (left) and compound Z (right).

Molybdenum cofactor deficiency has been successfully diagnosed prenatally in families

where one affected child has already been identified.9 Early studies used cultured amniocytes
for quantitation of sulfite oxidase activity. While absence of sulfite oxidase activity in the
amniocytes proved to be an accurate marker of an affected fetus, the low activity of this
enzyme in control amniocytes and the length of time required to culture cells in sufficient
numbers for assay precluded rapid diagnosis. More recently, the assay of the same enzyme
activity in uncultured chorionic villus biopsy material has been shown to give accurate and
more timely assessment of the status of the fetus at risk. During the course of prenatal
diagnosis studies, it was established that normal liver tissue, obtained as early as 15 weeks
gestational age, contains nearly adult levels of sulfite oxidase, whereas no activity is present
in the same tissue from an affected fetus. These high levels of activity emphasize the critical
metabolic role of this molybdoenzyme and its pterin cofactor in human development.

377
 80 20 Ill

g
G)

60 15
G)
::J u
~ 40 f 10
0
:I
20 u:: 5

5 10 15 20
60r-------------------~
IV

40
::J
oct
E
20

5
Elution Time {min) Elution Time {min)

Figure 4. HPLC elution chromatograms showing quantitation of urothione (13.5 min peak) from a control
(I) and molybdenum cofactor deficient patient em
and of compound Z (6 min peak) from a group B (lll) and
group A (IV) patient. Urothione was chromatographed on a C-18 column in 15% methanol with absorbance
monitored at 380 nm. Compound Z was chromatographed on a C-18 column in 5% methanol, pH 2.

CONCLUSIONS

The biochemical studies summarized above indicate that the late steps in molybdopterin
biosynthesis are the same in man and microorganisms. Indeed, the identification of functional
converting factor in group A patients, who are unable to synthesize the molybdenum cofactor
due to a block early in the pathway, raises the possibility that exogenously supplied precursor
Z could be of some therapeutic value to these patients. The increased stability of this pterin
relative to that of molybdopterin and the demonstrated ability of this cyclic phosphate
derivative to pass through the cell membrane in vitro strengthen the hypothesis. However,
additional study will be required to establish more effective stabilization conditions, to further
evaluate the precise isomeric configuration of the natural product, and to establish a synthetic
route for production of the necessary quantities of pure material.
Further investigations into early steps in the pathway will be needed to establish whether
group A patients exhibit defects at the same or at different enzymatic steps and whether the
cofactor pterin is synthesized de novo or elaborated from a preformed dietary intermediate.

REFERENCES

1. J.L. Johnson, W.R. Wand, K.V. &yagopalan, M. Duran, F.A. Beemer, and S.K. Wadman, Proc. Natl.
Acad. Sci. U. S. A. 77:3715 (1980).
2. J.L. Johnson and S.K. Wadman, in: "The Metabolic Basis of Inherited Disease," 6th edition, C.R. Scriver,
A.L. Baudet, W.S. Sly, and D. Valle, eds., McGraw-Hill Book Co., New York (1989).
3. S. Reiter, H.A. Simmonds, N. ZOllner, S.L. Braun, and M. Knedel, Clin. Chirn. Acta 187:221 (1990).
4. K.V. Rajagopa1an and J.L. Johnson, J. Bioi. Chern. 267:10199 (1992).
5. J.L. Johnson, M.M. Wuebbens, R. Mandell, and V.E. Shih, J. Clin. Invest. 83:897 (1989).
6. V.E. Shih, I.F. Abroms, J.L. Johnson, M. Carney, R. Mandell, R.M. Robb, J.P. Cloherty, and K.V.
Rajagopalan, New Engl. J. Med. 297:1022 (1977).
7. J.L. Johnson and K.V. Rajagopalan, Proc. Natl. Acad. Sci. U. S. A. 79:6856 (1982).
8. J.L. Johnson, M.M. Wuebbens, and K.V. Rajagopalan, 1. Biol. Chern. 264:13440 (1989).
9. J.L. Johnson, K.V. Rajagopalan, J.T. Lanman, R.B.H. Schutgens, A.H. van Gennip, P. Sorensen, and
D.A. Applegarth, J. Inherited Metab. Dis. 14:932 (1991).

378
MOLYBDOPTERIN BIOSYNTHESIS IN MAN.
PROPERTIES OF THE CONVERTING FACTOR IN
LIVER TISSUE FROM A MOLYBDENUM
COFACTOR DEFICIENT PATIENT

Jean L. Johnson and K. V. Rajagopalan

Department of Biochemistry
Duke University Medical Center
Durham, NC 27710

INTRODUCTION

Molybdenum cofactor deficiency is an inborn error of metabolism resulting from an

inability to synthesize functional molybdopterin, the organic pterin component of the
molybdenum cofactor. 1 Cofactor deficient patients exhibit combined deficiencies of the three
known molyboenzymes in man, sulfite oxidase, xanthine dehydrogenase and aldehyde
oxidase. Molybdopterin biosynthesis requires a multistep pathway, and patients with defects
at two different points in the pathway comprise two identified complementation groups. 2 The
terminal step in molybdopterin biosynthesis is carried out by a protein, termed converting
factor, that uses as its substrate a desulfo, cyclic phosphopterin, precursor Z. 3 The reaction
catalyzed by the converting factor and the structures of precursor Z, molybdopterin and the
molybdenum cofactor are outlined in Figure 1.

l .~
H2N N
~N

N
H
m ° OH

'-p/
o.p; 'o-
Converting Factor
defective in group B patients
H
2
o H
HN~Nry=y-CHOH-CH,OPo,=
N~ ..N)l ~ ) SH SH
MOL YBDOPTERIN
PRECURSOR Z /
not made in group A patients
0 H

H(XN)jC=\-CHOH-CH20PO,=
H2N N N S"-. /S
H .1' Mo'\. MOLYBDENUM COFACTOR
0 0

Figure 1. Structures of precursor Z, molybdopterin and the molybdenum cofactor. The reaction catalyzed
by the converting factor is indicated.

Chemistry and Biology of Pteridines and Folates, Edited by

I.E. Ayling et al., Plenum Press, New York, 1993 379
 As indicated in Figure 1, patients in the B complementation group lack the function of
the converting factor. They accumulate and excrete precursor Z in the urine. Patients in the A
complementation group exhibit a block in an earlier step in the molybdopterin biosynthetic
pathway and are unable to synthesize precursor Z but express a functional converting factor.
While information on the nature of the converting factor protein and the reaction catalyzed is
available from study of analogous molybdopterin mutants of Escherichia coli, a detailed
understanding of the physico-chemical properties of the converting factor in man is an
important basis for probing the molecular defect(s) in this activity in the group B
molybdenum cofactor deficient patients. Such a study is facilitated by the availability of
tissues from group A cofactor deficient patients, since endogenous molybdopterin in the
tissues does not interfere with the assay of molybdopterin formed in vitro by the converting
factor.

METHODS

Liver tissue was obtained post mortem from a patient with molybdenum cofactor
deficiency and stored at -70 °C. The classification of this patient as group A was based on the
absence of urothione, the metabolic degradation product of molybdopterin,4 and precursor Z
in the urine.
Converting factor was assayed as outlined in Figure 2. The Neurospora crassa nit-1
mutant was grown and harvested as described previously.5 A crude extract of the mycelia,
prepared by homogenizing 1 g in 2 ml nit-1 buffer (1 00 mM potassium phosphate, pH 7.4,
with 5 mM EDTA and 1 mM dithiothreitol) with 20 J.ll of 100 mM phenylmethylsulfonyl
fluoride, was centrifuged for 5 min at 14,000 rpm using an Eppendorf 5415 centrifuge. The
supernatant fraction served as the source of precursor Z and of apo nitrate reductase.
Incubation mixtures of 100 J.ll total volume were prepared using 10 J.ll of 0.5 M sodium
molybdate, 30 J.ll nit-1 extract, and up to 60 J.ll of a converting factor source. After incubation
at 4 oc for 3 hr, the substrates for nitrate reductase (NADPH, FAD, and nitrate) were added
and the amount of nitrite generated in a 30 min room temperature incubation was quantitated
by a colorimetric procedure. 5 The assay was shown to be linear with respect to the amount of
liver converting factor sample added. In addition, the nit-1 reconstituting activity in the liver
tissue sample was shown to be dependent on the presence of precursor Z in the nit-llow
molecular weight fraction, demonstrating that the activity observed was due to converting
factor and not attributable to low levels of residual molybdopterin in the liver extract.

nit-1 EXTRACT:
PrecursorZ
Apo Nitrate Reductase

(CONVERTING FACTOR ) ----+-

3 hr, 4 oc
( MOLYBDATE )

Figure 2. Assay of converting factor activity. The Neurospora crassa nit-1 extract, containing precursor Z
and apo nitrate reductase, is incubated with a source of converting factor. During this incubation period,
precursor Z is converted to molybdopterin, and the molybdopterin, in tum, reconstitutes the apo nitrate
reductase. A subsequent incubation in the presence of nitrate reductase substrates generates nitrite which is
quantitated by a colorimetric assay.

380
RESULTS AND DISCUSSION

Partial Purification of the Human Liver Converting Factor

Liver tissue (2.6 g) was homogenized in 5 ml of nit- I buffer and 45 ml H20 using a
motor driven ground glass homogenizer. The homogenate was centrifuged at 30,000 g for 20
min, and the supernatant fraction was treated with 43 g ammonium sulfate per 100 ml. After
stirring on ice for 10 min, the mixture was centrifuged as before, and the pellet was
resuspended in 50 mM potassium phosphate, pH 7.4, with 1 mM dithiothreitol. The sample
was freed of excess ammonium sulfate by passage through a calibrated column of Sephadex
G-25 equilibrated with the same buffer and then applied to an 8 ml column of DEAE-
cellulose (Whatman DE52) in the same buffer. The activity was eluted from the column using
the phosphate, dithiothreitol buffer with 0.15 M NaCl (see Figure 3).

Gl 4
u
:; 3
J:l
0
Ill
2
J:l
<
2 4 6 8 10 12 14
Fraction
Figure 3. Elution profile of human liver converting factor chromatographed on
DEAE-cellulose. Fractions of 6 ml were collected and monitored for activity using a
60 f!l aliquot in the nit-1 assay and measuring the nitrite complex color formation at
540 mn (e) and for protein at 280 mn (o).

Estimation of Molecular Mass

The material in fraction 6 from the DEAE-cellulose column was concentrated 3-fold
using an Amicon Centricon 10 unit, and 1 ml was applied to a Superose 12 gel filtration
column (Pharmacia) equilibrated in the phosphate, dithiothreitol buffer. The column was
eluted with the same buffer, and fractions of 1 ml were collected and assayed for converting
factor activity. The elution profile is shown in Figure 4 and compared to an equivalent profile
obtained using converting factor partially purified from the E. coli chlAJ mutant. In contrast
to the E. coli converting factor activity, which eluted as a sharp, symmetrical peak, the liver
converting factor profile was broad with a distinct trailing shoulder, suggestive of a
dimer/monomer equilibrium. Estimation of the apparent molecular masses of the two liver
converting factor forms using molecular mass standards (Figure 4) suggests a molecular
mass of 52 kDa for the dimer and 26 kDa for the monomer. The E. coli converting factor
runs with an apparent molecular mass of 42,000-43,000 as reported earlier.6 A second run of
human liver converting factor on Superose 12 using a slightly more dilute sample confirmed
the apparent dimer/monomer equilibrium with a somewhat more defined peak of the
monomeric species, as expected.
Earlier studies on the E. coli converting factor have established that it exists as a loosely
associated complex of 16 kDa and 10 kDa subunits.? The subunit stoichiometry of the
complex as present during gel filtration chromatography has not been established. However,
the apparent monomeric molecular weight of the human liver converting factor at 26 kDa is
interesting in that it could reflect the sum of the masses of the two E. coli subunits.

381
 E II)
100
1: II)
0 1.0 ftJ

"'
10
_(ig....
::iio
Gl
CJ 10
1: 0.5 ~ )(
ftJ 6.5 kDa
...
Gl
.a 0
0 :iii
II)
.a 0.0 1
< 15 20 25 30 35 40 1.3 1 .5 1 .7 1.9
Fract io n Ve I Vo

Figure 4. Left: Elution profiles of human liver (e) and E. coli ch!Al (o) converting factors
chromatographed on Superose 12. Right: Estimation of the molecular mass of the human liver converting
factor dimeric and monomeric forms by gel filtration using a calibrated column of Superose 12.

Sensitivity to Sulfhydryl Reagents

The converting factor from E. coli has been shown to be sensitive to sulfhydryl reagents
including N-ethylmaleimide, N-bromobimane, and iodoacetamide.6 The human liver enzyme
was inactivated by incubation with N-ethylmaleimide as shown in Figure 5. Incubation with
10 mM reagent for 15 min led to 93% inhibition of activity. The human liver enzyme was
also sensitive to iodoacetamide treatment (data not shown). These results provide strong
evidence for the role of a reactive sulfur on the human liver converting factor in the formation
of the molybdopterin dithiolene function and support the conclusion that the converting factor
protein serves as the direct sulfur donor for molybdopterin biosynthesis.

E 1.2
1: 0 10!11
0 • 20).11
"'
10 0.8 l!lil 40!11
Gl
(.1
1:
ftJ 0.4
.a
0
II)
.a 0.0
< Buffer NEM+DTT NEM/DTT

Figure 5. Effect of N-ethylmaleimide on human liver converting factor. The

enzyme samples (90 IJ.l) were incubated with 20111 of nit-I buffer, with 20 Ill of a
1:1 mixture of 100 mM N-ethylmaleimide and 100 mM dithiothreitol, or with 10111
of 100 mM N-ethylmaleimide for 15 min at 4 oc. The excess reagent in the latter
sample was destroyed by the addition of 10 111 of 100 mM dithiothreitol, and aliquots
of 10, 20 and 40111 were assayed for remaining activity using the nit- I assay.

REFERENC ES

1. J .L. Johnson and S.K. Wadman, in: "The Metabolic Basis of Inherited Disease," 6th edition, C.R. Scriver,
A.L. Baudet, W.S. Sly, and D. Valle, eds., McGraw-Hill Book Co., New York (1989).
2. J.L. Johnson, M.M. Wuebbens, R. Mandell, and V.E. Shih, J. Clin. Invest. 83:897 (1989).
3. K.V. Rajagopalan and J.L. Johnson, J. Bioi. Chern. 267:10199 (1992).
4. J.L. Johnson and K.V. Rajagopalan, Proc. Nat/. Acad. Sci. U. S. A. 79:6856 (1982).
5. M.E. Johnson and K.V. Rajagopalan, J. Bacterial. 169:117 (1987).
6. M.E. Johnson and K.V. Rajagopalan, J. Bacterial. 169:110 (1987).
7. D.M. Pitterle and K.V. Rajagopalan, J. Bacterial. 171:3373 (1989).

382
CLONING OF A EUKARYOTIC MOLYBDENUM
COFACTOR GENE

Puloma Kamdar, Michael E. Shelton,

and Victoria Finnerty

Department of Biology
Emory University
Atlanta, GA 30322

INTRODUCTION

Molybdenum requiring enzymes have been identified in virtually every species. All
molybdoenzymes, with the single exception of nitrogenase, require a molybdopterin cofactor
for catalytic activity. Mutations leading to a simultaneous loss of all molybdoenzyme
activities have been identified in organisms including, E.coli (1), higher plants such as
Nicotiana and Arabidopsis (2,3), as well as Drosophila (4,5), and humans (6). Such
mutations identify genes involved in the synthesis and/or activiation of the cofactor. Much
of the current interest in MoCF genes stems from the crucial roles of the molybdoenzymes.
One example is nitrate reductase, which is the key enzyme in nitrogen assimilation by plants.
At least 90% of the total nitrogen assimilated by plants is from the mineral nitrogen, mostly in
the form of nitrate (7). A mitochondrial molybdoenzyme, sulfite oxidase (SO) catalyzes the
terminal step in the oxidative degradation of sulfur containing amino acids and is
responsible for the detoxification of sulfite (8). Complete lack of sulfite oxidase activity
leads to a buildup of toxic sulfites which (perhaps combined with a deficiency in sulphates)
results in a severely debilitating human genetic defect involving abnormal neurological
development.
Although cofactor mutations have been identified in many eukaryotic organisms, there
is no information about their molecular structure or patterns of expression. The major
difficulty in studying the eukaryotic cofactor genes has been the lack of a reasonable strategy
for obtaining molecular clones. However, Drosophila provides an excellent model to
investigate the structure and function of the eukaryotic genes. There are four
molybdoenzymes activities in the fly: xanthine dehydrogenase, aldehyde oxidase, pyridoxal
oxidase, and sulfite oxidase (7). In Drosophila, three loci, maroon-like, low xanthine
dehydrogenase, and cinnamon have been identified as cofactor genes because these mutants
display a loss of two or more of the molybdoenzyme activities. The present study focuses on
the structure and pattern of expression of the cinnamon (cin) gene. Evidence that portions of
the cin gene may be conserved in eukaryotes will also be discussed.

Chemistry and Biology of Pteridines and Palates, Edtted by

J.E. Ayling et al., Plenum Press, New York, 1993 383
RESULTS
Mutations for cin are maternal effect zygotic lethals which map cytogenetically at the
tip of the X chromosome (9). A clone (TG-11), from an adjacenrlocus was used to walk
towards cin (10). The most distal cloned region is shown below as phage MES-1.

cin n:l
Distal Proximal
R R
I I
R
I
D/05•22•1 ~
I I I I I I I I
s H B s B H BH s B = llamiD
H = Hind III
R = EcoRI
••• TG 11··· S=SaJI

2.2 0.5 2.0 3.8 4.0

MES-1
Ht.okb

cDNAs
cM19B cM3SA cM17A cM2B

In order to focus on a possible cin coding region, EcoR 1 restriction fragments from MES-1
were used to select embryonic eDNA clones. The approximate location of 4 of these clones
based upon sequence data is depicted on the diagram. One mutant, cinill, carries a 400 bp
insertion as indicated. A deletion Df(l) 05-22-1, which does not complement cin, has its
proximal breakpoint within the cloned region, in the vicinity of the ciniTI insertion.
Therefore the illustrated genomic region is in or very near to cin.
When the cM17a eDNA is used as a probe on northern blots, the mutant ciniTl shows
a message approximately 400 bp larger than the 2.5 KB wild type mRNA. Another
mutant, cinC44, shows an apparent lack of that 2.5 KB message. These mutants thus served
to demonstrate that cM17a must represent at least part of the cin coding sequence.
To obtain a better understanding of the cin expression pattern, the cM17a eDNA was used to
probe a developmental northern blot of wild type mRNA. The results show a
developmentally regulated pattern of transcription for the 2.5 KB message, where
transcriptional activity is highest early in development. Transcription decreases in the later
embryonic stages and remains constant through the larval and pupal stages. There is
evidence of an additional, slightly smaller maternal message as expected for a gene which
functions during the ftrst few hours of embryonic development.
The cM17a eDNA was sequenced in order to obtain some clues concerning structural
features and possible function from its predicted amino acid sequence. The GenBank
database revealed homology with chlE, one of the E. coli MoCF genes (11 ). The region of
interest includes 117 contiguous residues. The homology is found in two regions.

Cin cM17a ~RNQ!-TPqiq FpS~TTMLTE 4-YYF9FNCMHTCVLSQSFQRTKESL~LFEVV Q~C!l<NY~MqDKpFV~VV,lD!-QF

E. coli chlE P~PWOOoiYi:irioo..AmWQWrnV INLG iirillnP~EAnSQmvVi:ssoovsvaiW¥rK:Tir:EaG-

Cin cM17a
.. ...... ...
RIHCGRVNIKPGKPMTFASRKDKYFFGLPGNPVSAFVTFH
.... .... . ...
.. ...........
E. coli chlE EIAFWKLAIKPGKPFA FGKLSNSWFCGLPGNPVSATLTF-

384
Region 1 is 77 amino acids, 24 identities and 19 conservative substitutions, totaling 55%
homology. Region 2 is 40 amino acids containing 20 identities and 6 conservative
substitutions, totaling 65% homology. Region 1 has two significantly hydrophobic regions,
while region 2 is strongly hydrophilic. Region 2 has 4 proline residues which are frequently
found in flexible regions and are important in stablizing the 3D conformation of proteins
(12). Such features lead us to speculate that this portion of the peptide may represent a
conserved binding site, possibly for a precursor of the molybdenum cofactor. Although the
homology does not necessarily imply that both genes carry out the same function, their
similar biochemical phenotype and lack of MoCF activity suggests that this may prove to be
the case (13).
Interestingly, cM17a is also homologous to a rat protein, gephyrin, which associates
with the mammalian inhibitory glycinergic receptor (GlyR) and binds tubulin (14). Gephyrin
apparently acts to cluster and anchor glycinergic receptors to the postsynaptic membrane.
The carboxy terminal region of homology between cin and gephyrin involves the same
residues as does the homology between cin and chlE.

Cin cM17a

Gephyrin
........... ..... .. .... .. .. ... ..... .. .... .. ..... ....... .. .. .......
VICSGGVSMGDKDFVKSVLE---DLQFRIHCGRVN IKPGKPMTFASRK-------DKYFFGLPGNPVSAFVTFH LFA LPAIR

I ITSGGVSMGEKDYLKQVLDI DLHAQIHFGRVFMKPGLPITFATI.DI DGVRKI I FALPGNPVSAVVTCNLFVVPALR

This region is 77 amino acids, 44 identities and 9 conservative substitutions, totaling 68%
homology. The underlined region is a putative membrane associated helix predicted by the
algorithm of Eisenberg et al (15). In the amino terminal region there is a second area of
homology which does not include homologies previously seen with the prokaryotic chlE,
suggesting that this could be a region important for the eukaryotic function.

Gephyrin
... .. . . . . . .. .... ................. .. .. . .
Cin cM17a ~~~KDI;QQ~~~~T!'~!?!'~~:::~~!RQu:'"!'<?QLSMYITI.E

IVPDE IEEIKET U DWCDEKELNLILTTGGTGFAPRDVTPEATKEVI EREAPGMALAMLMG

.- .. . ...... . ...... .. .....-......

..... ..
Cin cM17a SIKQ TQYAALSRGLCG!AGNTI.!LNLPGSEKAVKECFQTISALLPHAVHLI

Gephyrin SL1'iVTPLGMLSRPVCGIRGKTU INLPGSKKGSQECFQFILPALPHAI DLL

This region is 111 amino acids, 61 identities and 13 conservative substitutions, totaling 66%
homology. There are 6 conserved proline residues in this region, and it is possible that the
homology represents a structural conservation rather than a functional one.
Since the MoCF structure appears to be identical among eukaryotes it is reasonable to
expect that portions of those enzymes which bind the MoCF might also be conserved
(16,17). In order to explore this idea, the cM17a eDNA was used to probe southern blots
containing DNA from a variety of organisms (Figure 1). This "zooblot" analysis reveals
conservation at the DNA level in phylogenetically unrelated species such as yeast and
arabidopsis. This evidence, combined with the homology to chlE and gephyrin, suggests
that functionally important portions of the MoCF genes may be conserved. This indicates that
there may be future possibilities for using the information derivied from Drosophila to
understand the MoCF genes in other eukaryotic organisms.

385
 23.1-

9.4-

6.6 -

4.4-

2.3 -
2.0 -

Figure 1: Southern blot probed with cM17a. The filter was washed under high stringency conditions, i.e.,
wash in 2xSSC, 0.1% SDS at room temperature for 15 minutes, followed with a wash in 0.1xSSC, 0.1%
SDS at 65 C for 45 minutes. Yeast DNA restricted with BamHl. All other DNA was cut with EcoRl.

Acknowledgements
This research was supported by NSF grant MCB 8916915.

References
1. Johnson ME and RAjagopalan KV (1987) J Bacterial 169:117-125.
2. Mendel RR and Muller SJ (1979) Mol Gen Genet 117: 145-153.
3. Brassksma FJ and Feenstra WJ (1982) Physiol Plant 54: 351-360.
4. Warner CK, and Finnerty V (1981) Mol Gen Genet 184: 92-96.
5. Bentley MM and Williamson JH (1979) Can J Genet Cytol21: 457-471.
6. Johnson JL and Wadman SK (1989) In: Inherited Basis of Metabolic Disease. Ed. JB
Stanbury and JB Wyngaarden. New York, McGraw/Hill.
7. Warner RL and Kleinhofs A. (1992) Physiol Plant 85:245-252.
8. Rajagopalan KV and Johnson JL (1992) J Bioi Chern 267: 10199-10202.
9. Lefevre G (1981) Genetics 99:461-480.
10. Fleming RJ, DeSimone SM and White K (1989) Mol Cel Bioi 9: 719-25.
11. Nohno T, Kasai Y, Saito T (1988) J Bact 170:4097-4102.
12. Branden C and Tooze J (1991) Introduction to Protein Structure. Garland Publishing
Co., NY.
13. Stivaletta LA, Warner CK, Langley Sand Finnery V (1988) Mol Gen Genet 213: 505-
512.
14. Prior P, Schmitt B, Grenningloh G, Pribilla I, Multhaup G, Beyreuther K, Maulet Y,
WemerP,LangoschD,KirschJandBetzH (1992) Neuron 8:1161-1170.
15. Eisenberg D, SchwarzE, Komaraomy M and Wall R (1984) J Mol Bioi 179: 125-142.
16. Nason A, Lee KY, PanS, Ketchum PA, Lamberti A and DeVries J (1971) PNAS 68:
3242-3246.
17. Johnson Jl, Bastian NR, Rajagopalan KV (1990) PNAS 87: 3190-3194.

386
DESIGN AND SYNTHESIS OF INHffiiTORS OF FOLATE-DEPENDENT
ENZYMES AS ANTITUMOR AGENTS

Edward C. Taylor

Department of Chemistry, Princeton University

Princeton, New Jersey 08544

Tetrahydrofolate coenzymes play critical roles in a host of cellular one-carbon

transfer reactions. Among these are the de novo biosynthesis of both purine and
pyrimidine nucleotides (and thus both ATP and DNA), the methylation of homocysteine
to methionine, the interconversion of serine and glycine, and the catabolism of certain
amino acids. Dihydrofolate reductase (DHFR) plays a central role in the majority of
these metabolic reactions, for it is required for the regeneration of tetrahydrofolate from
dihydrofolate, and thus is responsible for maintaining a constant cellular supply of this
critical coenzyme.1 Inhibitors of DHFR thus have the potential to be potent
antiproliferative agents, since they interfere with many cellular processes critical for cell
growth. The deleterious consequences with respect to normal cells are certainly
responsible in part for the extreme toxicity of DHFR inhibitors such as methotrexate, and
severely limit their clinical usefulness in cancer chemotherapy. 2-6

Some years ago we undertook a program of chemical synthesis aimed at the

preparation of inhibitors of folate-dependent enzyme systems other than DHFR. One
such enzyme is glycinamide ribonucleotide formyltransferase (GAR FTase), which
mediates the conversion of glycinamide ribonucleotide to its N-formyl derivative (this
newly-introduced carbon atom eventually becomes the 8-carbon atom of the final purine
ring system). This formyl group is transferred from the cofactor for this process, 10-
formyl-5,6,7,8-tetrahydrofolate (note that the resulting deformylated cofactor is still in
the tetrahydro form, so regeneration of the 10-formyl derivative does not require DHFR
(Fig. 1). The inhibitor which we had designed at the time for the GAR-to-FGAR process
was 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF).7 The absence of nitrogen
atoms at positions 5 and 10 would make this compound incapable of conversion to
substrates which might serve as surrogate cofactors for any of the metabolic
transformations involving the normal folate cofactors; the absence of a 4-amino group
suggested that this compound would not be an inhibitor of DHFR; as a
tetrahydropyridine, DDATHF would be expected to be stable to oxidation; and its
structural resemblance to the natural folate cofactors suggested that it might be acceptable
as a substrate for normal folate transport systems, as well as for folylpolyglutamate
synthetase (Fig. 2).

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Aylmg et al., Plenum Press, New York, 1993 387
 As is well known by now, DDATHF proved to be the first representative of a new
class of extremely potent folate antimetabolites that are active antitumor agents due to
their effects on de novo purine biosynthesis. 8-12 Since DDATHF is not a DHFR
inhibitor, it is fully active against tumors resistant to methotrexate due to amplification of

The de novo Purine Biosynthesis Pathway

The GAR-to-FGAR Step

0
~OOH ~NHCHO

I
0
HN:J:~
I y"
NNH
ACONH-t-H
C~CH2COOHPO
~NH

FGAR
H2 N~N N) HO OH
H

Purines
Fig.l

the DHFR gene.9,13 The remarkable effectiveness of DDATHF as a chemotherapeutic

agent results from a combination of factors which include its efficient conversion within
the cell to polyglutamates by folylpolyglutamate synthetase (FPGS), leading to its
retention and slow release by the kidney and liver, and its ability to enter cells by
pathways involving both the reduced-folate and the folate-binding membrane protein
transport systems. DDATHF is now in Phase 2 clinical trials; initial results of some of the
Phase 1 trials have been published.14

The focus of my lecture today is not, however, the antitumor profile of DDATHF,
but rather some of the chemistry involved in its initial and subsequent syntheses, our
explorations of structure-activity relationships which have led to some fascinating new
heterocyclic chemistry, and the preparation, as a consquence of these SAR studies, of a
new and promising inhibitor of thymidylate synthase which is now in Phase I clinical
trials.

388
 ~OOH

oNH-f-H
CH2CH 2COOH

5,10-Dideaza-5,6,7,8-tetrahydrofolic Acid (DDATHF, Lometrexol)

Fig. 2

Since much of our early synthetic work has already been summarized
elsewhere,7' 15 I will mention here only one of our much improved DDATHF
syntheses,16 since it will serve to introduce a methodology based on palladium chemistry
which has proven to be exceptionally versatile in solving a host of diverse synthetic
problems. Thus, the key feature in this new and much shorter synthesis of DDATHF
(Fig. 3) is a palladium-catalyzed C-C bond coupling reaction - a kind of modified "Heck"
reaction 17 - which we used to form both the C6-C9 bond and the C10-aryl bond. We
started with malondialdehyde tetramethylacetal, which was first hydrolyzed with HCl to
the dialdehyde, and then brominated to give bromomalondialdehyde, which was
condensed directly with 2,4-diamino-6(1H)-pyrimidinone. The resulting very insoluble
product was then pivaloylated. This was a critically important step, for the pivaloyl
protecting/acylating group confers remarkable solubility upon an otherwise almost totally
intractable, rock-like substance.l8 Palladium-catalyzed coupling of this 6-bromo-2-
pivaloyl-5-deazapterin with trimethylsilylacetylene was followed by a second palladium-
catalyzed coupling, this time with diethyl 4-iodobenzoylglutamate. The triple bond and
the pyridine ring were then reduced in a single step, and the protecting groups were
removed to give DDATHF. The subsequent resolution of DDATHF into its 6R- and 6S
diastereomers, and the selection of the former for clinical trials, have been fully described
elsewhere.11

We have now prepared and evaluated a great many analogs of DDATHF, with
variations in Rings A and B, in the bridge region joining the tetrahydro-5-deazapterin and
benzoyl moieties, and in the benzoyl aromatic ring itself. Today I have selected from
these many analogs only a few which, from the point of view of a synthetic organic
chemist and long-time pteridinologist, are of particular interest because of the synthetic
methodologies developed for their synthesis.

389
 A New Synthesis of DDATHF
The Palladium-mediated C-C Coupling Approach

MeOYYOMe H+, thenBr1 0~Br

MeO OMe H.)

H,Nl)J
pivalic anhydride I
pyridine f
0
TMS-acetylene HN~ Br
PdC)z[Ph3Ph
Cui, then KF M~CCONH
h...NJl.)
N

0 _ ~OOEt

HN~c=cQ-coNH .. ~·H
h...Jl.) CH CH C00Et 2 1
Me 3CCONH N N

(HI~ CONH- !•H

COOEt

CH1CH1COOEt

Dr. GeorgeS. K. Wong

DDATHF
Fig.3

For example, we were particularly interested in the incorporation into the

DDATHF structural framework of a substituent at the folate C-10 position. The natural
cofactor for the GAR FTase enzyme is 10-fonnyltetrahydrofolate, and a potentially more
effective inhibitor than DDATHF itself, with its unadorned bridge, would be an analog
bearing a functionalized substituent at C-10 which might simulate the formyl grouping in

390
the natural cofactor. We have outlined in Fig. 4 a synthesis of the 10-hydroxymethyl
analog of DDATHF, 19 and it is noteworthy because of the two Pd-

+
HC:CCH:z()THP
/

COOEt
ONH~H
CH2CH:zCOOEt

COOEt
ONH~H
CH2CH 2COOEt

COOH
ONH- ~ •H
CHzCHzCOOH

Cheol-min Yoon Fig. 4

catalyzed C-C coupling reactions utilized in its preparation. Thus, the ubiquitous 6-
bromo-2-pivaloyl-5-deazapterin 18a was condensed with the tetrahydropyranyl-protected
derivative of propargyl alcohol, using palladium chloride/triethylamine/triphenyl-

391
phosphine/cuprous iodide in acetonitrile as solvent. Propargyl alcohol itself failed to
undergo this coupling reaction using a variety of palladium catalysts, bases and
phosphorus ligands. Reduction of the alkyne to the ill-alkene was carried out
satisfactorily with hydrogen in the presence of palladium/barium sulfate using freshly
distilled synthetic quinoline as the catalyst poison. These conditions were not our first
choice, but it turned out that other poisoned catalysts, normally used for the controlled
reduction of alkynes, were totally ineffective. The resulting cis-olefin was then coupled
with diethyl 4-iodobenzoylglutamate in the presence of palladium acetate, and the
coupled product was subjected to catalytic reduction, without significant allylic
hydrogenolysis, using platinum oxide as the catalyst and glacial acetic acid as the solvent.
Acidic removal of the THP protecting group and base removal of the pivaloyl and ester
groupings then gave 10-hydroxymethyl-DDATHF.

Although we have not pursued this compound any further, it is worth noting that
the mixture of four diastereomers produced in the above process was an extremely potent
cytotoxic agent, with an IC50 of 0.005 11g/ml. In earlier work we had prepared 10-
methyl-DDATHF, again as a mixture of four diastereomers, and although it was about
10-fold less reactive than the above 10-hydroxymethyl derivative, all of its cytotoxic
activity was apparently due to only one of the four diastereomers.20 This may also be the
case for 10-hydroxymethyl-DDATHF, which would make it the most active analog we
have thus far prepared. At this point, however, we have not attempted a separation of the
four diastereomers.

Homo-DDATHF, in which the bridge has been extended by one methylene group,
presented a different synthetic challenge. Our first synthesis of this analog, which proved
to be fully as active as DDATHF itself, is outlined in Fig. 5.21 We started with a
palladium-catalyzed C-C coupling between 1-butyn-4-ol and methyl4-bromobenzoate.
Reduction of the triple bond and pyridinium chlorochromate (PCC) oxidation of the
primary alcohol gave the 4-arylbutyraldehyde derivative shown. This was converted to
its Knoevenagel condensation product with malononitrile. In contrast to the starting
aldehyde, which could not be formylated under a wide range of reaction conditions, this
1,1-dicyanomethylene aldol product now possesses a much more acidic a-methylene
group, and can readily be formylated with triethyl orthoformate. Condensation of this
intriguing intermediate, which is in effect an activated malondialdehyde equivalent, with
2,4-diamino-6(1H)-pyrimidinone led directly and in excellent yield to the 5-deazapterin
derivative shown here. This ring-annulation product was then converted by a series of
standard operations to homo-DDATHF.

The course of this perhaps unexpectedly facile ring annulation deserves special
comment. A suggested mechanism is shown in Fig. 6. The concept involved is one
which we termed "carbonyl group activation". The first step is Michael addition by the
nucleophilic 5-position of the pyrimidine ring (as a primary enamine) to the
malondialdehyde equivalent, and it should be noted that replacement of -CH=O by
-CH=C(CN)z certainly enhances the electrophilic activity of the former aldehyde carbon.
Elimination of ethanol then leads to a substrate for another Michael addition, this time by
the pyrimidine-4-amino group to the a,P-unsaturated dinitrile. Once again, replacement
of -CH=O by a -CH=C(CN)z unit has facilitated the second Michael addition. Aroma-
tization then results from loss of malononitrile, which therefore, at least in principle,
could be viewed as a catalyst for this remarkably facile ring annulation process.

A conceptually different route to homo-DDATHF is shown in Fig. 7. Here we

utilized the oxazoline shown at the upper left, which had been prepared earlier by A. I.
Meyers from the Grignard reagent derived from 2-(4-bromophenyl)-4,4-

392
 Synthesis of HomoDDATHF (HDDATHF)
Route 1

_
HO(CH2hC: CH + Br
-o--.:::
""""
C02Me
___...
HO(CH:i) 3C :c
- o - C 02Me
-'7

COOH
CONii• : .. H
CH2CH2COOH
HDDATHF
PhiUip M. Harrington
Fig.S

Phillip M. Barrington Fig. 6

393
dimethyloxazoline and allyl bromide.22 This readily available intermediate was
subjected to acidic hydrolysis to reveal the corresponding benzoic acid, which was then
converted to the peptide with diethyl L-glutamate via its acid chloride, using
DMAP!Et3N. Carbon-carbon coupling with 6-bromo-2-pivaloyl-5-deazapterin, using (Q-
tolyl)3P, Pd(OAc)2, Cui and Et3N in acetonitrile, gave a mixture of cis- and trans-olefins
which was converted to homo-DDATHF by reduction and subsequent hydrolysis.23

Synthesis ofHomoDDATHF (HDDATHF)

Route 2

COOEt
CONH. . ~•H
.f.
CH2CH2COOEt

COOH
CONH~H
~H2 CH~OOH
HDDATHF

Dr. Joe Shih (Lilly)

PhillipM. Harrington
Fig. 7

Homo-DDATHF was almost an order of magnitude more potent as an inhibitor of

GAR FTase than DDATHF itself, although it exhibited about the same cytotoxicity (ICso
= 0.008 Jlg/ml) as DDATHF (ICso =0.007 J.Lg/ml) in the whole-cell assay (human T-
cell-derived lymphoblastic leukemia cells, CCRF-CEM). This decrease in cytotoxicity as
compared to its enzyme inhibitory activity may possibly be attributed to factors such as a
decrease in membrane transport, or perhaps insufficient intracellular polyglutamation,
both of which play important roles in eliciting the biological activity of antifolates.

394
 Synthesis ofNor-DDATHF
0

)Jr
HN
~
I
MejXONH :o..N

o H},to
HN~
e~CONH~NJlNJ
H

0 + 0

HN~I
~--Jl __ ) Me~CONH
~~
HNj(ro_l ~ CON~H
~OOEt
Me3CCONH N N N N .& .:
H H CH2 CH2COOEt

0 t
HN~ COOEt

Me~CONH
~,.)l""'~
N N U-coN~H
.:
CH 2CH 2COOEt

t
Nor-DDATHF

Dr. Joe Shih (Lilly)

Phillip M. Harrington
Fig. 8

Since the addition of a methylene group to the bridge region of DDATHF had not
altered its biochemical profile, what would be the consequence of removing a methylene
group? Fig. 8 outlines our synthesis of what we have termed "nor-DDATIIF".23 Once
again, the synthesis starts with 6-bromo-2-pivaloyl-5-deazapterin. Palladium-catalyzed

395
coupling with styrene led to the styryl derivative shown, which was subjected to
ozonolysis to give the corresponding 6-carboxaldehyde18a (our intermediate for an
earlier synthesis of 5-deaza-5,6,7,8-tetrahydrofolic acid, 5-DATHF18c). This compound
was converted in a single step to the novel exocyclic olefin shown here by treatment with
sodium cyanoborohydride in a 1: 1 mixture of acetic acid and ethanol. We assume that
this exocyclic olefin was formed through a reductive elimination process involving a
[3.3]-sigmatropic rearrangement of a presumed alkoxyborohydride intermediate.
Palladium-mediated coupling with diethyl 4-iodobenzoylglutamate gave the fully
aromatized nor-DDA THF precursor shown, and this was reduced and deprotected to give
our target compound. This modification in the DDA THF skeleton was disastrous for
biological activity, for this one-carbon bridged analog proved to be at least 4-5 fold less
potent than DDATHF, both in enzymatic inhibition and in whole-cell cytotoxicity.

As we have pointed out before, the asymmetric center at C-6 in DDATHF, which
had been introduced by catalytic reduction of the pyridine ring, posed a special problem
when DDATHF was initially synthesized and developed. The two diastereomers had to
be separated by a novel fractional crystallization process through a crystalline d-1 0-
camphorsulfonate diethyl ester salt intermediate.24 The disadvantages of this process are
obvious in that the resolution could be undertaken only at a late stage of the synthesis,
and it was also relatively inefficient. It therefore seemed attractive to try to bypass the
problems associated with the introduction of this C-6 chiral center by eliminating it
completely, and our initial approach was to delete either the 5- or the ?-methylene
grouping to give a series of what we have termed "open-chain" DDATHF analogs (see
Fig. 9).

"Open-Chain" Ring B Analogues ofDDATHF

"C-5 desmethylene"
COOH
CONH--+-H
=
CH2 CH 2COOH

"C-7 -desmethylene"

Fig. 9

Our general synthetic approach to a series of 7-desmethylene "open-chain"

analogs (which are now single enantiomers) is outlined in Fig. 10, and we have used this
strategy for the preparation of a large number of analogs with differing substituents on the
pyrimidine ring and the bridge region, and with variously substituted aryl and
heterocyclic rings replacing the phenyl ring.25-27 Once again, the ubiquitous C-C Pd-
catalyzed coupling approach was used to prepare the starting mesyloxy derivative.
Coupling of butyn-4-ol with methyl 4-bromobenzoate, a reaction we have mentioned

396
earlier, gave the alkyne shown, and this was reduced, mesylated, and the latter derivative
then used to alkyl ethyl cyanoacetate. Condensation of this a-substituted cyanoacetate
with guanidine in DMF at room temperature resulted in formation of a 5-substituted
pyrimidine which could be easily converted on to our "open-chain" DDATHF analog.
This intriguing simplified DDATHF analog, which is clearly much less rigid than
DDATHF itself, was a surprisingly good inhibitor of GAR FTase as shown by reversal
studies, although it was some 8-fold less inhibitory against CCRF-CEM leukemic cells
than DDATHF. Its inhibitory activity against GAR FTase, taken together with previous
work which showed that replacement of the -NH grouping of DDATHF with a methylene
group resulted in a 100-fold increase in Ki, certainly emphasizes the importance of a
hydrogen bond between the N-8 hydrogen ofDDATHF, or the almost equivalent amino
group in the 4-position of this "open-chain" analog, with an active site residue on GAR
FTase.

Related "open-chain" analogs have received considerable attention from other

research groups; particular mention should be made of the Burroughs-Wellcome open-
chain analog (which they have termed 5-DACTHF) of our 5-deaza-5,6,7 ,8-tetrahydrofolic
acid.28

"C-7 Desmethylene Open-chain" Analogue ofDDATHF

COOH
CON~H
i.
CH2CH2COOH

Phillip M. Harrington

Fig.lO

Encouraged by the activity shown by so many of these 7-desmethylene "open-

chain" analogs of DDATHF, we prepared a number of representatives of the isomeric
series of 5-desmethylene derivatives (Fig. 9) through the reaction of 2-pivaloylamino-4,6-
dichloropyrimidine with the appropriate amino intermediates.29 Perhaps surprisingly,
these "open-chain" derivatives were almost completely devoid of cytotoxic activity, and

397
thus stand in remarkable contrast to the highly active isomeric 7-desmethylene "open-
chain" series.

Although we did not realize it at the time, we had reached an unexpected turning
point in our investigations of potential antifolates. It has already been emphasized that
the penultimate step in the majority of our DDATHF syntheses involved reduction of the
aromatic pyridine ring of our synthetic intermediates. This ring reduction results in the
formation of a mixture of two diastereomers which differ in chirality at C-6. Both the 6-S
and 6-R diastereomers (the latter was selected for clinical trial) were potent and nearly
equiactive inhibitors of de novo purine biosynthesis, 12 althou~h they possessed slightly
different activities towards different neoplasms in vivo. 0 It was, of course, of
considerable interest that the above enantiomerically homogeneous "open-chain"
DDATHF analogs, in which the C-6 chiral center had been removed by excision of the C-
7 methylene group, were almost as active as DDATHF itself in vitro. Since studies of
model compounds had previously revealed that an -NH grouping attached to the
pyrimidine C-4 position was mandatory for activity against GAR FTase, 11 we were
particularly interested in examining analogs possessing the following characteristics: (1) a
left-hand heterocyclic moiety which would simulate the rigidity of the bicyclic 6-6 ring
system of DDATHF, (2) the hydrogen-bonding -NH grouping corresponding to the
pyrimidine 4-amino group or the 8-NH of DDATHF, but (3) no chiral center at the
position joining the A/B rings to the ethano bridge. Since the DDATHF precursor with
an aromatic pyridine ring was completely inactive as a cytotoxic agent (it does not
possess an NH grouping at position 8), we were left with consideration of possible 6-5
ring systems.

Results obtained from our first choice of a purine ring system were not
encouraging. The guanine analog ofDDATHF was prepared31 as shown in Fig. 11 from
dimethyl 4-ethynylbenzoylglutamate (an intermediate utilized by us earlier in one of our
DDATHF syntheses16), and 2-pivaloyl-8-bromoguanine, which was available by
bromination of 2-pivaloylguanine with bromine in acetic acid. Although only
dimerization of the acetylene was observed under the usual coupling conditions utilizing
palladium chloride/cuprous iodide/triphenylphosphine and triethylamine, we found that
smooth coupling could be achieved using palladium acetate rather than the chloride, and
1.9 equivalents of the acetylene. The coupling product was then reduced catalytically,
and the protecting groups were removed under basic conditions. This guanine analog of
DDATHF turned out to be essentially inactive as a cell growth inhibitor. In passing, it
should be noted that nrnr studies on the penultimate pivaloylated dimethyl ester showed it
to exist as a 2:1 ratio of the 9-NH and the 7-NH tautomers.

Our next attempt to satisfy the criteria defined above for a DDATHF analog
lacking a chiral center at the point of attachment of the ethano bridge, but preserving both
the rigidity of the bicyclic ring system of DDATHF, and the critically important ring B
-NH grouping, turned out to be a striking and very surprising success which has since
dominated our research directions.32 Our objective this time was the pyrrolo[2,3-
!;!Jpyrimidine analog shown in Fig. 12.

398
 Contraction of Ring B to an Imidazole - A Guanine
Analogue of DDATHF

~OOMe COOMe
• ~CONH-1-H CONH_l_H
HC2CSiMl!:J +~ CHzCH2C~OMe ~ ~HzCHzCOOMe
1 Hc:c~

HNCN
k __ jl __ .!l
Me~CONH N N C2C-0 COOMe
~
IH
CONH-+-H
11

HN\!-N l:u,cu,cooMe

HNkNJlN~
2 H llA ~OOH
CONH-:-H
!
Dr. Zen-yu Chang CH 2CHzCOOH
Dr. Dietmar Kuhnt

Fig.ll

Structure ofLY231514
~OOH
CONH- A•H
CH2CH2COOH

Fig.12

399
 Contraction of Ring B to a Pyrrole - Synthesis
ofLY231514

NCCH 2 CO~t + BrCH 2CH(OEth Et02C)/" CH(OEth

NC

0 +
HN~CH2CH(OEth
H2N
h_Jl
N NH2

COOH
CONH-+-H
!
CH2CH2COOH

LY231514

Dr. Dietmar Kuhnt

Fig.13

The left-hand 2-amino-4(3Jj)-oxo-7H-pyrrolo[2,3-.d]pyrimidine (7-deazaguanine)

moiety was prepared in a straightforward manner starting with alkylation of ethyl

400
cyanoacetate with bromoacetaldehyde diethyl acetal in the presence of potassium
carbonate (Fig. 13).33 The resulting a-alkylated cyanoacetate was cyclized with
guanidine/sodium ethoxide to a 2,4-diamino-6(1H)-pyrimidinone, which was then treated
with 0.5 N hydrochloric acid to effect hydrolysis of the acetal protecting group, and acid-
catalyzed cyclization of the resulting aldehyde with the pyrimidine 4-amino group.
Pivaloylation was then followed by treatment with 2.2 equivalents ofN-iodosuccinimide,
which gave the 5,6-diiodo derivative. This could be regioselectively monodeiodinated
with zinc in acetic acid to the desired 2-pivaloylamino-5-iodo-4(3H)-oxo-7H-pyrrolo[2,3-
.d]pyrimidine (more conveniently referred to as 2-pivaloyl-7-iodo-7-deazaguanine). This
compound was then coupled, under the usual conditions of palladium catalysis, with
dimethyl4-ethynylbenzoyl-L-glutamate. Selective hydrogenation of the triple bond was
achieved with hydrogen and 3% palladium-on-charcoal catalyst in a mixture of
methylene chloride and methanol. The target pyrrolopyrimidine was obtained in the
usual way by sodium hydroxide deprotection and saponification.

To our delight, this compound proved to be an extremely active inhibitor of tumor

growth, both in vitro and in vivo. To our surprise, however, it proved to be almost totally
inactive against GAR FTase. Instead, reversal studies showed that this new compound
was exhibiting its cytotoxic effect due to inhibition of thymidylate synthase!

Although details of the biological evaluation of this compound (now known as

LY231514) have been published,32 I show in Fig. 14, for purposes of emphasis, a
comparison of the antitumor activity of this new pyrrolopyrimidine antifolate with that of
the well-known quinazoline TS inhibitor, CB3717, against the L5178Y/TK_/HX_ tumor.
The indicated doses of LY231514 and CB3717 were administered to groups of mice
bearing a subline of the L5718Y lymphoma lacking expression of thymidine and
hypoxanthine-guanine phosphoribosyltransferase. Complete inhibition of tumor growth
by LY231514 was achieved, as can be seen, at doses from 12.5 mg/kg to 200 mg/kg, with
an excellent therapeutic index. By comparison, CB3717 was almost inactive at its
highest tolerated dose. Our pyrrolopyrimidine also turned out to be very effective against
two human colon xenografts, and has now entered clinical trials.

100

80
c
.2
:e
.J:
60
E

c;;d
E
Q)
40
~
Q)
c..
20

0
i i0 iOO
Dose (mg/kg/day)

Fig.14

401
 It may be worth speculating at this point about the reasons for this remarkable
turn of events, whereby a simple change in structure of ring B of DDATHF has switched
the target of inhibition almost completely from GAR FTase to TS. The well-known
reaction pathway for the conversion of dUMP to dTMP is mediated by thymidylate
synthase, which requires the folate coenzyme 5,10-methylene-5,6,7,8-tetrahydrofolate as
the one-carbon donor and the reducing agent. Modeling studies suggest a surprising and
non-obvious structural similarity between our new pyrrolopyrimidine inhibitor and the
natural cofactor, although an alternative working hypothesis is that LY231514 is binding
to TS as a structural analog of dihydrofolate, thus preventing recycling of the cofactor
through eventual reduction by DHFR.

Synthesis of the C-6 Regioisomer of LY231514

~OOH
CONH-t-H
Dr. Rajendra Chaudhari
CH2 CH2COOH
Dr. Hemantkumar H., Patel
Wendy B. Young
Fig.15

It will of course be noted that the -CH2NH-bridge in dihydrofolate is separated

from the pyrimidine ring by two atoms (N-5 and C-6), but that the ethano bridge in
LY231514 is separated from the pyrimidine ring by only one atom. It was therefore of
considerable interest to examine the isomeric 6-substituted 2-amino-4(3.H)-oxo-7H-
pyrrolo[2,3-g]pyrimidine (8-substituted 7-deazaguanine) isomer ofLY231514 in which
the -CH2CH2- bndge remains connected to the pyrrolopyrimidine ring system through an

402
sp2 carbon, but in which two atoms now separate the bridge from the pyrimidine ring, as
in dihydrofolate itself. This isomeric pyrrolopyrimidine derivative was prepared from 4-
carbomethoxycinnamic acid as shown in Fig. 15. Catalytic reduction to the P-
arylpropionic acid, conversion to its acid chloride, and treatment with diazomethane
followed by hydrochloric acid gave the chloroketone shown. This was condensed with
2,4-diamino-6(1H)-pyrimidinone in a mixture of sodium acetate and methanol. The
resulting 6-substituted 2-amino-4(3B.)-oxo-7B.-pyrrolo[2,3-d]pyrimidine (8-substituted 7-
deazaguanine) was then converted by hydrolysis to the carboxylic acid, followed by
peptide formation and eventual saponification, to the 6-isomer of LY23154. Despite its
(perhaps far-fetched) structural resemblance to dihydrofolate, this compound turned out
to be an extremely poor inhibitor of cell growth, and to be essentially inactive both
towards TS and GAR FTase.

Synthesis of the 5,6-Dihydro Derivative of the

6-Regioisomer of LY231514

CH2 CH:zCOOEt
0

COOH
CONH• I·H
Dr. Carsten Spanka CHzCH:zCOOH
Wendy B. Young Fig.16

However, suppose the pyrrole ring in this inactive isomer were now reduced? In
this case, a formerly inactive compound would now be converted into an analog of
DDA THF in which the 7-methylene group of the latter had been excised, and C-6 joined

403
to N-8. This new potential DDATHF analog was successfully prepared as shown in Fig.
16. 2-Pivaloylamino-4(3H)-oxo-7H-pyrrolo[2,3-.d]pyrimidine (2-pivaloyl-7 -deazaguan-
ine) was converted to its 7-ethoxycarbonyl derivative with three equivalents of sodium
hydride and one equivalent of ethyl chloroformate. In contrast to 2-pivaloylamino-4(3H.)-
7!!-pyrrolo[2,3-d.]pyrimidine itself, this ?-protected derivative underwent smooth
monobromination with N-bromosuccinimide at 0 °C in methylene chloride to give
exclusively the 6-bromo derivative. Subsequent coupling, reduction of both the triple
bond and the pyrrole ring, and final deprotection completed this synthesis of the 5,6-
dihydro derivative of the 6-isomer ofLY231514.

Biological evaluation now showed that we once again had in hand an active
antitumor agent, and reversal studies revealed that the target of inhibition was once again
GAR FTase. In other words, this compound is indeed a DDATHF analog, as predicted.
On the other hand, the dihydro derivative of LY231514, which was also an active
cytotoxic agent, was shown to be targeted against TS, since reversal studies indicated that
its cytotoxicity was reversed by thymidine. 35 It can thus be seen that very minor changes
in structure can determine whether the target of inhibition is TS or GAR FTase.

We have, of course, been very interested in the consequences of replacing the

pyrrole N-H grouping with other heteroatoms. Today I will mention only one attempted
excursion into such systems which led to an unexpectedly facile ring transformation with
very practical consequences. The corresponding fused furan was an obvious candidate,
and we attempted the preparation of such an analog by the route shown in Fig. 17. A
palladium-catalyzed reaction of allyl alcohol with ethyl4-iodobenzoate led directly to the
~-arylpropionaldehyde shown. This compound was smoothly converted in a single
operation to the corresponding hydroxymethylketone by reaction with formaldehyde in
the presence of triethylamine and 1-ethylbenzothiazolium bromide. Condensation of this
hydroxymethylketone with malononitrile then gave the expected 2-amino-3-cyano-4-
substituted furan intermediate.

There are innumerable examples in the literature of the conversion of Q.-

aminonitriles to fused 2,4-diaminopyrimidine systems by reaction with guanidine.34
Reaction of our Q-aminonitrile under the standard guanidine cyclization conditions,
however, led directly and in excellent yield to a pyrrolo[2,3-d.]pyrimidine, NOT to the
expected furo[2,3-Q]pyrimidine. It will certainly be noted that this ring transformation/-
annulation sequence represents a simple synthetic approach to Takeda's 2,4-diamino-
pyrrolo[2,3-g]pyrimidine DHFR inhibitors. 36

Although we have no definitive evidence on the mechanism of this intriguing

transformation, several possible pathways can be suggested. As outlined in Fig. 18,
initial Michael addition of guanidine to the 2-position of this furan Q-aminonitrile could
be followed not by loss of ammonia, as in the customary Q-aminonitrile/guanidine ring
annulation reactions, but rather by ring opening to generate an intermediate aldehyde
which then closes back on what was originally the 2-amino group of the starting furan. A
subsequent intramolecular amine-to-nitrile addition then completes formation of the
observed 2,4-diamino-5-substituted-7H-pyrrolo[2,3-g]pyrimidine (2,6-diamino-7-
substituted-7-deazaguanine). Alternatively, deprotonation of the furan 2-amino grouping
by guanidine could be followed by ring opening to a malonontrile derivative which
should then undergo a normal pyrimidine/pyrrole ring annulation sequence.

404
 Attempted Synthesis of the Furan Analog of LY231514
A Remarkable Ring Transformation/Annulation Sequence

C02Et
+ ~OH
I

CH 2(CN)z
base

EtO~anidine
C02Et

Dr. Hemantkumar H. Patel

Fig.17

405
 a NC R

H2 NC(=NH)NHz~ H N O
~
a

~2
b

guanidine
...
Fig.l8

In closing, I would like to pay a sincere and grateful tribute to my many dedicated
and talented students and collaborators whose work I have summarized today. The
names of those who have carried out the synthetic work are given in the Figures; a listing
of all those whose earlier efforts contributed so much to the development of DDATHF is
given in ref. 7. The important contributions of Prof. G. Peter Beardsley to the very early
stages of this project are also gratefully acknowledged. I cannot emphasize too strongly
the many vital contributions from my associates at Eli Lilly & Company, and in particular
Drs. Joe Shih and Charles J. Barnett in the area of organic chemistry, and Dr. Gerald B.
Grindey, who has been responsible for all of the pharmacology and pre-clinical
evaluation of our synthetic antifolates. All of the FPGS evaluations and many related
biochemical studies on these new antifolates were carried in collaboration with Prof.
Richard G. Moran of USC.

References

1. R. L. Blakley, "The Biochemistry of Folic Acid and Related Pteridines," North-

Holland Publishing Co., Amsterdam (1969).
2. F. M. Sirotnak, J. J. Burchall, W. B. Ensminger, and J. A. Montgomery, eds., "Folate
Antagonists as Therapeutic Agents, Vols. 1 and 2," Academic Press, New York (1984).
3. F. M. Sirotnak, Prog. Cancer Res. Ther. 28:77 (1984).
4. D. W. Fry and R. C. Jackson, Cancer Metastasis Rev. 5:251 (1987).
5. J. I. DeGraw, P. H. Christie, W. T. Colwell, and F. M. Sirotnak, J. Med. Chern. 35:320
(1992).

406
6. For extensive reviews of the chemistry of folic acid, aminopterin, methotrexate and
analogs as antitumor agents, see: (a) D. C. Palmer, J. S. Skoticki, and E. C. Taylor,
"Progress in Medicinal Chemistry," G. P. Ellis and G. B. West, Eds; Elsevier Science
Publishers; Holland (1988); Vol. 25, p. 85. (b) Rosowsky, A. "Progress in Medicinal
Chemistry," G. P. Ellis and G. B. West, Eds; Elsevier Science Publishers; Holland
(1989); Vol. 26, p. 1.
7. For leading references and a full discussion of the background and development of
DDATHF, see E. C. Taylor, J. Heterocyclic Chem. 27:1 (1990).
8. R. G. Moran, E. C. Taylor, and G. P. Beardsley, Proc. Amer. Assoc. Cancer Res.
26:231 (1985).
9. G. P. Beardsley, E. C. Taylor, C. Shih, G. A. Poore, G. B. Grindey, and R. G. Moran,
Proc. Amer. Assoc. Cancer Res. 27:259 (1986).
10. G. P. Beardsley, B. A. Moroson, E. C. Taylor, and R. G. Moran, J. Bioi. Chem.
264:328 (1989).
11. S. W. Baldwin, A. Tse, L. S. Gossett, E. C. Taylor, A. Rosowsky, C. Shih, and R. G.
Moran, Biochemistry 30:1997 (1991).
12. R. G. Moran, S. W. Baldwin, E. C. Taylor, and C. Shih, J. Bioi. Chem. 264:21047
(1989).
13. G. Pizzomo, B. A. Moroson, A. R. Cashmore, E. C. Taylor, and G. P. Beardsley,
Proc. Amer. Assoc. Cancer Res. 29:281 (1988).
14. (a) C. Young, V. Currie, L. Balder, B. Trochanowski, 0. Eton, R. Dyke, and R.
Bowsher, Proc. Amer. Assoc. Cancer Res. 31:177 (1990). (b) R. Nelson, F. Butler, W.
Dugan, Jr., C. Davis-lung, M. Stone, and R. Dyke, Proc. Amer. Soc. Clin. Oncol. 9:293
(1990). (c) 0. Pagani, C. Sessa, J. deJong, H. Kern, S. Hatty, H. Schmitt, and F. Cavalli,
Proc. Amer. Assoc. Clin. Oncol. 11:109 (1992).
15. E. C. Taylor, P. J. Harrington, S. R. Fletcher, G. P. Bearsley, and R. G. Moran, J.
Med. Chem. 28:914 (1985).
16. E. C. Taylor and G. S. K. Wong, J. Org. Chem. 54:3618 (1989).
17. R. F. Heck, "Palladium Reagents in Organic Syntheses," Academic Press, Orlando,
FL (1985).
18. See, for example, (a) E. C. Taylor and C.-m. Yoon, Synth. Commun. 18:1187 (1988).
(b) E. C. Taylor and P. S. Ray, J. Org. Chem. 52: 3997 (1987). (c) E. C. Taylor, J. M.
Hamby, C. Shih, G. B. Grindey, S.M. Rinzel, G. P. Beardsley, and R. G. Moran, J. Med.
Chem. 32:1517 (1989).
19. C.-m. Yoon, Ph.D. Thesis, Princeton University (1989).
20. E. C. Taylor, G. S. K. Wong, S. R. Fletcher, P. J. Harrington, G. P. Beardsley, and C.
J. Shih, "Chemistry and Biology of Pteridines," B. A. Cooper and V. M. Whitehead, Eds.;
de Gruyter, Berlin (1986), pp. 61-64.
21. E. C. Taylor and P.M. Harrington, J. Org. Chem. 55:3222 (1990).
22. A. I. Meyers, Ace. Chem. Res. 11:375 (1978).
23. C. Shih, G. B. Grindey, E. C. Taylor, and P.M. Harrington, Biorg. Med. Chem. Lett.
2:339 (1992).
24. This separation, the first of its kind in folate chemistry, was developed by Dr. C. J.
Shih of Eli Lilly & Company; see ref. 1.
25. E. C. Taylor, P.M. Harrington, and C. Shih, Heterocycles 28:1169 (1989).
26. C. Shih, L. S. Gossett, J. F. Worzalla, S.M. Rinzel, G. B. Grindey, P.M. Harrington,
and E. C. Taylor, J. Med. Chem. 35:1109 (1992).
27. E. C. Taylor, T. H. Schrader, and L. D. Walensky, Tetrahedron 48:19 (1992).
28. (a) E. C. Bigham, S. J. Hodson, W. R. Mallory, D. Wilson, D. S. Duch, G. K. Smith,
and R. Perone, J. Med. Chem. 35:1399 (1992). (b) J. L. Kelley, E. W. McLean, N. K.
Cohn, M. P. Edelstein, D. S. Duch, G. K. Smith, M. H. Hanlon, and R. Perone, J. Med.
Chem. 33:561 (1990). (c) G. K. Smith, S. D. Banks, E. C. Bigham, N. K. Cohn, D. S.
Duch, M.P. Edelstein, R. Perone, M. H. Hanlon, L. S. Heath, J. Humphreys, J. L. Kelley,
V. Knick, E. W. McLean, R. J. Mullin, S. Singer, H. R. Wilson, and J. Houghton,

407
"Chemistry and Biology of Pteridines, 1989," H.-Ch. Curtius, S. Ghisla, and N. Blau,
Eds.; de Gruyter, Berlin (1990), pp. 1015-1022.
29. E. C. Taylor, P. Gillespie, and M. Patel, J. Org. Chern. 57:3218 (1992).
30. C. Shih, G. B. Grindey, P. J. Houghton, and J. A. Houghton, Proc. Arner. Assoc.
Cancer Res. 29:283 (1988).
31. E. C. Taylor, D. Kuhnt, and Z.-y. Chang, J. Org. Chern. 56:6937 (1991).
32. E. C. Taylor, D. Kuhnt, C. Shih, S. M. Rinzel, G. B. Grindey, J. Barreda, M.
Jannatipour, and R. G. Moran, J. Med. Chern. 35:4450 (1992).
33. (a) J. Davoli, J. Chern. Soc. 131 (1960). (b) U. Liipke, and F. Seela, Chern. Ber.
110:1462 (1977).
34. E. C. Taylor, and A. McKillop, "The Chemistry of Cyclic Enaminonitriles and .Q.-
Aminonitriles," Wiley-Interscience, New York (1970).
35. C. J. Barnett, T. M. Wilson and G. B. Grindey, "Synthesis and Antitumor Activity of
LY288601, the 5,6-Dihydro Analog of LY231514", Poster presented at the lOth
International Symposium, Chemistry and Biology of Pteridines and Folates, Orange
Beach, Alabama, March 21-26, 1993. These workers prepared the 5,6-dihydro derivative
of L Y231514 by a different route .
36. T. Miwa, T. Hitaka, H. Akimoto and H. Nomura, J. Med. Chern. 34:555 (1991).

408
SYNTHESIS AND ANTITUMOR ACTIVITY OF LY288601,
THE 5,6-DIHYDRO ANALOG OF LY231514

Charles J. Barnett*, Thomas M. Wilson, and Gerald B. Grindey

Lilly Research Laboratories, A Division of Eli Lilly and Company
Lilly Corporate Center, Indianapolis, Indiana 46285-4813 U.S.A.

The series of deaza analogs of folic acid has been a rich source of compounds of in-
terest as potential antitumor drugs.! The mode of action of these compounds is, however,
related to structure in ways not yet fully understood. For example, DDATHF2 is a specific
inhibitor of glycinamide ribonucleotide formyl transferase (GARFT)3 while the related
pyrrolo[2,3-d]fyrimidine-based analog, LY231514, has been found to inhibit thymidylate
synthase (TS). Both (6R)-DDATHF (lometrexol)5 and LY2315I46 have shown promising
in vivo antitumor activity against a variety of murine and human tumor cell lines and are
currently undergoing clinical evaluation. LY28860 I may be viewed as a hybrid structure
which possesses both the ring saturation ofDDATHF and the 6,5-heterocyclic ring system
of the pyrrolo[2,3-d]pyrimidine-based LY231514. LY288601 was first described by
Akimoto and coworkers7 (as Takeda T-41440) but only limited cytotoxicity data has been
reported. 8 We report here a convenient alternate synthesis of LY28860 1, the results of cell
culture cytotoxicity and reversal experiments, and in vivo antitumor evaluation of this
compound in comparison with DDATHF and LY231514.

DDATHF LV231514

LV288601
(T-41440)

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 409
 Pd(O)
...

!
~OH

diethyl malonate
TiCI 4/Py, THF

EtO~~ CQzBut
2 I
DBU Et02C ~ ~
72% (3 steps)
3

1. TFA
...
2. guanidine

!
(64%)
7
H2N-L-Giu(OEt}2
chlorodimethoxytriazine
NMM, DMF (52%)
0 yO:zR

~lCOR 2

NaOH, r - 8 R = Et
HCI ~ LY288601 R = H
(85%)
Scheme 1. Synthesis of LY288601.

Synthesis

Our preliminary studies indicated that the preparation of a 5,6-dihydro analog of

L Y231514 by partial reduction would be difficult due to the remarkable stability of its 2-
amino-3, 4-dihydro-4-oxo-7H-pyrrolo[2,3-dJpyrimidine chromophore toward both catalytic
and alkylsilane reduction conditions. Rather than attempt to modify the heterocyclic sys-
tem so as to make it more amenable to reduction, we took a different arcproach to the
problem as illustrated in Scheme 1. 9 Thus palladium(O) mediated coupling o of tert-butyl-
4-iodobenzoate (1) with allyl alcohol provided aldehyde (2) which was condensed with di-
ethyl malonate in the presence of the titanium(IV)-pyridine complexII to give 3. Michael
addition of 3 with nitromethane afforded nitrodiester ( 4), obtained in 72% overall yield
from 1. Catalytic hydrogenation of 4 (5% Pd/C EtOH) provided lactam (5) which was
readily converted to the thiolactam (6) by heating with phosphorus pentasulfide in THF.
Brief treatment of 6 with trifluoroacetic acid effected selective cleavage of the tert-butyl
ester group and the resulting free acid gave rise to 7 when subsequently heated with
guanidine. To our knowledge, this is the first example of the synthesis of a 5,6-dihydro-

410
pyrrolo[2,3-dJpyrimidine by cyclization of an a.-carboalkoxythiolactam with guanidine.
Condensation of 7 with diethyl-L-glutamate (chlorodimethoxytriazine 12 method) followed
by basic hydrolysis afforded the target structure. LY28860 1 was obtained from the syn-
thesis as a mixture of diastereomers epimeric at C-5. All biological results reported are
based on studies of the diastereomeric mixture.

Biological Evaluation

Compound LY288601 was screened for cytotoxic activity against CCRF-CEM cells
in culture and found to have ICso = 0.09 Jlg/ml, as compared to 0.009 Jlg/ml for
LY2315144 and 0.004Jlg/ml for (6-RS)-DDATHF.13 The cytotoxic activity ofLY288601
was reversed (Figure) by addition of thymidine, whereas hypoxanthine had no effect, sug-
gesting that this compound is an inhibitor of thymidylate synthase. In contrast to
DDATHF, it does not appear to have any effect on de novo purine biosynthesis in this
model.

100
-e-
\ .' --- •
l Y2BB601

LY2BBB01 + S11M Thymid1ne

'•
80 '\.. --+ - LY288801 + 100 11M Hypoxanthine
ec '•
.....__
~-._
-
·-· ·-•-·-·•
0 60
(.)
0
c --
~ 40
al
c..

20

0
0.003 0.01 0.1 1 10 100
Cone. of LY288601 (119fml)

Figure 1. Comparison of cytotoxicity of LY288601 toward CCRF-CEM cells alone, with

added thymidine, and hypoxanthine.

The results of preliminary enzyme inhibition studies indicated that LY288601 is a

relatively weak inhibitor of TS CKi 5.5 JlM).14 It thus appears that the TS inhibition
activity revealed by the reversal study is due to one or more metabolites of LY288601,
most probably arising from polyglutamation. It should be noted in this regard that the
pentaglutamate of L Y231514 has been estimated to be about 100 times more active as
against TS than the parent drug.4

Although LY288601 demonstrated cytotoxic activity in cell culture studies, it was

found to be inactive in vivo at doses up to 200 mg/kg (daily X 8) when tested in mice
bearing implanted (thymidine kinase deficient) L5178Y/TK-tHX-lymphoma. By compari-
son, LY231514 exhibited complete inhibition of tumor at doses as low as 12.5 mg/kg when
tested in this tumor model and dose schedule.4

References

1. For a recent overview of the status of antifolate research related to antitumor

therapy see: E. M. Berman and L. M. Werbel, J. Med Chern. 34:479 (1991) and
references cited therein.
2. E. C. Taylor, P. J. Harrington, S. R. Fletcher, G. P. Beardsley, and R. G. Moran, J.
Med Chern. 28:914 (1985).

411
3. R. G. Moran, S. W. Baldwin, E. C. Taylor, and C. Shih, J. Bioi. Chem. 264:21047
(1989).
4. E. C. Taylor, D. Kuhnt, C. Shih, S.M. Rinzel, G. B. Grindey, J. Barredo, M.
Jannatipour, and R. G. Moran, J. Med Chem. 35:4450 (1992).
5. C. Shih, G. B. Grindey, P. J. Houghton, and J. A. Houghton, Proc. Am. Assn. for
Cancer Research, 29:abstr. 1125 (1988).
6. G. B. Grindey, C. Shih, C. J. Barnett, H. L. Pearce, J. A. Engelhardt, G. C. Todd, S.
M. Rinzel, J. F. Worzalla, L. S. Gossett, T. P. Everson, T. M. Wilson, M. E.
Kobierski, M.A. Winter, J. R. Bewley, D. Kuhnt, E. C. Taylor, and R. G. Moran,
Proc. Am. Assn. for Cancer Research, 33:abstr. 2451 (1992).
7. H. Akimoto, H. Takenori, and T. Miwa, European Patent Appl. 334 636 A2 (1989).
8. ICso 0.32j.!.g/ml vs KB cells. H. Akimoto, T. Hitaka, T. Miwa, K. Yukishige, T.
Kusanagi, and K. Ootsu, Proc. Am. Assn. for Cancer Research 32:abstr. 1938
(1991).
9. Full experimental details of the synthetic work presented here will be published
separately. C. J. Barnett and T. M. Wilson, Heterocycles, in press.
10. E. C. Taylor, P. Gillespie, and M. Patel, J. Org. Chern. 57:3218 (1992).
11. W. Lehnert, Tetrahedron Lett. 4723 (1970).
12. Z. J. Kaminski, Tetrahedron Lett. 26:2901 (1985).
13. G. P. Beardsley, B. Moroson, E. C. Taylor, and R. G. Moran, J. Bioi. Chem.
264:328 (1989).
14. R. G. Moran, personal communication. We thank Professor Moran for the TS
inhibition measurement on LY28860 1.

412
SYNTHESIS AND PRELIMINARY BIOWGICAL EVALUATION OF ANAWGUES
OF 5,8-DIDEAZAISOFOLIC ACID AND ITS 2-DESAMIN0-2-METHYL
DERIVATIVE CONTAINING FLUORINE AT POSITION FIVE

John B. Hynes, 1 Oliver S. Fetzer, 1 B. Shane, 2 and James H. Freisheim3

1Department of Pharmaceutical Sciences, Medical University of South Carolina,

Charleston, South Carolina 29425
2Department of Nutritional Sciences, University of California, Berkeley, CA 94720

3Department of Biochemistry and Molecular Biology, Medical College of Ohio,

Toledo, OR 43699

INTRODUCTION

The folate analogue 5,8-dideazaisofolic acid (IAHQ), la, has demonstrated antitumor
activity against a variety of mammalian tumor cells both in vitro and in vivo. 1-5 Its
derivative, 2-desamino-2-methyl-5,8-dideazaisofolic acid, lb, was found to be 20- and 44-
fold more cytotoxic toward MCF-7 and L1210 cells in culture, respectively. 6 In order to
evaluate the effects of other substituents located at position five of the quinazoline nucleus
upon antitumor activity, we have prepared 5-fluoro-5,8-dideazaisofolic acid, 2a, and
desamino-2-methyl-5-fluoro-5,8-dideazaisofolic acid, 2c, together with their 9-methyl
derivatives, 2b and 2d. This report describes the chemistry and preliminary biological
evaluation of these new compounds.

METHODOLOGY

Hog liver folylpolyglutamate synthetase (FPGS) was purified to homogeneity as

described previously_ 7 The specific activity of the purified enzyme with (6S)-H.tPteGlu as
the substrate was 123 units/mg of protein at saturating substrate concentrations. One unit
equals 1~-tmol ofH4PteGlu2 formed/hr. Enzyme activity was measured by the incorporation
of [14 C] glutamate into products using unlabeled folate or folate analogue as the substrate.
The assay conditions used were the same as those employed earlier. 8 L1210 leukemia cells
sensitive to MTX were grown in suspension culture according to the literature procedure. 9

For the preparation of 5-fluoro-5,8-dideazaisofolic acid, 2a, 6-amino-3,4-dihydro-5-

fluoro-4-oxo-2-(pivaloylamino)quinazoline was resynthesized and separated from its 8-
amino isomer as recently described. 10 This intermediate was condensed reductively with
di-tert-butyl N-(4-formylbenzoyl)-L-glutamatd in the presence of Raney nickel to give the
fully protected folate analogue. The pivaloyl group was then removed by refluxing in a
mixture of hydrochloric acid and tert-butanol in order to prevent transesterification of the
tertiary butyl ester groups. Final deprotection was accomplished in trifluoroacetic acid to

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayhng et al., Plenum Press, New York, 1993 413
afforded the target compound, 2a, in modest yield. Compound 2c was prepared in
analogous fashion from 6-amino-3,4-dihydro-5-fluoro-2-methyl-4-oxoquinazoline.10•11 The
methylation reactions were conducted as described earlier. 1

Table 1. Comparison of the cytotoxic effects of analogues of 5,8-dideazaisofolic acid with

their 5-fluoro counterparts against L1210 leukemia cells.

No. R X .Y lso.I!M
1a NH 2 H H 3.21
2a NH2 F H 3.0
2b NH2 F CH3 17.7
1b CH3 H H 0.073 1
2c CH3 F H 0.83
1c CH3 H CH3 0.060
2d CH3 F CH3 1.3
MTX 0.004
Reported previously, reference 6.

Table 2. Comparison of the kinetic constants of analogues of 5,8-dideazaisofolic acid with

their 5-fluoro counterparts for hog liver folylpolyglutamate synthetase. 1

R X .Y .K.n..MM Y.max•umol/hr/mg Yma/K.m:

NH2 H H 21 3 923 283

NH2 F H 6.7 69 64
NH 2 F CH3 42 23 3.5
CH3 H H 8.64 1044 764
CH3 F H 4.8 68 88
CH3 H CH3 3.2 67 130
CH3 F CH3 22 68 19
1Standard error of the mean K.,<±20%, Vmax<±lO%.
2Relativeto results for (6S)-H4PteGlu normalized to 100.
3Reported previously, reference 8.
4Reported previously, reference 6.

414
DISCUSSION

Each of the newly synthesized 5-fluoro analogues was evaluated for cytotoxicity against
L1210 leukemia cells in culture and the results are presented in Table 1. It will be seen
that 2a has the same level of antitumor activity as IAHQ. However, the introduction of
a 9-methyl substituent into 2a cause a 6-fold decrease in cytotoxicity. For the 2-desamino-
2-methyl analogues, it will be noted that the presence of a 5-fluoro substituent has a highly
detrimental effect on activity, since 2b and 2d are 11- and 22-fold less cytotoxic than their
unfluorinated counterparts, 1b and 1c.

The kinetic constants obtained for the 5-fluoro analogues, 2a-2d, using hog liver FPGS
are compared with their 5-unsubstituted counterparts in Table 2. While 5-fluoro-5,8-
dideazaisofolic acid, 2a, is a 2-fold more efficient substrate than IAHQ, methylation at
position 9 causes a large decrease in Vmax/K.n making 2b a much poorer substrate than
IAHQ. For the 2-desamino-2-methyl analogue, 1b, the introduction of a 5-fluorine, 2c,
improves substrate activity as measured by K,... However, when a 9-methyl substitutent
is present, the introduction of a 5-fluorine has a markedly negative effect upon substrate
activity as measured by either ~11 or VmaJI<o,.

REFERENCES

1. J.B. Hynes, Y.C.S. Yang, J.E. McGill, S.J. Harmon, and W.L. Washtien, J. Med.
Chern. 27:232 (1984).
2. D.J. Fernandes, J.R. Bertino, and J.B. Hynes, Cancer Res. 43:1117 (1983).
3. J.B. Hynes, A.B. Smith, and G.R. Gale, Cancer Chemother. Pharmacal. 18:231
(1986).
4. K.-Y. Tsang, J. B. Hynes, and H.H. Fudenberg, Chemother. 28:276 (1982).
5. J.J. McGuire, A.F. Sobrero, J.B. Hynes, and J.R. Bertino, Cancer Res. 47:5975.
6. R.L. Hagan, D.S. Duch, G.K. Smith, M.H. Hanlon, B. Shane, J.H. Freisheim,
and J.B. Hynes, Biochem. Pharmacal. 41:781 (1991).
7. D.J. Cichowicz and B. Shane, Biochemistry 27:504 (1987).
8. D.J. Cichowicz, J.B. Hynes, and B. Shane, Biochem. Biophys. Acta 957:363
(1988).
9. J.I. McCormick, S.S. Susten, and J.H. Freisheim, Arch. Biochem. Biophys.
212:311 (1981).
10. J.B. Hynes, S.K. Singh, O.Fetzer, and B. Shane, J. Med. Chern. 35:4078 (1992).
11. J.B. Hynes, A. Tomazic, C.A. Parrish, and O.S. Fetzer, J. Heterocyclic Chern.
28: 1357(1991).

415
SYNTHESIS AND BIOLOGICAL EVALUATION OF ANALOGUES OF 5,8-
DIDEAZAISOFOLIC ACID (IAHQ) MODIFIED AT POSITIONS 2, 4 AND 9

John B. Hynes, 1 Robert L. Hagan 1 , Barry Shane, 2 and James H. Freisheim3

1Department of Pharmaceutical Sciences, Medical University of South Carolina,

Charleston, SC 29425
2Department of Nutritional Sciences, University of California, Berkeley, CA 94720
3Department of Biochemistry and Molecular Biology, Medical College of Ohio,

Toledo, OH 43699

INTRODUCTION

The compound 5,8-dideazaisofolic acid (IAHQ), 1a, was found to posses modest
antitumor activity against a variety of human and murine tumor cells both in vitro and in
vivo. J-s Its lack of potency was attributed to slow influx into target cells as demonstrated
with eH] IAHQ in the presence of HCT-8 human colon adenocarcinoma cells in culture. 6
The analogues 2-desamino-5,8-dideazaisofolic acid, 1b, and 2-desamino-2-methyl-5,8-
dideazaisofolic acid, 1c, were prepared subsequently and found to be 6-and 44-fold more
cytotoxic toward L1210 cells than 1a7•8 This activity enhancement correlated well with
increased affinity for the reduced folate transporter as measured by competition for the
uptake of [3H] MTX into MOLT-4 cells. 8

This paper represents a logical extension of the earlier work and includes the
preparation of the 9-CH3 and 9-CHO derivatives of 1b and 1c. In addition, the 4-amino
modifications of 1b and 1c, 2b and 2c were synthesized for the first time. These two
analogues of 5,8-dideazaisoaminopterin, 2a, were also modified by the introduction of a
methyl or formyl group at position 9.

Each of the newly synthesized compounds was evaluated for antitumor effects against
the growth of L1210 leukemia cells in vitro and the results are presented in Table 1
together with values obtained earlier for 1b, 1c, and MTX. In addition, these analogues
were evaluated as substrates for hog liver folylpolyglutamate synthetase (FPGS) and the
kinetic constants are shown in Table 2.

METHODOLOGY

Hog liver FPGS was purified to homogeneity as described previously. 9 The specific
activity of the purified enzyme with (6S)-fftPteGlu as the substrate was 123 units/mg of

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 417
protein at saturating substrate concentrations. One unit equals 1 ttmol of H4PteGlu2
formed/hr. Enzyme activity was measured by the incorporation of [14C] glutamate into
products using unlabeled folate or folate analogue as the substrate. The assay conditions
used were the same as those employed earlier. 10 L1210 leukemia cells sensitive to MTX
were grown in suspension culture according to the literature procedure. 11

The 2-desamino-5,8-dideazaisoaminopterin, 2b, and 2-desamino-2-methyl-5,8-

dideazaisoaminopterin, 2c, were synthesized in an analogues fashion with 2-amino-5-
nitrobenzonitrile serving as the common starting material. Cyclization with formamide
gave 4-amino-6-nitroquinazoline in good yield, while the use of acetamide resulted in the
formation of 2-methyl-4-amino-6-nitroquinazoline in fair yield. Catalytic hydrogenation
of these two nitro compounds yielded the corresponding 6-amino derivatives in good yield.
The 6-amino intermediates were condensed reductively with di-tert-butyl N-(4-
formylbenzoyl)-L-glutamate1 to afford the penultimate di-tert-butyl esters, which were then
deblocked in the presence of trifluoroacetic acid to give 2b and 2c. The methylation and
formylation reactions were conducted as described previously.'

-o-
Table 1. Cytotoxic effects of analogues of 5,8-dideazaisofolic acid and 5,8-
dideazaisoaminopterin modified at positions 2 and 9 toward L1210 leukemia cells.

O y COOH la R NH 2 , Y H
HN~~CH2 ~ ' gNHCH lb R H, Y H

Ri.,)~~
lc R CH 2 , Y H
(bH2hCOOH

H H 0.53 1
H CH3 0.21
H CHO >100
CH3 H 0.073 1
CH3 CH3 0.060
CH3 CHO 18.1

N~L,-o'\ gNH~~oH 2a
2b
2c
R
R
NH 2 , Y
H,
R == CH3, Y
Y

Rl.,NA/ - (CH2l2COOH

H 4.3
CH3 0.044
CHO 8.3
H 1.5
CH3 0.18
CHO >100
0.004
Reported prevwusly; reference 8.

418
Table 2. Kinetic constants of analogues of 5,8-dideazaisofolic acid and 5,8-
dideazaisoaminopterin modified a positions 2 and 9 for hog liver folylpolyglutamate
synthetase. 1

y Ym.x.mol/h/mg

H 21 3 923 283
H 124 624 31 4
CH3 2.1 63 187
CHO 63 59 5.9
H 8.64 1044 764
CH3 3.2 67 130
CHO 131 116 5.5

H 443 lOP 143

H 12 80 44
CH3 10 36 21
CHO 21 35 10
H 15 85 36
CH3 21 83 24
CHO 21 81 24
1Standard error of the mean K,< .±20%, Vmax<.±lO%.
2Relative to results for (6S)-H4PteGlu normalized to 100.
3Reported previously, reference 10.

4Reported previously, reference 8.

DISCUSSION

Ten new analogues of IAHQ have been synthesized and fully characterized. Each
compound was evaluated for cytoxicity toward L1210 cells in culture. As will be seen in
Table 1, methylation at position 9 of either lb or lc results in only modest increases in
cytoxicity, while 9-formylation causes dramatic decreases in antitumor potency. The 4-
amino derivatives, 2b, and 2c, are 8- and 20-fold less potent than their 4-oxo counterparts.
However, 9-methylation of 2b and 2c enhances activity by approximately 100-fold and 10-

419
fold, respectively. Thus, 2-desamino-9-methyl-5,8-dideazaisoaminopterin is the most
cytotoxic of the IAHQ analogues studied (150 = 0.044 ~tM).

All of the compounds were evaluated as substrates for FPGS and the results are
presented in Table 2. It will be seen that methylation at position 9 of 1b and 1c causes
substantial increases in substrate activity as measured by Vmax/Km, with 2-desamino-9-
methyl-5, 8-dideazaisofolic acid being the most efficient substrate of the compounds studied.
Both of the 9-formyl modifications were significantly less efficient substrates than their
parent compounds.

Turning to the 4-amino derivatives, it was shown earlier that 5,8-

dideazaisoaminopterin, 2a, was approximately 2-fold less effective as a substrate for FPGS
than IAHQ. 10 As will be seen in Table 2 its 2-desamino and 2-desamino-2-methyl
derivatives, 2b and 2c, are 2- to 3-fold more efficient substrate than the parent compound
as measured by Vm./K111 • However, in this series either methylation or formylation has a
negative effect upon substrate activity.

In spite of the fact that 2-desamino-9-methyl-5,8-dideazaisoaminopterin is not a highly

efficient substrate for FPGS, it is the most potently cytotoxic of the compounds studied.
It is believed that this compound should be evaluated in greater detail as a potential
antitumor agent, in particular since its mechanism for killing cells has not been determined.

REFERENCES

1. J.B. Hynes, Y.C.S. Yang, J.E. McGill, S.J. Harmon, and W.L. Washtien, J. Med.
Chern. 27:232 (1984).
2. D.J. Fernandes, J.R. Bertino, and J.B. Hynes, Cancer Res. 43:1117 (1983).
3. J.B. Hynes, A.B. Smith, and G.R. Gale, Cancer Chemother. Pharmacal. 18:231
(1986).
4. K.-Y. Tsang, J.B. Hynes, and H.H. Fudenberg, Chemother. 28:276 (1982).
5. J.J. McGuire, A.F. Sobrero, J.B. Hynes, and J.R. Bertino, Cancer Res. 47:5975
(1987).
6. A.F. Sobrero, J.J. McGuire, and J.R. Bertino, Biochem. Pharmacal. 37:997 (1988).
7. J.B. Hynes, S.A. Patil, R.L. Hagan, A. Cole, W. Kohler, and J.H. Freisheim, J.
Med. Chern. 32:852 (1989).
8. R. L. Hagan, D.S. Duch, G.K. Smith, M.H. Hanlon, B. Shane, J.H. Freisheim, and
J.B. Hynes, Biochem. Pharmacal. 41:781 (1991).
9. D.J. Cichowicz and B. Shane, Biochemistry 27:504(1987).
10. D.J. Cichowicz, J. B. Hynes, and B. Shane, Biochem. Biophys. Acta 957:363 (1988).
11. J.I. McCormick, S.S. Susten, and J.H. Freisheim, Arch. Biochem. Biophys. 212:311
(1981).

420
NEW THIOPHENE SUBSTITUTED 10-DEAZAAMINOPTERINS: SYNTHESIS
AND BIOLOGICAL EVALUATION

Ann Abraham*, J .J. McGuire+, J. Galivan*, B. Rao Vishnuvajjala,, and

M.G. Nair*

*university of South Alabama, Mobile, Alabama 36688

+Roswell Park Cancer Institute, Buffalo, New York 14263
*Wadsworth Cancer for Laboratories and Research, Albany, New York
12201
,National Cancer Institute, Rockville, MD 20852

ABSTRACT

Analogues of 10-deazaaminopterin (10-DAM) and 4-amino-4-deoxy-10-deazapteroyl-'Y-

methylene glutamic acid (MDAM) in which the benzene ring was replaced with a
thiophene ring have been synthesized and evaluated for their antitumor activity. These
analogues were N-{[5-(2,4-diamino-6-pteridinyl)ethyl]-2-thenoyl}-L-glutamicacid (1) and
N-{ [5-(2, 4-diamino-6-pteridinyl)ethyl]-2-thenoyl}-"1-methylene glutamic acid@.

INTRODUCTION

Methotrexate (MTX) which is one of the most potent inhibitors of human dihydrofolate
reductase (DHFR) continues to be the most widely used classical antifolate drug for the
chemotherapy of cancer. As part of a continuing program aimed at the development of
potentially more specific and less toxic antifolate drugs we reported the synthesis and
preliminary biological evaluation of certain non-polyglutamylatable inhibitors of DHFR.
Two of the most potent non-polyglutamylatable analogues in this series were 'Y-methylene-
10-deazaaminopterin (MDAM) and 'Y-methylene-1 0-ethyl-1 0-deazaaminopterin
(MEDAM). 1•2 Both MDAM and MEDAM exhibited superior activity relative to MTX
in inhibiting a number of human tumor cell lines in culture under identical conditions.
These results were some what surprising in view of the presumed notion that
polyglutamylation of classical antifolates is a requirement for eliciting antitumor activity.
Subsequent invivo t·~sting of MEDAM in mice revealed that it is dramatically less toxic
than MTX under identical experimental protocol. These results prompted us to examine
in detail the role of polyglutamylation of classical antifolates in cytotoxicity and the study
required the development of analogues with varying abilities of polyglutamylation, while
preserving the DHFR inhibition and transport properties similar to MTX. Replacement
of the phenyl ring of 10-deazaaminopterin (10-DAM) with a thiophene ring was judged to

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al.• Plenum Press, New York, 1993 421
be a good strategy in altering polyglutamylation of 10-DAM. The synthesis of 1 and its
')'-methylene analogues was carried out by procedures that will be published elsewhere.
In this paper, we describe the biological evaluation of 1, z, and their close analogues.

BIOLOGICAL EVALUATION AND DISCUSSION

Preliminary screening of compounds 1 and Z established that they were good inhibitors
of H35 rat hepatoma cell growth and CCRF-CEM human leukemia cell growth (Table 1).
Substitution of glutamate moiety of 1 with a ')'-methyleneglutamate resulted in - 5-fold
increase in IC50 in both cell lines. The IC50 of 1 was 1.3 fold lower than that of
methotrexate (MTX) and was only 3 times higher than that of 10-DAM in CCRF-CEM
human leukemia cell line. Compound Z was less potent than MDAM in inhibiting CCRF-
CEM leukemia cell growth (IC50 59 nM Vs 7.25 nM). Thus substitution of a thiophene
for benzene ring in this class leads to a small loss of potency even on continuous exposure.
Substrate activities of these analogues with purified FPGS from CCRF-CEM leukemia
cells showed that 1 was a fair substrate with a V/K ratio 1.3 times lower than
that of 10-DAM (Table II). Compound Zwas neither a substrate nor an inhibitor ofFPGS
as expected. Compounds 1 and 2 were evaluated as inhibitors of dihydrofolate reductase
(DHFR) (Table ill). Compound 1 inhibited human DHFR at equivalent potency to 10-
DAM; thus for DHFR inhibition thiophene substitution is conservative. Transport studies
showed that 1 competed 3.6 times more effectively than MTX for folinic acid transport.
In vitro studies led to the selection of 1 for further evaluation . It was evaluated in
NCI's human tumor, disease oriented in ~itro screen. 6 A broad range of sensitivities
among the various types of human tumors to the cytotoxic effects of 1 was observed.
Compound 1 showed enhanced activity relative to MTX against the growth of a number
of tumor cells, in spite of its comparable DHFR inhibition (Table IV).
In vitro antitumor evaluation of 1 against ip implanted P388 leukemia was carried out
in CD2F 1 mice. A 95% ILS was observed with 96mg/kg/dose given at a schedule Q2DX5
and it was nontoxic to the mice. (Table V). With 15 mg/kg/dose given at a schedule
Q2DX5, 100% ILS was obtained with MTX. A 48mg/kg/dose of 1 given by Q2DX5
schedule gave only a 40% ILS, but the same dose given at a Q1DX9 schedule gave a 70%
ILS.
Although we are unable to explain the observed differences in percent ILS of tumor
bearing mice at the same dose under different protocols, it would appear that 1 is cleared
more efficiently than MTX from murine tissues. Since 1 exhibits similar
polyglutamylation and enhanced transport relative to MTX, the striking reduction in host
toxicity (drug tolerance) may be due to enhanced efflux or deactivation of the drug by
metabolism. These possibilities as well as studies to optimize the therapeutic index of 1
using different protocols are being investigated.

Table I. Inhibition of Tumor Cell Growth3

COMPOUND IC50 ,nM

H35 Hepatoma CCRF-CEM Leukemia

15 10.2
2 76 59
MTX 10 13.5
10-DAM 3.4
MDAM 7.25

422
Table n. Substrate and Inhibitory activity with CCRF-CEM human leukemia cell FPGS4

Relative*
COMPOUND Km,J.!M Vmax Rei V/K Inhibition Substrate Activity

Aminopterin 4.5 100 22.2 100

10-DAM 35 89 2.5
1 45.0 84 1.9
2 <7% ~1.3%

Inhibition of FPGS was measured by adding 100J.!M of compound to the assay mixture containing 50J.!M
aminopterin as the substrate. *Substrate activity of 2 was measured at 100J.!M of 2 and is relative to the
activity of 50JLM aminopterin.

Table ill. Inhibition of Dihydrofolate Reductase5

COMPOUND

1 0.95
2 4.5
MTX 0.98
10-DAM 0.80
MDAM 1.30

Table IV.
Activity of 1 against the growth of selected tumor cells in culture

Cell line Log to Glso

MTX 1
Leukemia:
CCRF-CEM -7.57 <-8.00
HL-60(TB) -7.48 <-8.00
K -562 -7.6 <-8.00
MOLT -4 -7.59 <-8.00
SR -7.52 <-8.00
Non-small cell lung cancer:
A549 (ATCC) -7.52 -7.11
NCI -H460 -7.54 <-8.00
NCI -H522 -6.50 7.04
LXFL529 -7.46 -7.42
Small Lung Cancer:
DMS -114 -7.48 -7.36
DMS -273 -7.51 -7.83
Colon Cancer:
HCC -2998 -7.00 -6.77
HCT-116 -7.54 <8.00
HCT -15 -7.49 -7.44
HT -12 -7.51 <-8.00
KM -29 -7.43 -7.08
sw -620 -7.51 <-8.00
CNS Cancer:
SF -268 -7.27 -7.20

423
 Table IV Continued

SF -539 -7.48 -7.38

I -251 -7.17 -7.17
Melanoma:
M19-MEL -6.40 -7.22
Ovarian Cancer:
OVCAR-8 -7.51 -7.35
Renal Cancer:
786-0 -7.59 <-8.00
ACHN -7.39 -7.04
CAKI-1 -7.39 -7.48
SN12C -7.52 -7.47
U0-31 -6.43 -7.13
Prostate Cancer
PC-3 <-8.00

Table V. Antitumor activity of 1 against IP implanted P388 leukemia in mice

Compound Dose (mglkg) Schedule %ILS

MTX 15.0 Q2DX5 +100

1 24.0 Q2DX5 +30
1 48.0 Q2DX5 +40
1 48.0 Q1DX9 +70
1 96.0 Q2DX5 +95

0 COOH

HN
2 N
6)-CHC
N
"'2--()----c- ~ -)I
II
H
COOH
1: R= H ~ R = =CH 2 R

ACKNOWLEDGEMENT

This investigation was supported by NIH grants CA 27101 (MGN); CA 43500 (JJM)
and CA 25933 (JG).

REFERENCES

1. A. Abraham, J.J. McGuire, J. Galivan, Z. Nimec, R.L. Kisliuk, Y. Gaumont, and M.G. Nair, J. Med.
Chern. 34:222-227. (1991).
2. M.G. Nair, A. Abraham, U.S. Patent 4,996207 (1991).
3. S.D. Patil, C. Jones, M.G. Nair, J. Galivan, F. Maley, R.L. Kisliuk, Y. Gaumont, D. Duch. and R.
Ferone, J. Med. Chern.32:1284. (1989).
4. J.J. McGuire, W.E. Bolanowska, J.R. Piper, Biochern. Phannacol. 37:3931. (1988).
5. J.J. Mcguire, C.A. Russell, W.E. Bolanowska, C.M. Freitag, C.S. Jones, T.l. Kalman, Cancer
Research 50:1726. (1990).
6. R.W. Fuller, J.H. Cardellina II, Y. Kato, L.S. Brinen J. Clardy, K.M. Snader, M.R. Boyd, J. Med.
Chern. 35:3007. (1992).

424
EVALUATION OF THE ANTI-ARTHRITIC ACTIVITY AND AN ALTERNATE
SYNTHESIS OF A THIOPHENE-SUBSTITUTED 10-DEAZAAMINOPTERIN

Anjali Desai and M.G. Nair

Drug Development Laboratory, Cancer Center

Department of Biochemistry, University of South Alabama
Mobile, Alabama 36688

INTRODUCTION

The antifolate methotrexate (MTX) has become an established treatment in patients with
rheumatoid arthritis (RA). Although the mechanism of action of this drug in RA is still
unknown, its efficacy has been demonstrated in short-term placebo-controlled studies,
comparative trials, and open prospective studies as well as in animal models of rheumatoid
arthritis. In a recent long-term study the effectiveness of methotrexate in the treatment of
RA has been demonstrated in patients who have received the drug for more than seven
years 1. While a sustained clinical response is seen with long-term MTX treatment, the
most common reason for withdrawal from the drug is toxicity.
The efficacy of MTX and the newer dihydrofolate reductase inhibitor 10-
deazaaminopterin (10-DAM) in a murine model of autoimmune disease has recently been
reported2 . In a short term clinical trial 10-DAM has been shown to be as effective as
MTX in the treatment of human rheumatoid arthritis with enhanced beneficial effects
relative to MTX in controlling joint pain, swelling and morning stiffness3 •4 .
The synthesis of a new 10-DAM analog in which the benzene moiety is replaced by a
thiophene ring has recently been accomplished in this laboratory5 . This compound, N-{5-
(2,4-diamino-6-pteridinyl) ethyl]-2-thenoyl}-L-glutamic acid (1), exhibits comparable
dihydrofolate reductase inhibition but superior antifolate activity relative to methotrexate
in a number of human tumor cell lines. Therefore it was appealing to explore this
compound as a potential anti-arthritic agent. The synthetic scheme as well as the
evaluation of this thiophene antifolate in the rat adjuvant arthritis model is presented in this
report.

METHODS

Chemistry

The methods used for the synthesis of compound (1) is illustrated in scheme 1. Direct

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 425
lithiation and carboxylation of ~ gave aldehyde 1 that was elaborated to 1 employing a
modified Boon-Leigh strategy. The novelty of the chemistry is the introduction of the
glutamate moiety prior to the pteridine ring closure thereby avoiding glutamate coupling
to a pteroate analog and subsequent purification steps.

Biological evaluation

Heat-killed Mycobacterium butyricum was emulsified in Freund's incomplete adjuvant

(7.5 mg/ml). Female Lewis rats weighing approximately 175-200 g were injected with
0.1 ml of this adjuvant. Antifolates were dissolved in water with addition of sodium
bicarbonate (pH 7.5). One day post adjuvant immunization rats were treated orally by
gavage with the antifolate solution. Control rats received only bicarbonate solution by
gavage. The rats were treated on a repeated cycle of five days of drug administration
followed by two days without the drug. The hind paws of all animals were measured
around the ankle joint with a vernier caliper. By day 18 post adjuvant immunization all
of the control rats developed maximum swelling of their hind paws. The percentage
inhibition of swelling of the hindpaws by the antifolates tested was obtained from the
following formula6: %Inhibition = 100 [1-(a-x)/(b-y)], where y = mean hind paw
thickness of control rats efore adjuvant immunization; b = mean hind paw thickness of
control rats on day 18 post adjuvant immunization; x = mean hindpaw thickness of
antifolate-treated rats before adjuvant immunization; and a = mean hind paw thickness of
antifolate-treated rats on day 18 post adjuvant immunization.

RESULTS AND DISCUSSION

Previous studies reported from this laboratory revealed that both MTX and compound
1 exhibited comparable inhibition of dihydrofolate reductase and tumor cell growth in
vitro. Compound 1 was transported more efficiently than MTX into H 35 Hepatoma cells
and it exhibited superior antitumor activity in a number of human tumor cell lines.
Therefore, it was speculated that 1 might be a potential anti-arthritic agent.
Galivan et al. previously demonstrated the efficacy of low-dose methotrexate in rat
adjuvant arthritis7 . Our experiments confirmed these observations. A weekly dose of 625
p.g/kg of methotrexate was effective in inhibiting the footpad swelling associated with
adjuvant arthritis by 91 % (Table 1). However, to achieve comparable inhibition of
footpad swelling by compound 1, a 20 fold higher dose of the drug was required.
Although we cannot explain the high dose of 1 required for anti-arthritic activity at the
present time, it is conceivable that it is due to increased efflux from the cell or metabolic
deactivation of 1. However, it should be emphasized that 1 was tolerated by all animals
at higher doses with no apparent signs of toxicity relative to methotrexate while
preserving the anti-arthritic activity. In order to optimize the therapeutic index of 1 in this
model, studies with higher doses will be required. It is interesting to note that in in vivo
antitumor screens using CDF1 mice bearing P 388 leukemia, 1 exhibited comparable
activity to MTX at 8 times the dose of MTX. Since 1 appears to be less toxic than MTX
(high drug tolerance without toxicity) in mice, clearly additional studies with 1 are
warranted to evaluate its clinical potential as a less toxic anti-arthritic agent. Such studies
are in progress.

ACKNOWLEDGEMENT

This investigation was supported by grant CA 27101 from the National Cancer Institute.

426
 Table 1. Effect of antifolates on the footpad swelling
in Lewis rats injected with mycobacterial adjuvant

Drug Dose (mg/kg/week) % Inhibition of footpad swelling

Methotrexate 0.375 11.1 (±4.3)

Methotrexate 0.625 90.9 (±7.45)
Methotrexate 1.0 88.9 (±6.0)
Compound 1 3.125 14.0 (±4.8)
Compound 1 6.25 30.0 (±3.1)
Compound 1 12.5 88.6 (±6.2)

SCHEME-1

a) CH(OCH3)]/NH4Br/Me0H b) BuLi/C02/THF c) 6 N HCI d) 1-Phthalimido-3-

(triphenylphosphoranylidene)-2-propanone e) L-glutamic acid diethyl ester f)
Zn/CH3COOH/Jones Rgt. g) H2N-OH HCI h) H2N-HN2 i) 6-chloro-2,4-diamino-5-
nitropyrimidine j) TFA/1 N HCI k) Sodium dithionite 1) DMF/heat m) 0.1 N NaOH

REFERENCES
1. M.E. Weinblatt, B.N. Weissman, D.E. Holdsworth, P.A. Fraser, A.L. Maier, K.R. Falchuk, and J.S.
Coblyn, Arthritis and Rheumatism, 35:129-137 (1992).
2. J.E. Baggott, S.L. Morgan, L.E. Freeberg, B.B. Hudson, W.H. Vaughn, M.G. Nair, C.L. Krumdieck,
W.J. Koopman, R.E. Gay, and S. Gay, Agents and Actions, 35:104-111 (1992).
3. G.S. Alarcon, 0. Castaneda, M.G. Nair, M. Ferrandiz, W.J. Koopman, and C.L.Krumdieck, Annals
of the Rheumatic Diseases, 51:600-603 (1992).

427
4. G.S. Alarcon, 0. Castaneda, M. Ferrandiz, C.L. Krumdieck, and W.J. Koopman, Arthritis and
Rheumatism, 35:1318-1321 (1992).
5. A. Abraham, S.W. Li, J.J. McGuire, J. Galivan, B.R. Vishnuvajjala, and M.G. Nair, 1. of Medicinal
Chemistry, submitted (1993).
6. B.B. Newbould, Brit.J.Pharmacol., 21:127-136 (1963).
7. W.L. Welles, J. Silkworth, A.L. Oronsky, S.S. Kerwar, and J. Galivan, J. of Rheumatology 12:904-906
(1985).

428
LIPOPHILIC ANTIFOLATES AS CANDIDATES AGAINST

OPPORTUNISTIC INFECfiONS

J.R. Piper, 3 C.A. Johnson, 3 , C.A. Hosmer, 3 R.L. Carter, 3 , E.R.

Pfefferkorn,b S. E. Borotz,b and S.F. Queenerc

3Southern Research Institute, Birmingham, Alabama 35255

bDartmouth Medical School, Hanover, New Hampshire 03756
cindiana University School of Medicine, Indianapolis, Indiana 46202

INTRODUCfiON

The problems in treating immunocompromised patients infected by

Pneumocystis carinii and Toxoplasma gondii have prompted intense efforts to develop
improved therapy against these pathogens. 1•2 Lipophilic antifolates trimethoprim and
pyrimethamine (structures shown under Table 1) are used in combination with sulfa
drugs in current treatment regimens which inhibit the ability of the microorganism to
synthesize reduced folates. In such treatment, the antifolate inhibits dihydrofolate
reductase (DHFR), and the sulfa drug inhibits utilization by the microorganism of 4-
aminobenzoic acid in its vital biosynthesis of dihydropteroic acid. 2· 4 Adverse reactions
that frequently occur with these regimens often necessitate discontinuation of the
therapy. New agents or combinations of agents of greater therapeutic effectiveness in
terms of lower toxicity and shorter treatment periods are needed.
At least two recent developments indicating progress toward those needs are
noteworthy: First, the lipophilic agents trimetrexate and piritrexim are undergoing
clinical trials. 2•3 These compounds are similar in their inhibitory effects against DHFR
isolated from P. carinii and T. gondii, and each is a much more potent DHFR inhibitor
than pyrimethamine and trimethoprim. A disadvantage in these potent agents is that,
unlike trimethoprim, each is more inhibitory toward mammalian DHFR than the
DHFR from the microorganisms (see Table 1). Thus they offer no beneficial
selectivity, 4 but these compounds can be administered with leucovorin, which, due to
transport differences,protects host cells but not the cells of the microorganism. 2·5 A
particular application sought for trimetrexate is its use, along with leucovorin, in the
treatment of P. cminii pneumonia (PCP) in AIDS patients who are unable to take
standard trimethoprim-sulfamethoxazole therapy. In another development, the
hydroxynaphthoquinone atovaquone (also called 566C80) was approved for the

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 429
treatment of PCP. This agent, which does not act on folate systems, was recently
shown to display a significant synergistic effect in atovaquone-pyrimethamine or
atovaquone-sulfadiazine combinations in the treatment of murine toxoplasmosis. 6 The
results might indicate that combinations of atovaquone and agents more potent than
pyrimethamine could afford even greater therapeutic value.
Two 2,4-diaminopteridines that we prepared initially for antitumor testing, the
phenylthio compound 1 and the 1-naphthylamino compound 2, showed favorable
differential activity toward DHFR from P. carinii and T. gondii in comparison with
mammalian DHFR (see Table 1). Following these results, we began a project aimed
toward identifying therapeutically useful agents which might selectively inhibit DHFR
from P. cminii and T. gondii over mammalian DHFR.

MATERIAlS AND METHODS

Syntheses

The lipophilic candidates were selected from the general structural type shown
in Scheme 1. The target compounds were prepared by standard synthetic procedures
from key precursors reported from our laboratories. 7

SCHEME 1. General Structure of Target Antifolates.

Types of compounds prepared:

Ar
N N NR, S, CHR, phenyl, pyridyl, biphenyl,
CR N SO, so 2 naphthyl. qumolyl.
CR CH Substltuenls on aromatic group:
alkyl, halogens, OCtl3. CF 3 . CN,
Nt!2.

Key precursors:
N!!2 R

~~CH2Br
HzN~;l_vJ
R = t!, Ctl3 R = H, CH 3
Y = N. C!! Y = N, CH

Target compounds were prepared from the key precursors by three general
approaches: (1) displacement reactions of the bromomethyl compounds with
substituted aniline and thiophenol types, (2) reductive alkylation between 6-cyano
precursors and anilino compounds, and (3) the Wittig reaction of phosphoranes
derived from bromomethyl precursors to give olefinic intermediates followed by
hydrogenation.

430
Antipathogenic Evaluations

Of the 71 2,4-diamino-type candidates synthesized, 27 are derived from 5-

methyl-5-deazapteridine while 4 each are 5-deaza- and 5,8-dideazapteridine types; the
remaining 36 are of the intact pteridine-ring type. Most have been tested in a primary
evaluation program for their ability to inhibit T. gondii cell growth in vitro 8 and for
their inhibitory effect against DHFR from both T. gondii and P. carinii and also from
a mammalian source (rat liver). 4

RESULTS AND DISCUSSION

Approximately 34 of the 71 candidates are at least as inhibitory as

pyrimethamine toward DHFR from both T. gondii and P. carinii. Of the 34, 23 are of
the 5-methyl-5-deazapteridine type, 2 are derivatives of 5-deazapteridine, and 3 are 5,8-
dideazapteridine types. Only 6 of the intact pteridine types exerted DHFR inhibition
comparable with pyrimethamine, and each of those 6 is considerably less potent than
its 5-methyl-5-deazapteridine analogue bearing the same side chain. Eight candidates
inhibit P. carinii DHFR as strongly as piritrexim and trimetrexate while 12 are
essentially as potent as these agents against T. gondii DHFR. There is generally good
correlation between potent enzyme inhibitory activity and in vitro cell growth inhibition.
Table 1 lists test results from 10 typical candidates (structures 3-12) selected
from those having greater inhibitory activity than pyrimethamine against in vitro cell
growth and the enzyme from each of the pathogens. The 10-deaza compound 6, a
bridge homolog of piritrexim, shows noteworthy selectivity (98-fold) for DHFR from
T. gondii over mammalian DHFR. With the exception of 6, the results shown are
typical representatives of the candidates of the 5-deaza- and 5,8-dideazapteridine types.
In numerous examples, candidates carrying similar sidechains, but with substituents in
different positions, gave similar test results. There also appears to be considerable
latitude for variations in the bridging group between the heteroaromatic ring and the
lipophilic sidechain. We plan greater emphasis on 10-thia and 10-deaza, including 10-
alkyl-10-deaza, types of candidates. In examples to date, N 10-methyl derivatives of
active N 10-H compounds have not proved to be significantly different from the parent.

NH 2 ~CH3
N~CH2-QOCH 3
H N~u~ OCH3
2 N

Trimethoprim Pyrimethamine

NH2 CH3
3: R = -sc 6H40CH3-4
~~CH2 R 4: R = -NHC10H7-1
H2 N~;(J 5: R = -NHC 6 H3 (0CH 3 ) 2 -2,5
6: R = -CH 2C6H3 (0CH 3) 2 -2,5
3-11 7: R = -NHC 6 H2(0CH 3) 3 -3,4,5
8: R = -NHC 6 H3 -2-CH 3-5-0CH 3
9: R = -N(CR 3)C 6 H 3 -2-0CH 3 -5-CF 3
10: R = -NHC6 H2 -2,6-(0CH 3) 2 -4-C0 2CH 3

11· R = -N~Cl
12

431
TABLE 1. Comparative Inhibition Data vs. T. gondii Cell Growth and DHFR from P.
carinii, T. gondii, and Rat Liver: Known Lipophilic Antifolates and Selected
Candidates.

T. gondii cell ICso (JLM) vs. DHFRb

growth
Compound inhibn. Rat Selectivit Selectivity
IC50 (MM) 3 P. carinii Liver y T. gondii RL(fg
RL/Pc
Trimethoprim nd 12 133 11.1 2.73 48.7
Pyrimethamine 0.4 3.65 2.3 0.63 0.39 5.9
Trimetrexate ndc 0.042 0.003 0.071 0.010 0.29
Piritrexim ndc 0.031 0.0015 0.048 0.017 0.088
1 10 9.5 246 25.9 0.77 319
2 0.25 0.13 1.26 9.7 0.076 16.6
3 0.25 0.56 0.52 0.93 0.063 0.46
4 0.12 0.22 0.11 0.5 0.24 8.3
5 0.2 0.011 0.010 0.94 0.014 0.73
6 0.1 0.34 0.77 2.3 0.0079 98
7 1.0 0.013 0.0054 0.42 0.0027 2.0
8 0.03 0.038 0.15 3.9 0.023 6.3
9 0.06 0.093 0.23 2.5 0.038 6.2
10 0.2 0.083 0.04 0.48 0.014 2.9
11 0.02 0.11 0.082 0.74 0.03 2.8
12 0.01 0.6 0.073 0.12 0.075 1.0
3 Methods described in ref. 8. bMethods described in ref. 4. cva!ues not determined,
but activity against T. gondii exceeds that of pyrimethamine (refs. 2,3).

CONCLUSIONS

The results show the high potential for the discovery of improved lipophilic
antifolates. Future work in our program will be in the ring systems of piritrexim and
trimetrexate; that is, 5-deaza- and 5,8-dideazapteridine types with emphasis on 5-alkyl
derivatives. Several of the promising candidates gave preliminary results indicating
they could be superior to agents in use or in advanced stages of clinical development.
Studies now in progress include tests for efficacy against the pathogens in infected
mice.

432
ACKNOWLEDGEMENT

This investigation was supported by Grant No. U01-AI30279 (J. R. P. and E.

R. P.) and Contract NOl-AI-87240 (S. F. Q.) from NIAID, NIH.

REFERENCES

1. Sattler, F. R. and Feinberg, J. Chest 1992, 101, 451-457.

2. Parrillo, J. E.; Chabner, B. A; and Masur, H. Antimicrab. Agents Chemather. 1988,
32, 430-433.
3. Kovacs, J. A; Allegra, C. J.; Kennedy, S.; Swan, J. C.; Drake, J.; Parrillo, J. E.;
Chabner, B.; and Masur, H. Am. J. Trap. Med. Hyg. 1988, 39, 491-496.
4. Broughton, M. C. and Queener, S. F. Antimicrab. Agents Chemather. 1991, 35,
1348-1355.
5. Rosowsky, A; Freisheim, J. H.; Hynes, J. B.; Queener, S. F.; Bartlett, M.; Smith,
J. W.; Lazarus, H.; and Modest, E. J. Biachem. Pharmacal., 1989, 38, 2677-
2684.
6. Araujo, F. G.; Lin, T.; and Remington, J. S. 1 Infect. Dis. 1993, 167, 494-497.
7. Piper, J. R.; Malik, N. D.; Rhee, M. S.; Galivan, J.; Sirotnak, F. M. 1 Med. Chem.
1992, 35, 332-337.
8. Pfefferkorn, E. R.; Pfefferkorn, L. C. J. Parasital. 1978, 64, 486-492.

433
ANALOGUES OF CLASSICAL ANTIFOLATES BEARING NAPHTHOYL

IN PLACE OF BENZOYL

I. R. Piper, 3 C. A. Johnson, 3 , J. A. Maddry, 3 J. J. McGuire,b G. M.

Otter,c and F. M. Sirotnakc

3Southern Research Institute, Birmingham, Alabama 35255

bRoswell Park Cancer Institute, Buffalo, New York 14263
cMemorial Sloan-Kettering Cancer Center, New York, New York 10021

INTRODUCTION

Molecular graphics approximating the binding of methotrexate (MTX) to

human dihydrofolate reductase (DHFR) reveal a large open space in the protein
structure which could accommodate potential inhibitors bearing in their side chains
groups of greater bulk than the benzoyl of the normal folate or a classical antifolate.
Candidate structures having larger groups may be readily designed to retain
conformation needed for binding by key groups, such as the salt-bridge forming a-
carboxyl group of the glutamic acid part. These observations prompted us to
synthesize 4-amino-1-naphthoyl analogues of known antifolates in order to test the
indications that inclusion of such larger or different groups would not lower the tight
binding of MTX-related structural types to DHFR.

MATERIALS AND METIIODS

Molecular Modelling

Molecular graphics studies were performed using an Evans and Sutherland

PS390 graphics station (DEC VAX 6420 mainframe computer under VMS 5.4) using
the MACROMODEL Version 3.0 and SYBYL Version 5.41 software packages.
Coordinates for the human DHFR binary complex with folate 1•2 were obtained from
the Brookhaven Protein Data Bank.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 435
Syntheses

Synthetic routes are indicated in Scheme 1. Side chain precursor 1 was

synthesized by two independent routes: (a) peptide coupling of known 4-
[[(benzyloxy)carbonyl]amino]-1-naphthoic acid 3a with diethyl L-glutamate followed by
hydrogenolysis (ambient conditions, 10% Pd/C in dioxane), and (b) peptide coupling
of reported 4-nitro-1-naphthoic acid3b with diethyl L-glutamate and subsequent
reduction (ambient, Raney Ni in EtOAc; or, preferably, ammonium formate in MeOH
containing 10% Pd/C4.) The methylamino compound 2 was prepared from 1 by
treatment with dimethyl sulfate in DMF containingN,N-diisopropylethylamine, and the
tetrahydro derivative 3 resulted from inadvertent over-hydrogenation in an early
experiment on the preparation of 1 from its nitro precursor.
Precursors 4-10 were prepared as previously reported 53 except bromomethyl
compounds 5 and 6 were prepared from the corresponding hydroxymethyl compounds
7 and 8 by a modified procedure using 20-25% dry HBr in acetic acid at room
temperature instead of HBr in dioxane. (This modification affords bromomethyl
compound 5 and 6 as ether-insoluble solids associated with HBr and acetic acid. In
a typical preparation, the formulation estimated by elemental analysis and 1 H NMR
spectral data is 6·1.7HBr·0.5CH3C02H.) The quinazoline precursor 11 was also
prepared by a reported procedure. 5b
The target compounds 12b-19b were synthesized via their purified (silica gel
column chromatography) diethyl esters 12a-19a which were prepared using one or
more of the general methods shown in Scheme 1. Methods A, B, and D as shown in
Scheme 1 are known general methods. Method C, however, has not been used
previously in this connection. The process involves the in situ formation of
dibromotriphenylphosphorane which then reacts with 5 or 6 to produce a 6-
bromomethyl intermediate whose amino groups have been converted to
triphenylphosphinimino functions. After alkylation of the side chain, the amino groups
are regenerated by treatment with acetic acid. This method has been used in the
replacement of nucleoside hydroxyl groups with halogen. 6

TEST RESULTS AND DISCUSSION

Effects on DHFR, Influx into Cells, and Tumor Cell Growth

Results listed in Table 1 show that naphthoyl analogues 12b-18b proved to be

as potent as MTX in inhibiting DHFR from L1210 cells, and they are more potent
than MTX as inhibitors of cell growth in vitro of L1210, HL60, and S1807 (see Table
1). Also, their transport through L1210 cell membranes (influx) is more facile than
that of MTX. Influx relative to MTX varied from 2.5-fold greater for 14b up to 6-fold
greater for 17b. Only with the tetrahydro derivative 19b does limiting bulk tolerance
begin to cause diminishing antifolate effects.

436
Effects on Human Folylpolyglutamate Synthetase (FPGS)

Studies of these analogues as substrates and inhibitors of folylpolyglutamate

synthetase (FPGS) from CCRF-CEM cells reveal substrate activity in this series is
markedly reduced relative to classical benzoyl analogues. 8 Only 13b showed significant
substrate activity, and it was 4.5-fold less active than MTX and over 70 times poorer
than aminopterin. All of the analogues were only weakly inhibitory to FPGS. Thus
it appears that human FPGS has a restriction for both catalysis and binding with
regard to the bulk that can be tolerated in this region of 2,4-diamino antifolates.

Antitumor Efficacy

The pteridine analogue 13b and 5-methyl-5-deazapteridine analogues 16b and

17b were tested along with MTX in mice against the E0771 mammary
adenocarcinoma. Results are listed in Table 2. The maximum tolerated dose of 16b
was the same as MTX (6 mg/kg) while that of 17b was 4-fold greater and that of 13b
was 25-fold greater. Although 16b proved to be toxic at the day-14 observation of
tumor volume (4 deaths in 5 mice), each of the compounds tested proved more
effective than MTX in reducing the solid tumor volume.

Scheme 1. Routes to Naphthoyl Analogues 12b-19b.

10. X = CCH 3 • Y = N
r--- a senes: R = Et
r- 19a: R = El (by Method D)
11· X =
Y =CK L.. 1gb· R =K
L..- b series: R = H
Structure Method used in
~0. X y prepn of ester~
~
12 N N H A
13 N N CK 3 A
14 CK N H B, D
16 CH N CH3 c
16 CCH 3 N H B, C
17 CCH3 N CH3 B. C
16 CH CH H D

aMethods of synthests. A, 6-CH 2Br compound 4 and sidechain precursors 1 or 2 in DMAC; B, 8-CH2 Br compound
5 or 6 with 1 or 2 in DMAC containing MgO; C. 6-CHzOH compound 7 or 8 with Ph 3 P and CBr 4 in DMAC followed
by 1 or 2 and later treatment with AcOH; D, 6-CN compound 9 or 11 w1lh l or 3 in reductive condensallon
(Raney Ni, AcOH).

437
 Table 1. In Vitro Comparisons of Naphthoyl Analogues 12b-18b and
Tetrahydronaphthoyl Analogue 19b with MTX Against Ll210, HL60, and S180.a

L1210 cell Ll210 cell IC50, nM

DHFR influx
Compound inhibn. Ki (J.£M) L1210 HL60 S180
Ki (pM)
MTX 4.82 ± 0.6 3.93 ± 0.4 9.0 ± 1.0 8.1 ± 1.0 10 ± 3.0
12b 4.55 ± 0.5 0.92 ± 0.2 6.0 ± 1.6 4.6 ± 1.0 5.9 ± 2.2
13b 5.22 ± 0.5 1.04 ± 0.3 9.0 ± 1.0 3.3 ± 0.8 9.3 ± 2.0
14b 3.65 ± 0.6 1.52 ± 0.3 4.2 ± 1.0 1.4 ± 0.2 3.9 ± 0.2
15b 4.65 ± 0.6 0.78 ± 0.2 2.3 ± 0.6 1.5 ± 0.3 1.9 ± 0.1
16b 5.08 ± 0.5 1.31 ± 0.2 3.9 ± 0.1 0.74 ± 0.1 3.8 ± 0.1
17b 4.84 ± 0.6 0.66 ± 0.1 2.4 ± 0.4 0.56 ± 0.1 2.6 ± 0.1
18b 5.2 ± 1.0 0.9 ± 0.1 6.1 ± 0.6 0.65 ± 0.03 3.9 ± 0.4
19b 13.1 ±2 0.6 ± 0.1 16 ± 3 17 ± 1 33 ± 2
aMethods described in ref. 7.

Table 2. Antitumor Activity of Naphthoyl Analogues 13b, 16b, and 17b, Compared with
MTX Against the E0771 Mammary Adenocarcinoma.a

Day 7 Day 10 Day 14

dosageb Ave. T/Cct Ave. T/Cct Ave. T/Cct
Compound (mg/kg) tum ore (%) tum ore (%) tum ore (%)
(mm3) (mm3) (mm3)
Control 221 624 1732
MTX 3 180 81 624 100 1437 83
6 108 49 187 30 540 31
13b 100 82 37 268 43 839 48
150 62 28 144 23 493 28
16b 3 113 51 421 68 1048 60
6 87 39 42 7 -toxic-
17b 12 108 49 333 53 1048 60
24 87 39 119 19 493 28
a5 mice/test; methods described in ref. 7. bRx daily for 5 days starting day 3 after
implantation. cvol. (mm3) = 4/3 1rr3. ctT/C = treated/control.

438
CONCLUSION

Antitumor activity of these compounds is due to potent inhibition of DHFR

combined with facile transport into tumor cells. Efficient influx into cells appears to
compensate for poor intracellular polyglutamylation.
Computer-generated molecular graphics based on X-ray crystallographic data
can serve as an aid in designing inhibitors of DHFR, but drug design considerations
regarding cell membrane transport and intracellular polyglutamylation must rely for the
present on structure-activity relationships. [Supported by Grants CA25236 (J.R.P.),
CA43500 (J.J.M.), CA18856 (F.M.S.), and CA22764 (F.M.S.) from NCI, NIH).]

REFERENCES

1. Davies, J. F; Delcamp, T. J.; Prendergast, N. J.; Ashford, V. A.; Freisheim, J. H.;

and Kraut, J. Biochemistry 1990, 29, 9467-7479.
2. Schweitzer, B. 1.; Dicker, A. P.; and Bertino, J. R. FASEB Joumal1990, 4, 2441-
2452.
3. (a) Nakayama, T.; Okutome, T.; Matsui, R.; Kurumi, M.; Sakurai, Y.; Aoyama, T.;
and Fujii, S. Chem. Phann. Bull. 1984, 32, 3968-3980. (b) Leuck, G. J.;
Perkins, R. P.; and Whitmore, F. C. J. Am. Chem. Soc. 1929, 51, 1831-1836.
4. Ram, S. and Ehrenkaufer, R. E. Synthesis 1988, 91-95.
5. (a) Piper, J. R.; Malik, N.D.; Rhee, M.S.; Galivan, J., and Sirotnak, F. M. 1 Med.
Chem. 1992,35, 332-337. (b) Davoll, J. and Johnson, A.M. J. Chem. Soc. (C)
1970, 997-1002.
6. Verheyden, J.P. H. and Moffatt, J. G. J. Org. Chem. 1972, 37, 2289-2299.
7. Sirotnak, F. M.; DeGraw, J. 1.; Schmid, F. A.; Goutas, L. J.; and Moccio, D. M.
Cancer Chemother. Pharmacal. 1984, 12, 26-30.
8. Bolanowska, W. E.; Russell, C. A.; McGuire, J. J. Arch. Biochem. Biophysics. 1990,
281' 198-203.

439
SYNTHESIS AND BIOLOGICAL ACTIVITY OF
TRICYCLIC, CONFORMATIONALLY RESTRICTED
ANAWGS OF LIPOPHILIC PYRID0[2,3-d]-
PYRIMIDINE ANTIFOLA TES.

Aleem Gangjee, *1 Farahnaz Mavandadi, 1 and Sherry F. Queener

1Division of Medicinal Chemistry

Graduate School of Pharmaceutical Sciences
Duquesne University
Pittsburgh, PA 15282
2Department of Pharmacology and Toxicology

School of Medicine
Indiana University
Indianapolis, IN 46202

INTRODUCTION

We have reported a series of phenyl substituted 2,4-diamino-5-methy1-6-anilinomethy1

pyrido[2,3-d]pyrimidines as inhibitors of dihydrofolate reductase (DHFR) from
Pneumocystis carinii and Toxoplasma gondii. 1•2 These are organisms which cause
opportunistic infections in AIDS patients and are often fatal. Among the more potent and
selective inhibitors ofT. gondii DHFR which we have reported was compound 1, the N-10
methyl analog, which was 11 times as potent and 30 times as selective as trimetrexate
(TMQ) 2 an FDA approved second line agent for P. carinii. Against P. carinii DHFR
compound 1 was 3 times as potent and 8 times as selective as 2.
In an attempt to study the influence of conformational restriction about r 1 and r2 on
potency and selectivity of 1 we designed the tricyclic analogs 3-6. Molecular modeling
using SYBYL3 and its SEARCH and MAXIMIN 2 options indicated that bridging the 5-
CH3 and N-10 of 1 in a 5-membered ring would allow for conformational rigidity and
orient the trimethoxybenzyl moiety in different yet suitable proximity to that attained by
the proposed DHFR bound conformation of 24 which was similar to the benzoyl group of
the bound conformation of the classical antifolate methotrexate (MTX). Molecular
modeling further indicated that the orientation of a trimethoxyphenyl moiety was predicated
almost entirely on the C-rin~ nitrogen and was not in close proximity to that of bound
TMQ, however, a methylene bridge between the tricyclic system and the trimethoxyphenyl

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 441
 OCH3
R= -\:tocH3
OCH3

SCHEMEl

- a

- b

d e
3 [5] + 6
I air oxidation t
orf ACH3
A= ~OCH 3
4 OCH3
a: THF/60°C/3 days; b: BrCH 2COOC 2HstDMF/60°C; c: NaOEVToluene;
d: Glacial CHaCOOH/120°C/12 hours; e: BH3/THF; f: MeOH/reflux;
g: Dowtherm-N240°C/2 hours.

moiety allowed for a much better overlap with bound TMQ and hence the
trimethoxybenzyl moiety was included as the side chain in preference to the
trimethoxyphenyl.

CHEMISTRY
The synthesis of the target compounds 3-6 was accomplished via the regiospecific
cyclocondensation of the P-ketoester 9 with 2,4,6-triaminopyrimidine (Scheme 1). Hurlbert
et al. 5 as well as reports from our laboratorf·7•8 have confirmed that /j-ketoesters condense
with appropriate 6-amino pyrimidines to afford regiospecifically angular, 5,6-disubstituted
pyrido[2,3-d]pyrimidines rather than the linear, 6,7-disubstituted analogs. Compound 9
was synthesized by the reaction of 3,4,5-trimethoxybenzylamine with ethylacrylate
followed by alkylation with ethylbromoacetate to afford 8 which on Dieckmann cyclization
gave the desired P-ketoester 9. Cyclocondensation of 9 with 2,4,6-triaminopyrimidine in

442
glacial acetic acid at 120° C for 12 hrs. afforded exclusively the lactam 3 in 55% yield,
while similar cyclocondensation in Dowtherm-A at 240° C for 2 hr. gave the
dehydrogenated lactam 4 as the only product in 40% yield. 1H NMR of 3 showed an
exchangeable (D20) lactam NH at o8.98 and three sets of methylene protons at o3.17,
3.78 and 4.43, as was previously reported for similar tricyclic compounds. 6 In contrast
the 1H NMR of 4 indicated a nonexchangeable olefinic proton at o 8.82 as we have
observed earlier, 8 and only two sets of methylene protons at o4.38 and 4.65. Both 3 and
4 were characterized via their 1H NMR, 13 C NMR, IR and elemental analysis. The lactam
3 was reduced using BH3/THF9 to afford a mixture of 5 and 6 in a 1: 1 ratio as indicated
by the 1H NMR and the mass spectrum of the mixture. All attempts to separate the
mixture resulted in the oxidation of 5 to 6. Thus only 6 was isolated as the pure reduced
lactam. This oxidation also occurs when the mixture is allowed to stand at room
temperature for an extended period. Conversion of the mixture to pure 6 was also
accomplished by reflux in methanol. The 1H NMR, 13C NMR, IR and elemental analysis
of 6 confirmed its structure. Compound 6 has been claimed in a patent10 via a different
synthetic route, however, no spectral or biological activity was reported.

BIOLOGICAL ACTIVITY AND DISCUSSION

The compounds 3-4 and 6 were evaluated as inhibitors of DHFR from P. carinii, 11
T. gondii 12 and rat liver (RL). Selectivity ratios were determined using RL DHFR as the
mammalian source. These IC50 values along with selectivity ratios are shown in Table I.
The inhibitory values for 1 and 2 are also included for comparison. The most potent of
the three target compounds against P. carinii DHFR and T. gondii DHFR was the
dehydrogenated lactam 4. More significantly this compound was about 93 times more
selective against P. carinii DHFR and about 110 times more selective against T. gondii
DHFR than 2. All three compounds were more selective than 2 against both forms of
DHFR. The significant increase in both potency and selectivity of 4 compared to 3
strongly suggested that partial planarity of the C-ring with the pyrido[2,3-d]pyrimidine
system was important. Thus, conformationally restricting r 1 and r 2 of 1 with partial
planarity of the C-ring as in compound 4 significantly increases selectivity against both P.
carinii DHFR and T. gondii DHFR compared to 1 and 2. Based on a comparison of 3 and
6 it appears that in the absence of planarity in the C-ring the lactam is somewhat
detrimental to potency as well as selectivity (except for T. gondii DHFR). A more
definitive assessment of the role of the lactam will be provided by the dehydrogenation of
6, which is currently underway.

Table 1. Inhibitory concentrations (IC5o) in ~-tM against DHFRs and selectivity ratios
for 3,4 and 6.
Selectivity Selectivity
ratio ratio
Compound Pc' RLb RL/Pc Tg" RL!Tg
3 94.5 42.9 0.45 9.0 4.8
4 3.1 20.3 6.5 0.62 32.7
6 12.6 11.7 0.9 5.3 2.2
1 0.013 0.007 0.58 0.0008 8.9
TMO 0.042 0.003 0.07 0.001 0.3
a: Pneumocystis carinii DHFR; b: Rat liver DHFR; c: Toxoplasma gondii DHFR.

443
ACKNOWLEDGEMENTS

This work was supported by a grant GM 40998 (AG) from The National Institutes
of General Medical Sciences, NIH and by a Public Health Service contract AI-87240
(SFQ) from the Division of AIDS, NIH.

REFERENCES

1. Gangjee, A.; Shi, J.; Queener, S.F. 5-Deaza Nonclassical Folates as Potent and
Selective Inhibitors of Dihydrofolate Reductase From Pneumocystis carinii and
Toxoplasma gondii, Presented at the 23rd National Medicinal Chemistry
Symposium. June 14-18, (1992), Buffalo, N.Y. Abstr.:61.
2. Vasudevan, A.; Gangjee, A.; Queener, S.F. Nonclassical 2,4-Diamino-5-methyl-5-
deaza Antifolates: Synthesis and Biological Activity. Presented at the 204th
American Chemical Society National Meeting, Washington, D.C. August 23-38,
(1992), Abstr. :MEDI 148.
3. SYBYL 5.5, TRIPOS Associates Inc.; St. Louis, MO 63144.
4. Sutton, P.A.; Cody, V. J. Med. Chern., 30:1843, (1987).
5. Hurlbert, B.S.; Ledig, K.W.; Stenbuck, P.; Valenti, B.F.; Hitchings, G.H. J. Med.
Chern. 11:703, (1968).
6. Gangjee, A.; Patel, J. J. Heterocyclic Chern., 25:1597, (1988).
7. Gangjee, A.; Donkor, I.O.; Kisliuk, R.L.; Gaumont, Y.; Thorndike, J. J. Med.
Chern., 34:611, (1991).
8. Gangjee, A.; O'Donnell, J.K.; Bardos, T.J.; Kalman, T.I. J. Heterocyclic Chern.,
21:873, (1984).
9. Degraw, J.I.; Christie, P.H.; Colwell, W.T.; Sirontnak, F.M. J. Med. Chern.,
35:320, (1992).
10. Watanabe, K.A. U.S. Patent 4,925,939, (1990).
11. Broughton, M.C.; Queener, S.F. Antimicrob. Agents Chemother. 35:1348, (1991).
12. Queener, S.F. Identification of Potent and Selective Inhibitors of Toxoplasma gondii
Dihydrofolate Reductase (DHFR) and Dihydropteroate Synthase (DHPS).
Thirty-first Interscience Conference on Antimicrobial Agents and Chemotherapy,
(1991), Abstr. 385.

444
NOVEL 2,4-DIAMINO-S-SUBSTITUTED FUR0[2,3-d]-
PYRIMIDINES AS POTENTIAL ANTIFOLATES.

Aleem Gangjee, *1 Rajesh Devraj, 1 Sherry F. Queener, and Roy L.

Kisliuk. 3

1Division of Medicinal Chemistry

Graduate School of Pharmaceutical Sciences
Duquesne University
Pittsburgh, PA 15282
2Department of Pharmacology and Toxicology

School of Medicine
Indiana University
Indianapolis, IN 46202
3Department of Biochemistry

Tufts University
Health Sciences Campus
Boston, MA 02111

INTRODUCTION

Opportunistic infections with Pneumocystis carinii and Toxoplasma gondii remain the
principal cause of death in patients with AIDS in the United States. For some time we
have been involved in the synthesis of antifolates in an attempt to provide more potent and
selective agents against dihydrofolate reductase from P. carinii and T. gondii than the
currently used antifolates for these infections. Our efforts have resulted in the synthesis
of bicyclic 6-6 ring fused analogs involving the pyrido[2,3-d]pyrimidines 1 and the
tetrahydroquinazolines. 2 In addition we have also synthesized 6-6-6 ring fused systems
involving the pyrimidonaphthyridines3 and the 6-6-5 pyrrolo fused pyrido[2,3-
d]pyrimidines.4 Of these analogs several have been found to be more potent and/or
selective than the currently approved antifolates, trimethoprim, pyrimethamine and
trimetrexate. In addition we have also observed significant antitumor activity in some of
these classical and nonclassical analogs.

NH2 X

N:)S~--o
HNAN 0
2
1 2. X = 3,4,5(0CH3b
3. X = 2,S(OCH3)2
4. X = 3,4(CI)2
5. X = 3,4,5(CI)3

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 445
 A logical extension of our 6-6 ring fused antifolates are the 6-5 ring fused
heterocyclics 1. Molecular modeling of 6-5 systems superimposed on 6-6 systems (Fig.
1). clearly indicates that 5-substituents of the 6-5 system lie close to the 5-substituent of
the 6-6 systems and that 6-substituents of the 6-5 systems lie between the 6 and 7-
substituents of the 6-6 system. Thus 6-5 systems may be 5 and/or 6-substituted. In
addition, since the B-ring is contracted, the 5- and or 6- substituent(s) could have one to
three atoms in the bridge to the phenyl moiety thus mimicking the known 6-6 antifolates.
In this paper we wish to report the nonclassical 5-substituted furo[2,3-d]pyrimidines 2-5
as the first in our series of classical and nonclassical bicyclic and tricyclic 6--5 ring fused
systems as potential antifolates.

Figure 1. Superimposition of a 6-5 ring fused system on a 6-6 ring fused system.

Though the furo[2,3-d]pyrimidine antifolates 2-5 are novel, other 6-5 ring fused
systems as potential antifolates have been reported by Roth et al. ,5 Rosowsky et al. 6,
Elslager et aP and others. A recent resurgence of interest in 6-5 ring fused classical
antifolates is evidenced by the significant inhibitory effects of pyrrolo[2,3-d]pyrimidines
against DHFR, thymidylate synthase and tumor cells reported by Miwa et al. 8 and Taylor
et al. 9 and the potent cytotoxic effects of the pyrazolo[2,3-d]pyrimidines.10

CHEMISTRY

The synthesis of the target compounds were carried out as shown in Scheme I.
Facile entry into the furo[2,3-d]pyrimidine ring system was provided by the reaction of
2,6-diamino-4-hydroxypyrimidine and 1,3-dichloro acetone in DMF at room temperature

SCHEME 1

NH2 X

6 +
b
N:):)~-o
H2NAN 0
7. X = 3,4,5(0CH3)3 2. X = 3,4,5(0CH3b
8. X = 2,5(0CH3)2 3. X = 2,5(0CH3)2
9. X = 3,4(CI)2 4. X = 3,4(CI)2
10. X = 3,4,5(CI)3 5. X = 3,4,5(CI)3

a. DMF, R.T., 24 hrs; b. DMSO, K2C0 3, R.T., 72 hrs.

446
for 24-28 hrs. to afford 6 based on a report by Secrist and Liu. 11 Purification of the
product, which falls out of solution, was carried out via column chromatography to afford
pure 6 in 65-68% yield. Compound 6 was not soluble in DMF or DMAc at room
temperature, thus, heating to 55° C was necessary to form a solution to which was added
3,4,5-trimethoxyaniline 7. The displacement reaction was not fast enough and extensive
degradation of 6 occurred. The reaction, however, proceeded smoothly in anhydrous
DMSO with 2 equivalents of anhydrous K2C03 at room temperature over a period of 72
hrs. to afford the target compound 2. Isolation of 2 from the reaction mixture was greatly
simplified by adding excess water and stirring the mixture at room temperature for 6-8 hrs.
during which time the product 2 separated. The crude product was further purified by
chromatography on silica gel using 5% methanol in chloroform as the eluent. Target
compounds 3-5 were similarly obtained by halide displacement of 6 by the appropriate
anilines 8-10 respectively.

Table 1. Inhibitory concentrations (IC5o) in JLM against DHFRs.

ComJlound Pc" RLb Tg" Led Humane

2 >4 >37 >4 >26 >26
3 >21 >21 >21 >31 > 15
4 >35 35.2 89.3 > 3 >15
5 8.3 25.6 > 3.9 >25 >25
a: Pneumocystis carinii DHFR; b: Rat liver DHFR; c: Toxoplasma gondii DHFR.
d. Lactobacillus casei DHFR; e. Recombinant human DHFR, from Dr. J. Freisheim.

BIOLOGICAL ACTIVITY AND DISCUSSIONS

The compounds were evaluated as inhibitors of DHFR from P. carinii, rat liver
(RL), T. gondii, L. casei and recombinant human DHFR and the results are indicated as
IC50 values in Table I. None of the nonclassical compounds 2-5 showed significant
inhibitory effects. However, the classical analogs 11 and 12 (the synthesis and inhibitory
effects of which will be reported shortlyl4) showed significantly greater inhibitory activity
against DHFR and tumor cells in culture and are currently undergoing preclinical screening
against tumor cells in culture by the National Cancer Institute. Clearly a nonclassical 5-
substituted anilino methyl system is not conducive to potent DHFR inhibition in the
furo[2,3-d]-pyrimidines, however, in the classical series a methylamino bridge does
provide for significant inhibition. We have now developed synthetic methodology for three
atom bridged analogs as well as the 6- substituted, 5,6-disubstituted, and ~-alkyl analogs.
Synthesis and biological evaluations of these compounds is currently underway and should
provide a better understanding of the structure activity relationship of furo[2,3-d]-
pyrimidines as potential inhibitors of folate metabolizing enzymes.

447
ACKNOWLEDGEMENTS

This work was supported in part by a grant GM 40998 (AG) from The National
Institutes of General Medical Sciences, NIH, AI 30900 (AG) from NIAID, NIH and by
a Public Health Service contract AI-87240 (SFQ) from the Division of AIDS, NIH.

REFERENCES

1. Gangjee, A; Shi, J.; Queener, S.F. 5-Deaza Nonclassical Folates as Potent and
Selective Inhibitors of Dihydrofolate Reductase From Pneumocystis carinii and
Toxoplasma gondii, Presented at the 23rd National Medicinal Chemistry
Symposium. Buffalo, N.Y. June 14-18, 1992. Abstr:61.
2. Zaveri, N.; Gangjee, A.; Queener, S.F. A Series of Nonclassical2,4-Diamino-6-
(aminomethyl)tetrahydroquinazoline Antifolates: Synthesis and Biological
Activity. Presented at the 204th American Chemical Society National Meeting,
Washington, D.C. August 23-38, 1992, Abstr:MBDI 130.
3. Shi, J.; Gangjee, A.; Queener, S.F. Conformationally Restricted, Tricyclic
Pyrimido[4,5-c][2,7]napthyridine Analogs of Trimetrexate. Presented at the
203rd American Chemical Society National Meeting, San Francisco, CA. April
5-10, 1992. Abstr:MBDI 28.
4. Gangjee, A.; Mavandadi, F.; Queener, S.F. Synthesis and Biological Activity of
Tricyclic, Conformationally Restricted Analogs of Lipophilic Pyrido[2,3-
d]pyrimidines Antifolate. This Symposium.
5. Roth, B. J. Med. Chern. (1969), 12:227.
6. Rosowsky, A.; Chaykovsky, M.; Chen, K.K.N.; Lin, M. and Modest, B.J. J. Med.
Chern. (1973), 16:185,188,191.
7. Blslager, B.F; and Davoli, J. "Lee. in Hetero. Chern.," (1974), 2:S-97.
8. Miwa, T.; Titaka, T.; Akimoto, H. and Nomura, H. J. Med. Chern. (1991),
34:555.
9. Taylor, B.C.; Kuhnt, D.; Shih, C.; Rinzel, S.M.; Grindey, G.B.; Barredo, J.;
Jannatipour, M. and Moran, R.G. J. Med. Chern., (1992), 35:4450.
10. Taylor, B.C. and Patel, H.H. Tetrahedron, (1992), 48:8089.
11. Secrist, III, J.A. and Liu, P.S. J. Org. Chern., (1978), 43:3937.
12. Broughton, M.C.; Queener, S.F. Antimicrob. Agents Chemother, (1991),
35: 1348-1355.
13. Queener, S.F. Identification of Potent and Selective Inhibitors of Toxoplasma gondii
Dihydrofolate Reductase (DHFR) and Dihydropteroate Synthase (DHPS).
Thirty-first Interscience Conference on Antimicrobial Agents and
Chemotherapy, (1991), Abstr:385.
14. Devraj, R.; Gangjee, A. and Kisliuk, R.L. Novel 2,4-Diamino-5-substituted
furo[2,3-d]pyrimidines as Potential Classical Antifolates. Presented at the
205th American Chemical Society National Meeting. Denver, CO. March 28,
- April2, 1993. Abstr:MBDI 19.

448
BICYCLIC CONFORMATIONALLY RESTRICTED
ANALOGS OF NONCLASSICAL PYRID0[2,3-d]
PYRIMIDINES AS POTENTIAL INHffiiTORS
OF DlliYDROFOLATE REDUCTASES

Aleem Gangjee, *1 Anil Vasudevan, 1 and Sherry F. Queener

Division of Medicinal Chemistry

1

Graduate School of Pharmaceutical Sciences

Duquesne University
Pittsburgh, PA 15282
2Department of Pharmacology and Toxicology

School of Medicine
Indiana University
Indianapolis, IN 46202

INTRODUCTION

Pneumocystis carinii and Toxoplasma gondii remain the major cause of morbidity and
mortality in patients with AIDS. We have recently reported the synthesis of a series of
2,4-diamino-5-methyl-6-substituted pyrido[2,3-d]pyrimidines of general structure 11·2 and
their selective and potent inhibitory activity against dihydrofolate reductases (DHFR) from
P. carinii and T. gondii. These compounds, like the clinically used nonclassical
antifolates, trimethoprim, pyrimethamine and trimetrexate (TMQ), bypass the folate uptake
mechanisms necessary for classical antifolates such as methotrexate and thus are able to
penetrate P. carinii and T. gondii organisms which appear to lack the folate uptake
systems. Compounds 1 were designed as hybrid molecules of the nonselective DHFR
inhibitors TMQ and piritrexim (PTX) to provide more potent and selective inhibitors of
P. carinii DHFR and/or T. gondii DHFR. The most potent and selective of the series of
compounds 1 was that with R1 = CH3 and R2 = 3' ,4' ,5'-trimethoxy which was several
times as potent and selective as the analog with R1 = H as well as TMQ and PTX against
both P. carinii DHFR and T. gondii DHFR. Larger R 1 substituents such as Et, i-Pr and
propargyl decrease potency (except R1 = i-Pr for P. carinii DHFR) with some loss of
selectivity towards both P. carinii DHFR (except R1 = i-Pr) and T. gondii DHFR. In an
attempt to study the effect of conformational restrictions of 7 3 of 1 to potency and
selectivity we have synthesized the methoxy indoline analogs 2-4, the corresponding indole

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 449
analogs 5-7 and the 6,7-dimethoxytetrahydroisoquinoline analog 8. In a previous report/
we had shown that restriction of r 3 , by means of an indoline, in the 2,4-diaminotetra-
hydroquinazoline series, does indeed provide increased potency and selectivity for T.
gondii DHFR compared to the unrestricted analog.

·~WaR
2&5.R=4-0CH3 N~N8
3&6. R=5-0CH3 H NAN~NJ f ~
2
4 & 7. R = 5,6(0CH3)2 - R
2·4 5-7

CHEMISTRY

Our general approach towards the synthesis of the target compounds was a
modification of the procedure reported by Piper et al. 4 for the synthesis of the common
intermediate 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile 10 (Scheme 1).
Improved yields of this intermediate were obtained on increasing the molar ratios of
guanidine hydrochloride as well as the reaction time (76% :literature4 58%). Compound
10 was reduced to the corresponding aldehyde 11 using Ni-Al alloyS in quantitative yield.
We have previously reported the reductive amination of acetylated 2,4-diamino
tetrahydroquinazoline-6-carboxaldehyde with a variety of secondary anilines and
benzylamines including indoline. 3 This method was employed with the 2,4-diamino-5-
methylpyrido[2,3-d]pyrimidine-6-aldehyde 11 for the synthesis of the target compounds
2-4. The desired indolines were obtained via reduction of the corresponding indoles in
acetic acid with NaCNBH3 • Initial attempts at the reductive amination of 4-methoxy, 5-
methoxy and 5,6-dimethoxy indolines with 11 in 5N HCl-MeOH/NaCNBH3 did not afford
the target compounds 2-4. We reasoned that perhaps limited solubility of 11 in MeOH was
responsible for the failure of the reaction. Thus, we converted the aldehyde 11 to its
acetate salt and carried out the reductive amination in MeOH with NaCNBH3 at a pH =
6, (adjusted with HCl) to afford the target compounds 2,3 and 4 in 30%, 32% and 36%
yields respectively. The IR, 1H NMR and elemental analysis of these compounds
confirmed their structure.
For the synthesis of the corresponding indole derivatives compound 11 was reduced
to the corresponding alcohol 12 using NaBH4 in MeOH. Compound 12 was brominated
with anhydrous HBr/Dioxane to afford the bromomethyl compound 13 as its hydrobromide
salt which was used immediately without purification in the displacement reaction. Initial

450
attempts at alkylating the methoxy indoles with 13 resulted in recovery of starting material,
presumably due to the rapid quenching of the indole anion by the hydrobromic acid present
in 13. However, when 13 was stirred in anhydrous DMF with excess triethylamine and
was then added dropwise to the indole anion generated in situ with NaH in DMF the halide
displacement occurred affording the corresponding indole derivatives 5-7. 6, 7-Dimethoxy-
1,2,3,4-tetrahydroisoquinoline was alkylated with 13 in anhydrous N,N-DMAC using
CsC03 to yield 8 in 70% yield.

SCHEMEl

H : c r CH3
2 N CHO
N
Ni-Al alloy.._ 1_
HCOOH ~N N
(Quant.) H2N
10 11
9

NaCNBH3
MeOH

Ct:J
R R

N,N-DMAC

CsC03 H-N~
~OCH3
NaH,DMF (JQ I

OCH3 H
H
8 5 ,6&7 2,3&4.

Table 1. Inhibitory concentrations (IC 5o) in ~tM against DHFRs and selectivity ratios for
2,3, 4 and 14.
Selectivity Selectivity
ratio ratio
Compound No. Pc" RL!Pc Tg" RL!Tg
2 0.29 0.15 0.52 0.048 3.1
3 0.25 0.17 0.68 0.057 3.0
4 0.41 0.23 0.56 0.049 4.7
14 0.044 0.0076 0.17 0.0088 0.86
TMQ 0.042 0.003 0.07 0.01 0.3
PTX 0.038 0.0015 0.04 0.011 0.14
a: Pneumocystis carinii DHFR; b: Rat liver DHFR; c: Toxoplasma gondii DHFR.

BIOLOGICAL ACTIVITY AND DISCUSSIONS

Preliminary biological activity of the target compounds 2-4 as inhibitors of DHFR

from P. carinii, 6 T. gondiP and rat liver (RL) are reported in Table I. Selectivity ratios
are also reported and were determined using RL DHFR as the mammalian source. The
appropriate values for TMQ, PTX and the conformationally unrestricted analog 14 (1, R1
= H, R2 = 3'4 '-dimethoxyl are also listed for comparison. Restriction of r 3 via
incorporation into an indoline ring leads to a decrease in potency against all three DHFRs.
Significantly, however, a distinct increase in selectivity was observed against both P.
carinii DHFR and T. gondii DHFR for compounds 2-4, compared to 14, TMQ and PTX.

451
This indicated that conformational restriction about r 3 is conclusive to selectivity. The
indole derivatives 5-7 and the tetrahydroisoquinoline analog 8 will be biologically evaluated
in the near future and the results should further define the effects of r 3 restriction with
respect to inhibition of DHFRs.

ACKNOWLEDGEMENTS

This work was supported by a grant GM 40998 (AG) from The National Institutes
of General Medical Sciences, NIH and by a Public Health Service contract AI-87240
(SFQ) from the Division of AIDS, NIH.

REFERENCES

1. Gangjee, A; Shi, J.; Queener, S.F. 5-Deaza Nonclassical Folates as Potent and
Selective Inhibitors of Dihydrofolate Reductase From Pnewnocystis carinii
and Toxoplasma gondii, Presented at the 23rd National Medicinal Chemistry
Symposium. Buffalo, N.Y. June 14-18, 1992. Abstr.:61.
2. Vasudevan, A.; Gangjee, A.; Queener, S.F. Nonclassical 2,4-Diamino-5-methyl-
5-deaza Antifolates: Synthesis and Biological Acitvity. Presented at the
204th American Chemical Society National Meeting, Washington, D.C.
August 23-28, 1992. Abstr: MEDI 148.
3. Zaveri, N.; Gangjee, A.; Queener, S.F. A Series of Nonclassical 2,4-Diamino-6-
(aminomethyl)tetrahydroquinazoline Antifolates: Synthesis and Biological
Activity. Presented at the 204th American Chemical Society National
Meeting, Washington, D.C. August 23-38, 1992, Abstr.:MEDI 130.
4. Piper, J.R.; McCaleb, G.S.; Montgomery, J.A.; Kisliuk, R.L.; Gaumont, Y.;
Sirotnak, F.M. J. Med. Chern., 29:1080-87, (1986).
5. Piper, J.R.; Montgomery, J.A.; Sirotnak, F.M.; Kisliuk, R.L. J. Med. Chern.,
31:264, (1988).
6. Broughton, M.C.; Queener, S.F. Pneumocystis carinii Dihydrofolate reductase used
to Screen Potential Antipneumocystis Drugs. Antimicrob. Agents
Chemother, 35:1348-1355, (1991).
7. Queener, S.F. Identification of Potent and Selective Inhibitors of Toxoplasma gondii
Dihydrofolate Reductase (DHFR) and Dihydropteroate Synthase (DHPS).
Thirty-first Interscience Conference on Antimicrobial Agents and
Chemotherapy, Abstr. 385, (1991).

452
SYNTHESIS, STRUCTURAL AND
BIOCHEMICAL CHARACTERIZATION OF
CYTOSTATIC METHOTREXATE-y-
GLUTAMYL-GLUTATHIONE CONJUGATES

Martin Kussmann, Dieter Wiehr, Thomas

Knepper, and Michael Przybylski

Faculty of Chemistry
University ofKonstanz
P.O. Box 5560
W-77 50 Konstanz, Germany

Introduction

The synthesis and pharmacological evaluation of methotrexate (MTX) derivatives with

the aim of obtaining modified cellular uptake and transport properties, and cytostatic activity
has been subject to intensive studies in the last yearsl. Among several approaches,
modifications at the y-glutamyl residue of MTX have been carried out both by covalent
attachment of oligopeptides2,3 and lipid moieties4, the latter adducts being able to form
defined supramolecular structures. Based on previous work by Leszcynska and Pfaff who
observed a significantly enhanced uptake of MTX in hepatocytes in the presence of
extracellular glutathione (GSH)5, we have synthesized a series of covalent MTX-glutathionyl
peptide conjugates. In the present study, an efficient synthetic pathway, structural and initial
biochemical characterization data of MTX-y-glutamyl-GSH derivatives are described. The
surprisingly high in vitro dihydrofolate reductase (DHFR) inhibitory activity found indicates
these derivatives to be potentially interesting new cytostatic antifolates3.

RESULTS AND DISCUSSION

The principal pathways for the synthesis of MTX-(y-glutamyl)-glutathione conjugates

are schematically shown in Figure 1.
Starting with the oxidized glutathione (GSSG) moiety, oligopeptide precursors with
different chain lengths (e.g. for MTX conjugates 1-4) were synthesized by stepwise
N-terminal prolongation with y-glutamyl residues using DCC/HOBT-ester activation and
Fmoc and tert. butylester protecting groups. The same coupling procedure can be directly
employed for the final condensation with 4-amino-NlO_methylpteroic acid (APA), resulting in
MTX-peptide adducts encompassing y-oligoglutamyl residues identical to the naturally
occurring intracellular metabolites3.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 453
 :
· 1 -
H~N~ :
COOIBu :
N--c
H O
N/'COOIBu

+ .: H 0
.: S H

~H 0 ~ (S H
H~N~N~N........,..COOtBu
APA : COOIBu : H 0

L J
l DCC,HOBt

·
~ COOIBu ~ H
1 -
Pleroy~N~
: H
:
0 :
N--c o
N/'COOtBu
S H
: : '
: H 0 : (S H
Pleroy~N~N~N........,..COOtBu

...
: COOIBu ~
n
H O

TFA-domgo r 1
TFA- cleavage,
DTI - reduction

~ COOH ~H O
Pleroy._;. N~ N..._)J...N/'COOH
: H 0 ~ l5 H
' I

: 0 : S H
.H
Pleroy~ N. ~~ . ~ N........,..COOH
, y - :-N n
: COOH ~ H O n = 2, 3
n 1 ..!
n = 2, 3
!·~
Figure 1. Scheme of synthetic pathways to MTX-y-oligoglutyrnyl-gluthathione conjugates, based on
corresponding oligopeptide precursors. y-Glutarnyl peptide building blocks were obtained by stepwise
N-terrninal prolongation using Frnoc and tert.butylester protecting groups.

454
 The final MTX-GSH conjugates 1-4 were obtained by subsequent reductive
dithiothreitol (DTT) cleavage and trifluoroacetic acid (TFA) deblocking in good yields (1 0-
15%, relative to the common glutathionylester precursor; Table 1). Their structures and
molecular homogeneity were established by 1H-NMR and UV-spectroscopy and, particularly,
by fast atom bombardment (FAB) mass spectra (Table 1). As exemplified by the spectrum of
the tert.-butylester precursor of MTX-conjugate 2 (Figure 2), FAB-MS in
3-nitrobenzylalcohol or glyceroVacetic acid matrices provide abundant molecular ions even
for high molecular weight derivatives, and structurally characteristic fragmentation at the
peroyl amide and peptide bonds7.

cootBu 0 cootBu 0 2195 MH+

N):~ NJN-~C~NH~~._,..A.Nif'cootBu
I ... CH.'=f
1,.. o cootBu 0 l_
H:!N..-;:N N _ _ _st-------r> A

Nt.xNt~-!2ii:~~HNY'f~~cootBu
H:!N~N N cootBu o cootBu 0)

D
C B

1099 AH+

DH+
2093

BH+ CH+
1887 2020

1200 1600 2000 2400 m/z

Figure 2. FAB mass spectrum of MTX-y-diglutamyl-gluthathione-tert.-butylester precursor of MTX-
conjugate 2. Major fragmentation pathways (arrows) are marked A, B, C, D.

Initial biochemical studies of the MTX-glutathionyl-conjugates were carried out by

characterization of their in vitro DHFR inhibitory activity. All peptide derivatives (1-4)
exhibit a remarkably high inhibition of chicken liver DHFR with ICso values 10-40 fold lower
compared to parent MTX (Table 1). The molecular basis for this high enzyme inhibitory
activity has not yet been evaluated, but is assumed to be related to the y-glutathionyl moiety
as suggested by molecular modelling studies of the DHFR-complexes with the MTX-peptide
conjugates. A potentially specific interaction of these conjugates will be characterized in
further studies, using defined chemical modifications ofDHFR.

455
 Table 1. Yields, structural data and biochemical characterization of MTX-y-
oligoglutamyl gluthathione conjugates 1-4.

Yielda MS 1H-NMRC IC5od

UV/E370
MH+b N10_CH) x1o-10

[%] mlz (%) [ppm] [mol/!]

[l/mol/cm]

1 15 1487 (12) 15530 3.2 4.51

2 14 1742 (25) 11730 3.2 8.71
3 ue 745 (100) 7280 3.2 14.3
4 we 873 (60) 6680 3.25 4.72

MTX 183

a Yield relative to the peptide precursor, Bis[L-y-glutamyl-(a-tert.-butylester)-L-y-glutaruyl-(a-tert.-

butylester) ]-L-cysteinyl-bis-[glycine-tert. -butylester].
b Fast atom bombardment (FABMS) or plasma desorption (PDMS) data.
c Data for W0methylpteroyl-group; chemical shifts are identical for conjugates 1-4.
dIn-vitro inhibitory activity against chicken liver DHFR. DHFR inhibition assays were performed
according to Bertino and Fischer (6).
e Incomplete cleavage to 3 and 4.

ACKNOWLEDGEMENTS

This work has been supported by grants from the Deutsche Forschungsgemeinschaft,
Bonn, FRG, and by the Fonds der chemischen Industrie.

REFERENCES

l. F.M. Huennekens, K.S. Vitols, and G.B. Henderson, Adv. Enzyme Related Areas Mol. Bioi. 47:313
(1987).
2. K.S. Vitols, Y.D. Montejano, H. Kuefner, and F.M. Huennekens, Pteridines, 1:65 (1989).
3. M. Przybylski, R Renkel, and P. Fonrobert, in: "Chemistry and Biology ofPteridines", B.A. Cooper and
V.M. Whitehead, eds, De Gruyter, Berlin 1986, 65.
4. T. Knepper, M. Przybylski M. Ahlers, and H. Ringsdorf, in: "Chemistry and Biology ofPteridines",
H. C. Curtius, S. Ghisla, N. Blau, eds, De Gruyter, Berlin 1990, 1280.
5. A. Lescynska and G. Pfaff, Biochem. Pharmacal. 34:1627 (1985).
6. J.R. Bertino and G.A. Fischer, Meth. Med. Res. 10:297 (1964).
7. M. Przybylski: in: "Chemistry and Biology ofPteridines", H. C. Curtius, S. Ghisla, N. Blau, eds., De
Gruyter, Berlin 1990, 140.

456
ACTIVATION BY PEPTIDASES AND CYTOTOXICITY OF
2-(L-a-AMINOACYL) PRODRUGS OF METHOTREXATE

H. T. Andrew Cheung, Zhen Dong, Luc Escoffier, Mary A. Smal and

Martin H. N. Tattersall

Departments of Pharmacy and Cancer Medicine

University of Sydney, Sydney, NSW 2006, Australia

The clinical use of methotrexate (MTX) in cancer chemotherapy is limited by its

toxicity towards normal cells. In principle, the side-effects may be minimised by
selectively generating the active drug at the tumour site from a latent form of the drug
(prodrug). Suitable prodrugs of MTX are the 2-(L-a-aminoacyl) derivatives, which are
not expected to bind tightly to the target enzyme dihydrofolate reductase (DHFRV Such
derivatives may be cleaved enzymatically to the active drug by the action of the
appropriate aminopeptidase. In a direct approach, selective generation of the active drug
may be achieved since there are reports of elevated levels of aminopeptidase activity
associated with certain tumour sites.2 An alternative approach is to target the
appropriate exogeneous enzyme by conjugation to tumour-specific antibodies (the
"ADEPT" strategy)Y The use of these two prodrug strategies requires that the
prodrugs are not cleaved prematurely before reaching the tumour site. This paper
describes studies aimed at determining which 2-(L·a-aminoacyl) derivatives of MTX are
suitable in either of the above two prodrug strategies.

MATERIALS AND METHODS

Materials. The following 2-(L-a-aminoacyl) derivatives of MTX were prepared as

described5•6 and were assayed by high pressure liquid chromatography (HPLC) (see
below) for MTX content(%): L-leucyl (Leu-MTX) (7.7% MTX), L-isoleucyl (Ile-MTX)
(0.5%), L-valyl (Val-MTX) (0.6%) and L-pyroglutamyl (Pyr-MTX) (14.1%).
Aminopeptidase M (AP-M) (EC 3.4.11.2) from porcine kidney and pyroglutamate
aminopeptidase (Pyr-AP) (EC 3.4.19.3) from Bacillus amyloliquefaciens were obtained
from Boehringer Mannheim and the Sigma Chemical Co. respectively; for these two
enzymes, activity is defined as: one unit will hydrolyse 1.0 pmol of L-leucine-p-
nitroanilide and 1.0 nmol of L-pyroglutamyl ~-naphthylamide respectively. Pure
dihydrofolate reductase from Lactobacillus casei was a gift from Dr. J. Feeney of the
National Institute for Medical Research, London. Methotrexate was provided by Lederle
Laboratories. All other reagents were from the Sigma Chemical Co.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 457
 HPLC analyses were carried out on an isocratic system consisting of a Spectra
Physics Isochrom LC pump, a Rheodyne injector fitted with a 20pl sampling loop, a
Spectra Physics Spectra 100 variable wavelength detector set at 300 nm and a Merck
5pm LiChrospher 100 RP-18 column (125mm x 4mm). The mobile phase was a mixture
of 0.1M phosphate buffer pH 7.3 and methanol (in ratios ranging from 68:32 to 80:20,
depending on the aminoacyl group) at a flow rate of 0.8ml/min. Areas under the peaks
were determined and the results expressed as a percentage of the total area.

Stability and Enzymic Hydrolysis of MTX Derivatives. MTX derivatives were

incubated at 37°C in a mixture of 1M phosphate buffer pH 7.3 and human serum, with
or without the appropriate aminopeptidase. Samples were withdrawn and analysed by
HPLC.

Inhibition of DHFR. Inhibition by MTX and its derivatives of DHFR-catalysed

NADPH-dependant reduction of dihydrofolate was measured as described by Mathews
et al/ but with 50mM TRlS pH 7.5/150mM KCl replacing 40mM phosphate.

In Vitro Cytotoxicity. Murine leukaemia L1210 cells were grown as suspension

cultures in RPMI-1640 medium (Flow Laboratories) supplemented with 10% non-
dialysed fetal calf serum (Commonwealth Serum Laboratories, Melbourne), 2mM L-
glutamine (Cytosystems) and 32pg/ml gentamycin (David Bull Laboratories). 24 Hours
before drug addition, cells were suspended in fresh medium at a concentration of 5x104
cells/ml, and all experiments were carried out on exponentially growing cells. Each drug
was administered as a single dose and remained in culture for the duration of the
experiment. Cell counts were made at 24 h and 48 h using phase-contrast microscopy to
discriminate between live and dead cells.

RESULTS AND DISCUSSION

Stability of Prodrugs

Results of stability studies on the prodrugs in serum-1M phosphate buffer pH 7.3

are shown in Table 1; these results were essentially identical to those obtained in buffer
alone (not shown), except for Leu-MTX which was substantially hydrolysed in serum.
All prodrugs synthesised initially contained some MTX, in most cases less than 1%.
This is believed to be a by-product of the deprotection step utilising trifluoroacetic
acid. 6 Very little MTX was formed from all prodrugs (except Leu-MTX) during the
incubation with serum, indicating that there are no aminopeptidases or other hydrolytic
enzymes in serum capable of acting on these substrates. However, all prodrugs except
Pyr-MTX decomposed slowly to a compound with an increased retention time. The
following observations were made regarding this decomposition product. Its ultraviolet
absorption was very similar to MTX and the nature of the aminoacyl group influenced
the retention time. While no decomposition occurred in 1M phosphate buffer pH 5.2,
decomposition increased with a decrease in buffer strength or an increase in pH. This
suggests that a free amino group is involved in the decomposition reaction. Attempts to
isolate and characterise the decomposition product have so far been unsuccessful.
All prodrugs released MTX when incubated with the appropriate enzyme (Table 1).
Hydrolysis of Leu-, Ile- and Val-MTX was rapid with porcine kidney aminopeptidase
(AP-M). Pyr-MTX hydrolysis using pyroglutamate aminopeptidase (Pyr-AP) was slower,
indicating that Pyr-MTX is a poor substrate.

458
 Table 1. Stability and enzymic cleavage of prodrugs

Prodrug Concentration Enzyme Composition'(%)

(M) (cone. in U/ml)

Prodrug MTX decomp.

Val-MTX 4.8 X 10"5 62.0 1.7 36.3

Val-MTX 4.8 X 10·5 AP-M (0.08) 29.8 43.0 27.1
Leu-MTX 1.0 X 10"5 42.7 24.1 33.1
Leu-MTX 1.0 X 10·5 AP-M (0.08) 5.1 81.5 13.4
Ile-MTX 7.8 x 10·4 61.4 4.7 33.8
Ile-MTX 7.8 x 10·4 AP-M (0.08) 23.3 44.0 32.7
Pyr-MTXb 6.9 X 104 75.0 25.0 0
Pyr-MTX 7.5 x 10·4 Pyr-AP (500) 53.8 46.1 0

• After a 60-min incubation in 2:10 serum- 1M phosphate buffer pH 7.3 at 37'C, unless otherwise stated.
b Composition refers to a 120-min incubation in 4:10 serum -buffer at 37'C.

Inhibition of DHFR

MTX and Val-MTX were found to inhibit DHFR with IC50 values of 4.7 x 10·8M
and 1.7 x 10·6M respectively. Since Val-MTX is contaminated by 0.6% MTX, the small
degree of inhibition observed is mostly due to contamination. Hence Val-MTX is a
very poor inhibitor of DHFR and is expected to be non-cytotoxic. After Val-MTX was
incubated with AP-M, 37% MTX was formed and the increase in the IC50 to 1.2 x 10·
1M correlates with the MTX concentration. Similar results were observed for Pyr-MTX.

In a separate experiment, Val-MTX was allowed to be partially converted to the

decomposition product and the resultant mixture was tested for inhibition of DHFR. The
mixture was found to be less inhibitory than before decomposition, indicating that the
decomposition product is even a poorer inhibitor of DHFR.

Cytotoxicity

Prodrug - enzyme combinations were tested for their capacity to inhibit the growth
of murine L1210 cells in vitro (Table 2). Prodrugs alone were approximately 10 times
less cytotoxic than MTX, except for Leu-MTX which was substantially cytotoxic. For
the other prodrugs, significant cytotoxicity was observed at high concentrations. Initial
contamination by MTX may partially cause this toxicity. Additionally, the prodrug may
be enzymatically hydrolysed intra- or extracellularily. At this stage, it is not known if
the prodrugs are internalised without prior metabolism.
Inclusion of the appropriate enzyme with the prodrug generally increased
cytotoxicities (Table 2). In the case of Pyr-MTX at 10·8M, the cytotoxicity reached that
of MTX. The increase in cytotoxicities of the other prodrugs was not as great, possibly
because these prodrugs are converted slowly to non-cytotoxic decomposition products.
There was no observed cytotoxicity caused by enzyme alone.

459
 Table 2. Effect of MTX-prodrugs on L1210 cell growth"

Drug Enzyme (cone. live cells (% control? at drug concentrations:

in U/ml

w-'M 10"7M 2 X 10"8M 10"8M

MTX 2.6 ± 0.6 6.6 ± 0.5 16.0 ± 5.4 35.3 ± 1.5

Leu-MTX 6.7 ± 0.8 10.0 ± 0.4 39.8 ± 2.2 NT"
Leu-MTX AP-M (0.005) 2.4 ± 0.6 8.1 ± 0.2 12.1 ± 1.7 NT
Val-MTX 9.0 ± 0.5 49.4 ± 17.6 116d NT
Val-MTX AP-M (0.005) 4.5 ± 0.2 12.7 ± 1.2 110±3 NT
Ile-MTX 9.5 ± 0.8 36.4 ± 3.9 78 ± 8.2 NT
Ile-MTX AP-M (0.005) 5.5 ± 0.3 7.9 ± 0.3 39.9 ± 1.5 NT
Pyr-MTX 4.6 ± 0.6 11.7 ± 1.2 NT 120 ± 20
Pyr-MTX Pyr-APR (50) 5.6 ± 0.9 9.1 ± 0.9 NT 33.9 ± 4.0

• After 48 hours; b mean of two determinations(± range); • not tested; d single determination only.

CONCLUSIONS

Our interim results indicate that 2-(L-a.-aminoacyl) derivatives of MTX (except Leu-
MTX) are potential prodrugs. Firstly, they are relatively non-cytotoxic and are not
significantly cleaved in serum. Whether or not these prodrugs are metabolised in vivo has
yet to be determined. Endogeneous intracellular aminopeptidases would be capable of
releasing MTX (except possibly from Pyr-MTX); however, this process would rely on the
prodrugs being transported into the cell. Secondly, exogeneous enzymes are available
which can release MTX and hence could be used in the ADEPT strategy. At present, Pyr-
MTX is the best candidate prodrug since it is very stable in serum. Although the other
prodrugs decompose slowly, it appears that the decomposition product is not cytotoxic.
The disadvantage of decomposition is that the concentration of the prodrug is decreased.
As for the contamination of the prodrugs with MTX, either an efficient method of
separation or an alternative deprotection method needs to be developed.

Acknowledgments: This work was supported by the Leo & Jenny Leukaemia &
Cancer Foundation and the National Health & Medical Research Council of Australia. We
thank Mr. Michael Costello for technical assistance.

References

1. J.H. Freisheim and D.A. Matthews, in: "Folate Antagonists as Therapeutic Agents, Vol.l", F.M. Sirotnak,
J.J. Burchall, W.D. Ensminger and J.A. Montgomery, eds, Academic Press, Orlando (1984).
2. B. Schlagenhauff, C. Klessen, S. Teichmann-Dorr, H. Breuninger and G. Rassner, Cancer 70:113 (1992)
and lit cited.
3. K.D. Bagshawe, Br. J. Cancer 60:275 (1989).
4. P.D. Senter, FASEB J. 4:188 (1990).
5. H.T.A. Cheung, D.K. Boadle and T.Q. Tran, Heterocycles 28:751 (1989).
6. H.T.A. Cheung, Z. Dong, M. Smal and M.H.N. Tattersall, Pteridines, 3:101 (1992).

460
EFFECT OF A NOVEL ANTIFOLATE, N"-(4-AMIN0-4-DEOXYPTEROYL)-N8-
HEMIPHffiALOYL-L-ORNITHINE (PT523), ON GROWffi OF H35 RAT
HEPATOMA AND HEPG2 HUMAN HEPATOMA CELLS

Myung S. Rhee1, John Galivant, Errol M. Tyobeka2, Matthew L.

Sherman2, and Andre Rosowsky2

1Wadsworth Center for Laboratories and Research

NYS Department of Health, Albany, NY 12201
2Dana-Farber Cancer Institute, Boston, MA 02115

INTRODUCTION

Structural modifications of folic acid have generated various derivatives that act at
different target sites. Well-known examples include the dihydrofolate reductase (DHFR)
inhibitor methotrexate (MTX), 1-3 the thymidylate synthase inhibitor 2-desamino-2-methyl-
W0-propargyl-5,8-dideazafolic acid, 4·5 and the glycinamide ribonucleotide formyl-
transferase inhibitor 5,1 0-dideaza-5, 6, 7, 8-tetrahydrofolic acid. 6•7 More recently, folate
analogues containing ornithine in place of glutamate have been synthesized as inhibitors
of folylpolyglutamate synthetase. 8•9 These ornithine-containing analogues showed weaker
activity than the corresponding glutamate-containing analogues on cell growth presumably
because of inefficient transport across the cell membrane. 9•10 In contrast, a novel derivative
of an ornithine-containing antifolate, N"-(4-amino-4-deoxypteroyl)-N-hemiphthaloyl-L-
ornithine (PT523), exhibits potent in vitro and in vivo antitumor activity.U
In the present study the effect of PT523 on cell growth was examined in cultured
H35 rat hepatoma and HepG2 human hepatoma cells.

MATERIALS AND MEffiODS

Materials. PT523 was synthesized at Dana-Farber Cancer Institute by Dr. H. Bader

according to the method described previously . 12 MTX was provided by Lederle
Laboratories (Pearl River, NY). [6-3H]Deoxyuridine was purchased from Moravek
Biochemicals (Brea, CA) and [14C]glycine from DuPont (Boston, MA). Tissue culture
media, horse serum and FBS were purchased from GIBCO (Grand Island, NY).

Cell Culture and Growth Inhibition. H35 rat hepatoma cells were grown in Swim's
medium containing 20% horse serum and 5% FBS, 13 HepG2 cells in Dulbecco's MEM

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 461
containing 10% PBS, 14 and human U937 myeloid cells in RPMI 1640 medium containing
10% FBS 15 as previously described.
For growth inhibition study, cells were seeded at a density of 1 x 104/200 ILL/well
in a 96-well plate (Coming Glass Works, Coming, NY) and incubated in the presence of
drug for 72 h or as indicated. Outgrowth was measured by the modified method16 of Finlay
et al. 17

Thymidylate and Purine Biosynthesis. [6-3H]Deoxyuridine or [l4C]glycine incorporation

into DNA was used as a measure of de novo thymidylate biosynthesis18 or purine
biosynthesis, 19 respectively.

Analysis of DNA Fragmentation. Genomic DNA was isolated by proteinase K and

RNAase A treatment as previously described, 20 and was separated on 2% agarose gels and
visualized by UV illumination after ethidium bromide staining.

RESULTS AND DISCUSSION

It was reported previously that PT523 inhibits DHFR as effectively as MTX. 12 In

spite of the fact that PT523 is unlikely to be -y-glutamylated, it exhibits greater antitumor
in vivo and in vitro activity than MTX. 11 In the present study, growth inhibition by this
compound was examined in monolayer cultures of H35 rat hepatoma and HepG2 human
hepatoma cells. Since MTX is the most widely utilized and studied antifolate, the
inhibitory activity of PT523 was evaluated by comparison with MTX. As shown in Table
1, PT523 inhibited cell growth with IC50 of 3 nM in H35 cells and 8 nM in HepG2 cells,
and thus was three fold more potent than MTX against both cell lines.

Table 1. Effect of PT523 on growth inhibition of H35 cells and HepG2 cells.

ICso (J.tM) •
H35 cells HepG2 cells

PT523 0.003 0.008

MTX 0.010 0.024

• the average of 2 or 3 experimen1s.

Since growth inhibition by antifolates is primarily due to inhibition of thymidylate

and/or purine biosynthesis, the effect ofPT523 on these metabolic pathways was measured
in exponentially growing cells. As shown in Table 2, both deoxyuridine and glycine
incorporation were extensively inhibited by PT523 in H35 and HepG2 cells. Protection
studies with thymidine, hypoxanthine and folinic acid were carried out. Cell growth in the
presence of PT523 at a >90% inhibitory concentration was restored to normal rates by
the presence of thymidine (20 1£M) plus hypoxanthine (50 1£M) or with folinic acid (50
J.LM). Neither hypoxanthine or thymidine alone could rescue the treated cultures (data not
shown). This verifies the incorporation study shown in Table 2 and implies that PT523 is
not a selective inhibitor of either thymidylate synthase or folate dependent reactions of de
novo purine biosynthesis alone. The tentative conclusion from these results is that DHFR
is the primary intracellular target site of this compound.

462
Table 2. Effect of PT523 on de novo biosynthesis of thymidylate and purine by H35 cells
and HepG2 cells.
Cells were exposed to drug for the last 4 h of a 72 h culture.

IC50 {J.tM)"
H35 cells HepG2 cells

WH]UdR
PT523 0.009 0.03 0.009 0.012
MTX 0.22 0.24 0.62 1.68

• the average of 2 or 3 experiments.

Due to its lack of a glutamate side chain, PT523 was not considered a likely
substrate for polyglutamylation. This was established by incubation with folylpolyglutamate
synthetase, ATP and [3H]glutamic acid which resulted in the complete absence of
glutamate incorporation (unpublished results). Yet, PT523 was clearly more potent than
MTX, which does form polyglutamates. One of the characteristics of the non-glutamatable
analogue of MTX, fluoro-MTX, is that its toxicity in short term treatment is vastly
reduced relative to MTX. 21 This is presumably because fluoro-MTX can not be
glutamylated and is poorly retained by the cell. When a 72 h exposure to MTX was
compared with a 2 h pulse, the latter condition required 80-fold more MTX to inhibit
growth by 50% (Table 1 & 3, ref 21). In an analogous experiment a 2 h pulse of fluoro-
MTX was 6000-fold less effective than a 72 h exposure. 21 When the same comparison was
made with PT523 in H35 and HepG2 cells, a 2 h pulse resulted in a 70-fold and 20-fold
increase in IC50 , respectively (Tables 1 & 3). Therefore, in spite of the fact that PT523
can not be glutamylated, it acts as a potent growth inhibitor even after an exposure time
of only 2 h. This suggests that there may be unidentified mechanism(s) for the cellular
retention of this compound or its growth inhibitory effects.

Table 3. Growth inhibition by pulse treatment with PT523.

Cells grown for 24 h were pulsed with drug for 2 or 24 h and then further incubated in drug-free medium
for an additional 48 h.

Drug exposure (h) H35 cells HepG2 cells

PT523 MTX PT523 MTX
2 (24-26) 0.213 0.78 0.174 1.48
24 (24-48) 0.016 0.037 0.049 0.15

• the average of 2 - 5 experiments.

It has been demonstrated that treatment of cells with MTX resulted in DNA
fragmentation/2•23 which was closely linked to cytotoxicity. 22 We have examined the effect
of PT523 on DNA fragmentation in human U937 myeloid cells. Similar DNA laddering
in the multimers of approximately 200 base pairs was observed with a ten-fold lower
concentration of PT523 (150 nM) than MTX (1500 nM) (unpublished results).

463
 In summary, the results of this study indicate that an unique analogue of aminopterin
with the glutamate side chain replaced by N8-hemiphthaloyl ornithine, PT523, exhibits a
greater potency than MTX on the growth of HepG2 human hepatoma and H35 rat
hepatoma cells. The 2,4-diamino structure of this compound, and its nearly equivalent
inhibition of glycine and deoxyuridine incorporation, suggest that dihydrofolate reductase
is the primary target site in intact cells. Although PT523 is unlikely to be glutamylated,
it appears to retain stronger inhibitory activity than MTX when used in short term
exposures. This result, coupled with the very low IC50 for growth inhibition, presents the
likelihood of facile cell entry and prolonged retention or activity once inside the cell.

Acknowledgements

This work was supported by NIH grants CA25933 (JG) and CA25394, CA19589 (AR).
The authors acknowledge the excellent technical assistance of Aiga Pupons.

REFERENCES
1. W.D. Ensminger, in: "Folate Antagonists as Therapeutic Agents," F.M. Sirotnak:, J.J. Burchall,
W.D. Ensminger, and J.A. Montgomery, eds., Academic Press. NY (1984).
2. J.R. Bertino, Cancer Res 39:293 (1979).
3. R.C. Jackson and G.B. Grindey, in "Folate Antagonists as Therapeutic Agents," F.M. Sirotnak:, J.J.
Burchall, W.B. Ensminger and J.A. Montgomery, eds., Academic Press, NY (1984).
4. L.R. Hughes, A.L. Jackman, J. Oldfield, R.C. Smith, K.D. Burrows, P.R. Marsham, J.A.M.
Bishop, T.R. Jones, B.M. O'Connor, and A.H. Calvert, J. Med. Chem. 33:3060 (1990).
5. S.D. Patil, C. Jones, M.G. Nair, J. Galivan, F. Maley, R.L. Kisliuk, Y. Gaumont, D. Duch, and
R. Perone, J. Med. Chem. 32:1284 (1989).
6. D.H. Boschelli, S. Webber, J.M. Whiteley, A.L. Oronsky, and S.S. Kerwar, Arch. Biochem.
Biophys. 265:43 (1988).
7. G.P. Beardsley, B.A. Moroson, E.C. Taylor, and R.G. Moran, J. Biol. Chem. 264:328 (1988).
8. S.A. Patil, B. Shane, J.H. Freisheim, S.K. Singh, and J.B. Hynes, J. Med. Chem. 32:1559 (1989).
9. A. Rosowsky, J.H. Freisheim, R.G. Moran, V.C. Solan, H. Bader, J.E. Wright, and M. Radike-
Smith, J. Med. Chem. 29:655 (1986).
10. J.J. McGuire, W.E. Bolanowska, and J.R. Piper, Biochem. Pharmacal. 37:3931 (1988).
11. A. Rosowsky, H. Bader, and E. Frei, Proc. Am. Assoc. Cancer Res. 32:325 (1991).
12. A. Rosowsky, H. Bader, and R.A. Forsch, Pteridines 1:91 (1989).
13. M.S. Rhee, M. Balinska, M. Bunni, D.G. Priest, G. Maley, F. Maley, and J. Galivan, Cancer Res.
50:3969 (1990).
14. J. Galivan, M.S. Rhee, D.G. Priest, M. Bunni, J. Freisheim, and J. Whiteley, in: "Purine and
Pyrimidine Metabolism in Man VII" R.A. Harkness, ed., Plenum Press, NY (1991).
15. M.L. Sherman, R.M. Stone, R. Datta, S.H. Bernstein, and D.W. Kufe, J. Biol. Chem. 265:3320
(1990).
16. J. Galivan, M.S. Rhee, T. Johnson, R. Dilwith, M.G. Nair, M. Bunni, and D.G. Priest, J. Biol.
Chem. 264:10685 (1989).
17. G.J. Finlay, B.C. Baguley, and W.R. Wilson, Anal. Biochem. 139:272 (1984).
18. D.S. Duch, M.P. Edelstein, S.S.W. Bowers, and C.A. Nichol, Cancer Res. 42:3987 (1982).
19. E. Cadman, C. Benz, R. Heimer, and J. O'Shaughnessy, Biochem. Pharmacal. 30:2469 (1981).
20. H. Gunji, S. Kharbanda, and D.W. Kufe, Cancer Res. 51:741 (1991).
21. J. Galivan, J. Inglese, J. McGuire, Z. Nimec, and J.K. Coward, Proc. Natl. Acad. Sci. 82:2598
(1985).
22. M.A. Barray, C.A. Behnke, and A. Eastman, Biochem. Pharmacal. 40:2353 (1990).
23. J.B. Kowk and M.H. Tattersall, Br. J. Cancer 65:503 (1992).

464
TUBULIN BINDING PROPERTIES OF TWO CHIRAL ISOMERS
WITH 1-DEAZA-7,8-DIHYDROPTERIDINE STRUCTURE*

Daniel Leynadierl, Vincent Peyrotl,

Marcel Sarrazinl Jose Manuel Andreu2,
Carol! Temple Jr~, Gregory Rener3, and
Claudette Briandl
lGroupe de Recherche sur les Interactions des
Proteines en Pharmacologie (GRIPP) Faculte de
Pharmacie, 27 Bd Jean Moulin, 13005 Marseille
FRANCE
2centro de Investigaciones Biologicas, CSIC,
Velazquez 144, 28000 Madrid, Spain
3southern Research Institute, Birmingham,
Alabama 35255, USA

INTRODUCTION

It has been shown that ethyl 5-amino-1,2-dihydro-2-

methyl-3-phenyl pyrido (3,4-b)pyrazin-7-yl carbamate (NSC
370147) inhibits microtubule formation, that this racemic
compound binds to tubulin at or near the colchicine site,
and that it enhances the binding of Vincristine to
tubulin 1 . It appeared of interest to evaluate the activity
of the R-enantiomer (NSC 613863) and the s-enantiomer (NSC
613862, CI 980). Hence, their interactions were examined at
molecular level. Furthermore, we compared the binding
properties of these new compounds to those of colchicine
and colchicine analogues, which are firmly established. The
influence of chirality on the polymerization of microtubule
proteins and tubulin is also discussed.

RESULTS

MICROTUBULE ASSEMBLY STUDIES

The s- and R-enantiomers inhibited the assembly of
microtubule proteins and purified tubulin in a dose
dependent manner and with substoichiometric concentrations.
With a tubulin concentration of 2 mgjml, the half-
inhibitory concentration was 1.5 ~M for R and 0.5 ~M for s.
For microtubule protein (1 mgjml) the values were 0.75 ~M
and 2.10 ~M for the S and the R-isomer, respectively. In

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 465
vitro, at low or high concentrations these compounds did
not disrupt preformed microtubules.

BINDING TO PURIFIED TUBULIN

NSC 613862 and NSC 613863 in the absence of tubulin

have a very weak fluorescence in the PG buffer, pH = 7.
Addition of tubulin to NSC 613863 (R) resulted in a large
increase in relative fluorescence (Fig. 1). In contrast the
fluorescence of the s-enantiomer was not greatly enhanced
in presence of tubulin (see inset of Fig. 1).

200

w
~
~ 150

~
;
w100

50

240 280 320 360 400 440 480 520 560 600 640
WAVELENGTH (nm)

Figure 1
Fluorescence changes produced by the tubulin-isomer interaction.
a) blank of 2 11M tubulin without ligand. b) fluorescence excitation
and emission spectra of 3 11M R-enantiomer without tubulin. c)
fluorescence excitation (emission at 470 nm) of 3 11M R and 2 11M
tubulin. d) fluorescence emission (excitation at 380 nm) of 3 11M R and
2 11M tubulin.
The inset shows (4-3) the bound fluorescence excitation and emission
spectra of 3 pM S-enantiomer, (2) fluorescence excitation and emission
spectra of 3 11M S-enantiomer without tubulin. (1) blank of 2 11M
tubulin without ligand.

Changes in the spectrofluorimetric properties of the

ligands can be measured as a function of total ligand
concentration. Binding parameters were calculated from
nonlinear least-squares regression analysis. S and R
binding measurements indicated one high affinity binding
site per tubulin heterodimer. The apparent binding
constants to tubulin dimer was (2.80 ± 0.50) x 10 6 M- 1 for
the s- and R-enantiomers.
The Rummel-Dreyer column gel permeation technique was
used to perform binding measurements. The binding
equilibrium constants were ( 5. 20 ± o. 86) x 10 6 M- 1 for s
and (3.88 ± 1.61) x 106 M- 1 for R. This direct procedure
revealed the presence of some low affinity binding sites
(Kapp ~ 104 M-~) for both enantiomers.
Knowing that NSC 370147 decreases the exten1: of
colchicine binding to purified tubulin 1 , we examined the
binding of our compounds to tubulin in presence of
podophyllotoxin and colchicine analogues. Our findings

466
indicated that podophyllotoxin and MTC displaced the two
enantiomers from their binding sites. The order of addition
of the reagents did not modify the results. To check
whether the R- and s-enantiomers bound to the same site, we
added the R-enantiomer to tubulin and recorded the relative
fluorescence. When the plateau was attained, an excess of
s-enantiomer was added and the time course of the
fluorescence change was followed. The effect of s-
enantiomer indicated that the R-tubulin complex was
dissociated in less than 20 minutes.

EFFECTS OF LIGAND BINDING ON THE CONFORMATION OF TUBULIN.

Since these new compounds bind to tubulin in the same
site of colchicine, and since colchicine induces a
structural change in tubulin, manifested essentially by an
appearance of the GTPase activity2 we examined this
activity in tubulin liganded to NSC 613862 and 613863.

Table 1. Ligand-induced GTPase tubulin activity

Ligand Concentration (~M) activity*

Colchicine 100 100

Isomer S 100 50
Isomer R 100 20
Podophyllotoxin 100 0

*GTP hydrolysis rate relative to the tubulin-colchicine complex.

Table 1 shows the measurements of the GTPase activity

induced in tubulin by the various ligands. The s-enantiomer
induced a greater GTPase activity in tubulin than did the
R-enantiomer. Podophyllotoxin developed no GTPase activity.

DISCUSSION

Inhibition of polymerization
Results indicated that the S and R enantiomers
substoichiometrically inhibited the assembly of
microtubules, as do colchicine, podophyllotoxin and
vinblastine. The s-enantiomer is a more potent inhibitor
than the R-enantiomer. Like colchicine analogues, they do
not disrupt preformed microtubules. In the same
experimental conditions (glycerol and high Mg
concentrations) vinca-alkaloids were reported to completely
disrupt preformed microtubules and to form a large number
of spirals3.

Binding characteristics
s- and R-enantiomer binding to tubulin as well as the
localization of their high affinity binding sites were
investigated. The apparent binding constants to tubulin

467
 heterodimer are higher than that of the colchicine
analogue 4 (MTC : Ka = 5 x 105 M- 1 ), but close to that of
colchicine5 (c.a. 107 M-1) or podophyllotoxin6 2 x 106 M-1.
Furthermore, binding measurements indicated that there is
one binding sitejtubulin dimer, as for colchicine and its
analogues.
The consequences of the binding to tubulin (GTPase
activity) and competition reactions indicate that the two
enantiomers may bind to a site common to colchicine and
podophyllotoxin.

FOOTNOTES

PG buffer = 10 mM sodium phosphate, 0.1 mM GTP, pH = 7.0.

NSC 613862 : S-enantiomer ; NSC 613863 : R-enantiomer.
MTC = 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-
cycloheptatrien-1-one.
* This work was supported by a joint French-Spanish Grant,
Spanish DGI Cyt Grant PB 87022 and grants from ARC, FNCLCC
and FEGEFLUC.

REFERENCES

1. Bowdon, B.J., Waud, W.R., Wheeler, G.P., Hain, R.,

Dansby,L., and Temple, C.Jr., 1987,
Comparison of l-2-dihydropyrido[3,4-b]pyrazines(l
-deaza-7-8-dihydro-pteridines) with several other
inhibitors of mitosis. Cancer Res. 47:1621-1626.

2. David-Pfeuty, Th., Simon, C., and Pantaloni, D., 1979,

Effect of antimitotic drugs on tubulin GTPase activity
and self assembly. J. Biol. Chem. 254:11696-11702.

3. Himes, R.H., Kersey, K.N., Heller-Bettinger, I., and

Samson,F., 1976,
Action of the vinca alkaloids vincristine, vinblastine
and desacetyl vinblastineamide on microtubules in
vitro. Cancer Res. 36:3798-3802.

4. Andreu, J.M., Gorbunoff, J.M., Lee, J.C., and Timasheff,

S.N., 1984,
Interaction of tubulin with bifunctionnal
colchicine analogues on equilibrium study.
Biochemistry 23:1742-1753.

5. Garland, D.L, 1978, Kinetics and mechanism of colchicine

binding to tubulin : evidence for ligand-induced
conformational change. Biochemistry 17:4266-4272.

6. Cortese, F., Battacharyya, B., and Wolff, J., 1977,

Podophyllotoxin as a probe for the colchicine binding
site of tubulin. J. Biol. Chem. 252:1134-1140.

468
EFFECTS OF FOLIC ACID ON PYRIMETHAMINE TERATOGENESIS IN RATS

Gen Kudo, Kunitoshi Tsunematsu, Minoru Shimoda,

and Eiichi Kokue

Department of Veterinary Medicine

Faculty of Agriculture
Tokyo University of Agriculture and Technology
Fuchu, Tokyo 183, Japan

INTRODUCTION

Pyrimethamine (PYR), an inhibitor of dihydrofolate reductase, has been known to

induce megaloblastic anemia in human. The dosing of folic acid (PteGlu) or folinic acid
(5-formyltetrahydrofolic acid) is recommended for the treatment of PYR-induced anemia.
PYR has been also known to be a potent teratogen1•2•3.4. Therefore, we studied the effects
of PteGlu and folinic acid on the embryotoxicity of PYR using rats and mice. The plasma
level of 5-methyltetrahydrofolic acid (5-CH 3-H4PteGlu) was also studied. 5-CH3-
HlteGlu is the principal active folate in plasma and its plasma level reflects folate status in
the body.

ANIMAL STUDY

A. Embryotoxicity of PYR

Rats: Wistar rats were used. PYR and folinic acid were administered in-feed and
PteGlu was administered in-feed or intraperitoneally (i.p.) for 5 days of day 11-15 preg-
nancy. The results were represented in Table 1. The PYR embryotoxicity was potentiated
by oral PteGlu and reduced by i.p. PteGlu or oral folinic acid. Frequent malformations
included cleft palate and brachygnathia.
Mice: ICR mice were used. PYR and PteGlu were administered by gavage as
suspension in 1% carboxyl methyl cellulose solution and folinic acid was injected i.p. for 7

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 469
days of day 9-15 pregnancy. The results were represented in Table 2. Also in mice the
embryotoxicity of PYR was potentiated by oral PteGlu.

Table 1. Effects of PteGlu and folinic acid on PYR embryotoxicity in rats.

Dose No. of Resorption Malformation Body Weight

(mg/kg/day) Dams (%) (%) of Fetus(g)

PYR1.6 5 3.8±5.6 o# 2.9±0.35#

PYR1.6+PteGlu 5 5.5±3.1 100* 2.2±0.44*
50p.o.
PYR3.6 5 7.0±7.7 100* 2.3±0.31*
PYR3.6+PteGlu 5 6.5±4.5 24±43# 2.8±0.36#
50i. p.
PYR3.5+folinic 5 4.2±7.2 o# 3.1±0.41#
acid120p.o.
PteGlu50 6 9.4±8.6 o# 2.9±0.24#
Control 4 8.6±5.4 o# 3.0±0.15#

Each value represents mean±S.D ..

* p<0.05 from PYR1.6. # p<0.05 from PYR3.6

Table 2. Effects of PteGlu and folinic acid on PYR embryotoxicity in mice.

Dose No. of Resorption Malformation Body Weight

(mg/kg/day) Dams (%) (%) of Fetus(g)

PYR50 11 18.1±28.6 18.5±30.4 1.2±0.19

PYR50+PteGlu 9 100*
50p.o.
PYR50+folinic 7 6.3±5.0* 2.1±3.7* 1. 3±0.14
acidlOp.o.
Control 8 1.9±3.4* 0.7±2.1* 1. 3±0.13

Each value represents mean±S.D ..

* p<O.OS from PYRSO

Plasma levels of 5-CH3-H4PteGlu in non-pregnant rats were determined after single

dosing of PYR with or without PteGlu and folinic acid. PYR and folinic acid were adminis-
tered by gavage and PteGlu was administered by gavage or i.p .. The plasma 5-CH3-
HlteGlu level was determined by the HPLC-ECD method 5 . Figure 1 shows the plasma
5-CH3 -H4 PteGlu concentration-time curves after dosing in rats. PYR decreased the
plasma 5-CH3-HlteGlu level. This decrease was potentiated by oral PteGlu and prevent-
ed by i.p. PteGlu or oral folinic acid. The extent of decrease of plasma 5-CH3-H4PteGlu

470
 --..- PYR3.6(mg/kg)
----- PYR1.6+PteGlu50p.o.
150 --o- PYR3.6+PteGlu50i.p.
-tr- PYR3.6+folinic acid120p.o.

100

15 20 25
Time(hr)
Figure 1. The plasma 5-CH3 -HlteGlu concentration-time curves after single administration of PYR
3.6mg!kg, PYR1.6+PteGlu50mg!kg(p.o.), PYR3.6+PteGlu50mg!kg(i.p.) and PYR3.6+folinic acid 120mg!kg
(p.o.) in rats. Each point represents mean±S.E. (n=4).

level corresponded to the development of the embryotoxicity. Similar results were ob-
tained in mice.

C. Plasma level of PYR

Effects of oral PteGlu on the pharmacokinetic profile of PYR were determined using
non-pregnant rats and mice to examine a possible mechanism of the potentiated embryo-
toxicity of PYR by PteGlu. The plasma PYR level was determined by the HPLC-UV
method 6 • The time course of plasma PYR level was not affected by oral PteGlu. Similar
results were obtained in mice.

DISCUSSION AND CONCLUSION

1) The embryotoxicity of PYR was potentiated by oral PteGlu in rats and mice. On
the other hand, it was reduced by i.p. PteGlu.
2) A good relation between the decrease of plasma 5-CH3-H4PteGlu level and the
embryotoxicity was demonstrated.
3) The pharmacokinetics of PYR was not affected by the concomitant oral PteGlu in
rats and mice.
We suggest that the potentiated embryotoxicity of PYR by oral PteGlu dosing resulted
from the decrease of plasma 5-CH3- HlteGlu in dams. It has been described that a large
amount of 5-CH3-H4 PteGlu is circulating in the enterohepatic cycle and it plays an
important role in folate homeostasis 7 • In addition it has been reported that PteGlu inhibits
the absorption of 5-CH3-H4PteGlu in the intestinal mucosa 8• Therefore, the potentiated

471
decrease of plasma 5-CH3-HlteGlu may be induced by the interruption of the entero-
hepatic circulation of 5-CH3-HlteGlu by PteGlu.

REFERENCES

1. J.B. Thiersch, Pro. Int. Cong. Chemother. 3: 367-372 (1964).

2. G.E. Sullivan and E. Tacacs, Teratology 4: 205-210 (1971).
3. J. Misawa, E. Kokue, T. Hayama et al., Toxicology Letters 10: 51-54 (1982).
4. Y. Yamamoto, M. Ohnishi, E. Kokue et al., Cong. Anom. 24: 83-87 (1984).
5. K. Tsunematsu, G. Kudo, M. Shimoda et al., Cong. Anom. 30: 113-120 (1990).
6. K. Tsunematsu, M. Shimoda and T. Hayama, Cong. Anom. 32:357-365 (1992).
7. S.E. Steinberg, C.L. Campbell and R.S. Hillman, J. Clin. Invest. 64: 83-88 (1979).
8. J. Selhub, G.M. Powell and I.H. Rosenberg, Am. J. Physiol. 246: G515-G525 (1984).

472
MUTATIONS OF HUMAN DIHYDROFOLATE REDUCTASE CAUSING
DECREASED INHffiiTION BY METHOTREXATE

Raymond L. Blakley, James R. Appleman, Srinivas K. Chunduru,

Takayuki Nakano, WilliamS. Lewis, and Susan E. Harris

Department of Molecular Pharmacology

St. Jude Children's Research Hospital
Memphis, TN 38101

INTRODUCTION

Oligonucleotide-directed mutagenesis has been used by many laboratories to obtain

information regarding the significance of the structural features of various enzymes. When
an amino acid residue, or group of residues, is deleted from the enzyme structure, or is
replaced by other residues by directed mutagenesis, inferences can be drawn regarding the
relation of that portion of the structure to the catalytic activity of the enzyme. This
approach has been applied extensively to the study of dihydrofolate reductase (DHFR) by
Benkovic and his collaborators.
The extension of this technique to the study of variants of human DHFR (hDHFR)
that are relatively insensitive to inhibition by methotrexate (MTX) has potential clinical
relevance for two reasons. The first is that mutations occurring in vivo to produce such
variants may conceivably account for some cases of neoplastic disease that are clinically
resistant to therapy with MTX, or that become so in the course of therapy. In support of
this concept are several reports of the selection from established mammalian cell lines of
cells that are resistant to MTX, and that contain a variant of DHFR instead of the wild-
type (WT) enzyme (Table 1). It is also well-documented that outbreaks of malaria and of
some bacterial infections that are resistant to antifolate therapy are due to mutations in the
gene for DHFR in the infectious organisms.
Such variants of hDHFR that are insensitive to MTX also have clinical relevance
because they may have potential as therapeutic agents. MTX therapy of forms of
neoplastic disease like osteogenic sarcoma, is limited by the toxicity of the drug to
sensitive host tissues, particularly the bone marrow and the intestinal tract. If the gene for
MTX-insensitive hDHFR could be transfected into bone marrow cells prior to MTX
therapy, this might assure the survival for host tissues at risk, during therapy with very
large doses of MTX. Evidence to support the feasibility of such a proposal has been
obtained by infecting murine bone marrow cells in vitro with recombinant retroviruses
containing the eDNA for a methotrexate-insensitive variant of murine DHFR. 5•6 Following

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 473
infection the transduced cells were transplanted into lethally-irradiated recipients. The
variant DHFR was expressed in spleen and bone marrow cells of recipients and their
progeny, and enhanced their survival as compared with controls, and protected them from
otherwise lethal doses of MTX.

Table 1. Variant DHFRs found in mammalian cell lines selected for resistance to
methotrexate

Species Substitution Reference

Mouse Leu22 -+ Arg Simonsen and Levinson 1

Hamster Leu22 -+ Phe Mel era et al. 2
Human Phe31 -+ Ser Srimatkandada et al. 3
Mouse Phe31 -+ Trp Mcivor and Simonsen4

Most of the side chains lining the active site cavity of DHFR are hydrophobic and the
four variants of mammalian DHFR that have been identified in cells resistant to MTX have
substitutions in two of these residues, Leu22 and Phe31 (Table 1). Other variants of mouse
or human DHFR that have decreased sensitivity to MTX as a result of substitutions for
active site hydrophobic residues have been obtained by directed mutagenesis. 7 •8 We are
producing variants of hDHFR by directed mutagenesis at codons for such hydrophobic
residues, and we are extensively characterizing those variants that have greatly decreased
affinity for MTX but nevertheless retain a relatively high turnover number (k.:.J, and
relatively low Ku, values for NADPH and dihydrofolate.

RESULTS

A total of 13 variants of hDHFR have been expressed in Escherichia coli M13

(pREP4) by the use of the expression vector pDS5/RBSII, Sphl (Sphi-Pstl) kindly provided
by Dr. Dietrich Stuber of Roffman-LaRoche. Substitutions have been made for Ile7 , Leu 22 ,
Phe31 and Phe34 • The side chains of these residues are close to the pterin ring of bound
folate in the crystal structure of hDHFR. 9 Phe34 is in van der Waals contact with the pterin
ring of folate. Its carbon E1 is 3.18 A from C8A of folate, and 3.31 A from N8. Phe31
is slightly more distant, but 04 of folate is 3.62 A from carbon D1 of Ph2 1 and 3.77 A
from carbon El. The side chains of Leu22 and Ile7 are still more distant. The side chain
of Leu22 is closest to 04 of folate but the nearest atoms of Leu22 are over 5 A away. The
oxygen and a-carbon of Ile7 are close (3.46 A and 3.86 A) to N8 of bound folate, but the
side chain is little more than 4 A from the 2-amino N of bound folate.
All of the mutant cDNAs for variant hDHFRs are well expressed in the system used,
and the enzymes have been purified to 95-100% homogeneity as judged by polyacrylamide
gel electrophoresis. When affinity chromatography was used in the purification, residual
tightly-bound ligand was removed by isoelectric focusing. All measurements were
performed at 20°, pH 7.65, in MATS buffer (25 mM MES, 25 mM Tris, 100 mM NaCl,
0.02% sodium azide).

Decreases in Affinity for Methotrexate

Of the 13 variants that we have prepared and studied to date, all except three have
significantly decreased affinity for MTX (at least 70 times lower than for WT). Affinity
was measured in two ways as appropriate for the characteristics of the enzyme. (1) Where

474
K,. for dihydrofolate could be determined without difficulty, K, was determined by fitting
data for MTX inhibition of steady state velocity to the appropriate equation for a tight-
binding inhibitor by the use of a modification of the CRICF program. 10 (2) Where
substrate inhibition prevents accurate determination of K,. for dihydrofolate (Phe34 and
some Leu 22 variants) we determined the ternary equilibrium dissociation constant, KJ, for
MTX binding to the enzyme.NADPH complex. Kd was obtained by measuring the
quenching by MTX of the fluorescence of the enzyme.NADPH complex, and by fitting the
data to the appropriate equation by the use of the program already mentioned.
The greatest decrease in MTX affinity found to date was for the Phe34 ~ Ser mutation
for which the ternary Kct is 228 nM. Since K, for MTX for WT is 0.0034 nM, 11 this
represents a 67,000-fold decrease in affinity for MTX in this variant. It is noteworthy that
this represents a much lower affinity for MTX than measured for those variants found in
resistant cell lines that we have investigated.

Mechanism for the Decrease in MTX Affinity

The higher Kd for MTX could be the result of a decrease in k.,., an increase in korr,
or both. There is also another possibility. The WT hDHFR.NADPH.MTX initially
formed isomerizes to a non-dissociating form, and this isomerization increases MTX
binding by a factor of 60. If mutations make this isomerization unfavorable this would
also contribute to elevation of Kct for the MTX complex.
In the case of at least one variant, k.,. is decreased by a factor of only 5, so that most
of the increase in Kd is due either to an increase in korr or to decreased isomerization, or
both. We have been able to find no evidence for isomerization of the ternary MTX
complex of this variant, so that loss of this isomerization step appears to be the major
cause of decreased affinity of MTX.

Effect of Mutations on Affinity for Dihydrofolate

MTX and dihydrofolate bind differently in the active site cavity, MTX having its
pterin ring rotated 180° (flipped over) compared with the way in which the pterin ring of
folate (and dihydrofolate) binds. This permits MTX to become protonated and make an
ionic interaction with the side chain carboxylate group of Glu30 • Folate and dihydrofolate
do not make such an ionic interaction. Nevertheless, when folate and dihydrofolate bind
to WT enzyme their pterin rings interact with hydrophobic side chains in the cavity in a
similar manner to MTX. It might therefore be expected that mutations greatly decreasing
the affinity for MTX will similarly decrease the affinity for dihydrofolate. Such a large
decrease in affinity for dihydrofolate, coupled with the fact that the concentration of
dihydrofolate is cells is very low (_~0.1 ~tM), would make the variant enzyme very
inefficient at maintaining levels of tetrahydrofolate and its derivatives.
The affinity of dihydrofolate for the DHFR.NADPH complex was measured in several
ways. (1) Although K"' is often a poor measure of substrate affinity, it provides a
reasonable approximation to Kct for the ternary substrate complex when the chemical
transformation step is very slow compared with the rate of substrate binding and
dissociation, and such is the case with these variants of hDHFR. Consequently, when
there was no substrate inhibition, so that K,. could be measured accurately, this parameter
was used as the estimate of Kct for binding to the DHFR.NADPH complex. (2) When
substrate inhibition prevented accurate determination of K,n and when Kd was large enough,
it could be measured by the dependence of the rate of product formation on dihydrofolate
concentration during transient state kinetics in single turnover experiments (NADPH
substoichiometric with respect to enzyme). These experiments were conducted by stopped-
flow measurement of the absorbance change at 340 nm as dihydrofolate binds and reacts.

475
(3) In cases of substrate inhibition where ~ for the ternary substrate complex was not
large ( -1 p.M), it can be obtained by examining the kinetics of dihydrofolate binding as
measured by stopped flow fluorimetry. This gives k,rr (rate constant for dihydrofolate
dissociation) and k,., (rate constant for dihydrofolate association). The ratio korrlk,n gives
the ternary Kct.
We have found that, in all the cases that we have examined, the decrease in affinity
of dihydrofolate for apoenzyme or for enzyme.NADPH complex is much less than the
decrease in affinity for MTX for the same variant. The maximum ternary ~ for
dihydrofolate observed was about 400-fold higher than for WT, but this increase is
associated with increases of 12,000 to 67,000 in the ternary ~ for MTX. For some
variants the affinity for dihydrofolate was actually increased.
~ values for binary complexes of dihydrofolate with some of the variant hDHFRs
were determined by titration of the enzyme fluorescence with dihydrofolate. It was found
that affinity of dihydrofolate for the apoenzyme was also decreased little or even increased,
even in the case of variants with large increases in ternary ~ for MTX.

Effect of Mutations on the Rate of the Chemical Transformation and on kcat

The rate constant (~heJ for the chemical transformation step can be measured in
single turnover experiments with a stopped-flow spectrophotometer. Change in absorbance
at 340 nm with time over a short time period is described by an exponential term with a
rate constant, k,bs· When either k,b, or the initial velocity of the change is plotted as a
function of dihydrofolate concentration, a rectangular hyperbola is obtained. Under
favorable conditions both the ternary Kct for dihydrofolate and ~hem can be obtained by
suitable treatment of the data. For all variants for which it has been measured ~hem is
much lower than the value of 1360 s· 1 for WT hDHFR. 12 Values range from 7 to 36 s· 1 •
Clearly, the interaction of the substrates with the hydrophobic residues that were varied
is critically important for the chemical transformation step, i.e., hydride and proton
transfer to dihydrofolate.
On the other hand, ~.1 is decreased little by most mutations and increased by several.
This result is compatible with the greatly decreased k.:hem produced by these mutations
because for WT hDHFR k.:hem (1360 s· 1) is much greater than 1<,.1 (11 s· 1). 12 The rate of the
catalytic cycle for the WT enzyme is therefore completely limited by the rate of
dissociation of the products, NADP and tetrahydrofolate (Figure 1).
As seen in Figure 1, the rate constant for dissociation of NADP from E.NADP. THF,
the ternary product complex is only about twice that for tetrahydrofolate dissociation from
this complex, so that the enzyme uses a pathway branching at this point. Since the
resulting binary complexes of NADP and tetrahydrofolate have low dissociation rate
constants, substrate binds to them before they can dissociate, and the result is the formation
of the abortive ternary complexes: E.NADP.DHF and E.NADPH.THF. Dissociation of
product from the E.NADPH.THF complex is faster than for the E.THF complex, but is
still relatively slow, and this dissociation contributes to limitation on the rate of the overall
reaction. NADP dissociation from E.NADP.DHF is so slow that at steady state much of
the WT enzyme accumulates as this complex. Although about 70% of product formation
at steady state occurs by the pathway indicated by bold arrows (Figure 1), dissociation of
NADP from E.NADP.DHF is a major limitation of the overall rate.
Although some of the mutations we have investigated decrease ~hem by a factor of
40-50, ~.1 is often increased by these mutations because of decreased formation of
E.NADP.DHF and/or an increased rate of NADP dissociation from it. For many of our
hDHFR variants (perhaps all of them) the chemical transformation contributes to overall
rate limitation, as we have shown in some cases by demonstrating that the overall reaction

476
Figure 1. Major branched catalytic pathways for wild-type hDHFR. Bold arrows indicate the faster of the
two pathways. Numbers against arrows indicate association rate constants (units, JLM- 1 s 1), or dissociation
rate constants (units, s· 1). Symbols are: E, wthDHFR; NH, NADPH; DHF, dihydrofolate; N, NADP; THF,
tetrahydrofolate.

is slower when NADPD replaces NADPH in the reaction. However, this limitation is
offset to a greater or lesser extent by the faster overall dissociation of products.
In the case of variants that show substrate inhibition by dihydrofolate, this inhibition
is due to increased accumulation ofE.NADP.DHF at high concentrations of dihydrofolate.
Phe34 variants show such behavior, and in their case we have also obtained preliminary
evidence that tetrahydrofolate dissociates much more rapidly than NADP from the ternary
substrate complex, so that E.NADP is formed. At low concentrations of dihydrofolate this
dissociates to give apoenzyme which can then bind substrates to start a new cycle.
However, at increasing concentrations of dihydrofolate, more and more E.NADP is
converted to E.NADP.DHF from which NADP dissociation is quite slow.

Effect of Mutations of Catalytic Efficiency: the "Best" Mutations

The quotient kc./Ku, is commonly regarded as a measure of enzyme catalytic

efficiency, since it is the factor by which low substrate concentrations must be multiplied
to obtain the rate of enzyme catalysis. When the 10 variants of hDHFR with markedly
decreased affinity for MTX were examined for catalytic efficiency, three were found to
have such low catalytic efficiency compared with WT ( <0.3% of WT), that it seems
unlikely that they could operate efficiently enough under physiological conditions to sustain
cell growth. These are the Phe34 variants. The Ile7 variant has an efficiency <0.7% of
WT so that its capability of supplying the needs of the cell are questionable. The
remaining six variants were ranked in order of the magnitude of the product K, (~./K,,).
Three of them have a higher value than that for the Phe31 - Ser variant found in an MTX-
resistant human colon carcinoma line3 and for the Leu 22-Phe mutant found in an MTX-
resistant mouse line. 1 The best has a value three times that of the Ser 1 variant, and 4.9
times that of the Phe22 variant.

477
Relation of Structural Changes to Function changes

Until the crystal structures of complexes of these variant hDHFRs with folate and with
MTX are solved, the extent of structural changes is largely a matter of speculation.
However, a number of conclusions seem plausible. Our results suggest that contact
between Phe34 and the pterin ring of bound folate, dihydrofolate or MTX is very important
for strong binding of these ligands, and for hydride transfer between NADPH and
dihydrofolate. It will be of interest to determine whether shrinking of the side chain of this
residue results in altered location of folate (and perhaps of NADPH also) in the active site
cavity.
Shrinking the side chain of residue 31 also decreases binding of MTX but binding of
folate and dihydrofolate is actually increased. A possible explanation may be that a fixed
water molecule occupies the space normally occupied by the phenyl ring of residue 31,
with a consequent alteration in the dielectric constant of the site, reducing the strength of
the ionic bond between Glu30 and N1 of bound MTX.
Effects of side chains that have been increased in size are most likely due to steric
effects, possibly resulting in significant movements of several components of the active
site, so that binding interactions become suboptimal.
It is significant that in all the replacements of Leu22 , Phe31 and Phe34 for which we
have information at this point, there is a drastic decrease in k.:hem· This suggests that all
these hydrophobic residues are important in promoting the formation of the transition state
in which hydride and proton transfer occurs.

Variants of hDHFR and Gene Therapy

Our results indicate that the potential exists to bioengineer variants of hDHFR that
should be superior to those isolated from resistant cell lines in their ability to confer
resistance to MTX on cells. Although trials of transfection of the eDNA for such variants
into cells have yet to be reported there seems no obvious reason why they should not be
as successful as those already reported for other variants. 5•6 Possibly some of them may
also be more protective of recipient animals against MTX in gene transfer experiments.

Acknowledgements

This work was supported in part by USPHS Research Grant CA 31922 from the
National Cancer Institute, by a John Sununu Postdoctoral Fellowship (to S.K.C.), and by
the American Lebanese Syrian Associated Charities.

REFERENCES

1. C.C. Simonsen and A.D. Levinson, Proc. Nat!. Acad. Sci. 80:2495 (1983).
2. P.W. Melera, J.P. Davide, and H. Oen, J. Bioi. Chern. 263:1978 (1988).
3. S. Srimatkandada, B.I. Schweitzer, B.A. Morenson, S. Dube, and J.R. Bertino, J.
Bioi. Chern. 264:3524 (1989).
4. R.S. Mcivor and C.S. Simonsen, Nucl. Acids Res. 18:7025 (1990).
5. D.A. Williams, K. Hsieh, A. DeSilva, and R.C. Mulligan, J. Exp. Med. 166:210
(1987).
6. C.A. Corey, A.D. DeSilva, C.A. Holland, and D.A. Williams, Blood 75:337
(1990).
7. J. Thillet, A. Josette, S.R. Stone, and R. Pictet, J. Bioi. Chern. 263:12500 (1988).
8. B.I. Schweitzer, S. Srimatkandada, H. Gritsman, R. Sheridan, R.
Venkataraghavan, and J.R. Bertino, J. Bioi. Chern. 264:20786 (1989).

478
9. J.F. Davis, T.J. Delcamp, N.J. Prendergast, V.A. Ashford, J.H. Freisheim, and
J. Kraut, Biochemistry 29:9467 (1990).
10. J.P. Chandler, D.E. Hill, and H.O. Spivey, Comp. Biomed. Res. 5:515 (1972).
11. J.R. Appleman, N. Prendergast, T.J. Delcamp, J.H. Freisheim, and R.L. Blakley,
J. Bioi. Chern. 263:10304 (1988).
12. J.R. Appleman, W.A. Beard, T.J. Delcamp, N.J. Prendergast, J.H. Freisheim, and
R.L. Blakley, J. Bioi. Chern. 265:2740 (1990).

479
CONFORMATIONAL ANALYSIS OF HUMAN DIHYDROFOLATE
REDUCTASE INHIBITOR COMPLEXES: CRYSTAL STRUCTURE
DETERMINATION OF WILD TYPE AND F31 MUTANT BINARY AND
TERNARY INHffiiTOR COMPLEXES

Vivian Cody, Andrzej Wojtczak, Thomas I. Kalman*, James H.

Friesheim+, and Raymond L. Blakley#

Medical Foundation of Buffalo, Buffalo, NY 14203, *state University of

New York at Buffalo, Buffalo, NY 14260, +Medical College of Ohio,
Toledo, OH 43699, and #st. Jude Children's Hospital, Memphis, TN,
38101

INTRODUCTION

Since dihydrofolate reductase (DHFR) is responsible for maintaining intracellular

folate pools in their biochemically active reduced state, its inhibition has been the target
of continuing antifolate development to improve selectivity, penetration into target cells,
and effectiveness against tumors with either intrinsic or acquired anti~-olate resistance 1.
For example, investigation of patients with methotrexate (MTX)-resistance has shown
that in some tumors the resistance is due to defects in cellular transport or
polyglutamation and that new, second generation, antifolate drugs such as 10-ethyl-1 0-
deazaminopterin (10-EDAM) or the lipophilic antifolates piritrexim (PTX) and
trimetrexate (TMQ), are more effective than methotrexate (MTXl In other tumors it
was found that DHFR encoded mutations which were responsible for the MTX
resistance. Studies of a highly MTX-resistant subline of the HCT-8 human colon car-
cinoma cell line showed that DHFR possessed a phenylalanine to serine mutation at
residue 31 3-5 . Kinetic studies further showed that the Ki for MTX was 100-fold higher
than wild type DHFR while a mutation of phenylalanine 34 to serine resulted in over
20,000-fold increase in the Ki for MTX. Similar changes in Ki were noted for
trimetrexate as well.
Recent data also reveal that MTX analogues in which the ')'-glutamate moiety was
replaced by an isosteric tetrazole group (MTXT), which is not polyglutamylated, induce a
response in leukemic cell lines in culture6. In addition, human leukemia CCRF-CEM
cells which are resistant to MTX, due to either increased levels of DHFR or decreased
uptake, are cross-resistant to MTXT indicating that DHFR is the primary target of the
tetrazole analogue.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 481
 To understand the observed variation in biological activity that results from
structural changes within a given series of antifolates whose substitutions probe specific
regions of the DHFR active site, we have carried out X-ray crystal structure
determinations of both wild type and mutant recombinant human DHFR as binary or
ternary complexes. These structural data provide information on the mode of inhibitor
binding to DHFR and on the role of active site residues in defining the mechanism of
action, especially as it relates to drug resistance in chemotherapy. To investigate the role
of active site residues on altered catalytic and inhibitory properties, the structures of a
series of F31 mutant complexes are also under investigation. We report the crystal
structure determination for the following wild type DHFR cofactor complexes with: (1)
methotrexate-y-tetrazole (MTXT), (2) arninopterin-y-phosphinothricin (AMP), (3) PTX,
(4) 4-oxo-5,X-dideaza-N10-propargylfolyltriglutamate (CB3717TG), and as F31 mutants
with (5) F31S MTX, (6) F31G 10-EDAM, (7) F31A TMQ, and (X) F31A binary complex
with CB3717 (Table 1, Fig. 1).

Table 1. Structural Data For Human DHFR Complexes

Complex Lattice(a,c) Resol Total CurrentR

Data
1. MTXT-NADPH 87.24,76.79 2.0A 14044 19.2%,8-2.0A
2. AMP-NADPH 87.22,76.94 2.3 7717 21.0, 8-2.3
3. PTX-NADPH 86.53,76.99 2.2 11604 18.0, 8-2.2
4. CB3717TG-NADPH ('?) 86.84,75.85 2.0 15184 18.3, 8-2.0
5. F31A-CB3717 86.84,76.53 2.1 10198 19.1 ,8-2.1
6. F31A-TMQ-NADPH 85.61,78.09 2.3 10814 21.3 ,8.2.6
7. F31G-10-EDAM-NADPH 87.03,76.76 2.2 13606 18.5 ,8-2.4
8. F31S-MTX-NADPH 87.15,76.92 2.1 17024 21.2 ,8-2.8
9. F31G-MTX-NADPH 87.25,77.09 2.2 10664
10. F31A-10-EDAM-NADPH 86.79,76.87 2.2 12398

FOlATE-DEPENDENT ENZVME INHIBITOR PROBES

DHFR Inhibitors

AMP CB3717 Tnglu

Figure I. Schematic diagram of DHFR inhibitors.

482
METHODS

Recombinant wild type human DHFR was obtained from Dr. Freisheim and the F31
mutants from Dr. Blakley. The antifolate samples MTXT and AMP were obtained from
Dr. Kalman, CB3717TG from ICI (UK), 10-EDAM from Dr. Bertino, PTX from
Burroughs Wellcome and TMQ from Park Davis (Ml). Crystals were grown with
NADPH and inhibitor incubated together with 2.7 mg/ml enzyme in O.lM potassium
phosphate buffer pH 6. 9 and 30% ammonium sulfate. Hanging drop experiments were
carried out at room temperature with 60% ammonium sulfate, O.lM phosphate buffer, pH
6. 9, in the wells and 10 ~-tl protein solution. Rhombohedral crystals grew in about 2-4
weeks. All complexes crystallize in the rhombohedral space group R3 and are
isomorphous with that reported for the R3 human folate binary complex 7 . Data for
CB3717TG was collected on a Siemens area detector at Wayne State University (with
special thanks to Jean Stuckey and Dr. Brian Edwards) and all others were collected on
the Rigaku RAXIS-IIc imaging plate area detector at the Medical Foundation of Buffalo.
Initial phases for the MTXT complex were calculated using the enzyme coordinates from
the human binary folate complex 7 and all others from the MTXT ternary complex X.
Refinement was carried out with the restrained-constrained least-squares program
PROLSQ in combination with the model building program FRODO on the graphics
graphics system.

RESULTS

Analysis of these crystal structure complexes reveals that their secondary structures
are similar to that of the isomorphous human DHFR folate binary complex 7 although
there is a different fold of the flexible loop near residues 40-47 and a cis-peptide link at
Pro-668. A suggested role of the flexible loop 40-46 in these enzymes has been to
communicate the effects of ligand binding and reversible protein unfolding during
activation 9. When the conformation of this loop is compared with that of the folate
binary structure 8, these data show that it has moved significantly away from the position
in the folate structure thereby removing the non bonded contacts with the loop at residues
100-IOS, also observed in the avian holo enzyme. However, in the triclinic human binary
complex 10, these interactions differ from those of the R3 data 7 in that the four N-
terminal residues were not observed in the electron density maps of the folate complex.
The proximity of these three loops to theN-terminus suggests that flexibility in this
region could permit communication with the active site and thus the binding interactions
of the inhibitors and substrates.
By far the most striking results are the unexpected binding interactions observed for
the lipophilic antifolates piritrexim and trimetrexate. Data for the ternary complex with
PTX reveals several differences in its active site interactions compared with MTXTX: the
diaminoquinazoline ring is oriented differently compared to the pteridine ring of other
antifolates, the PTX 5' -methoxy interacts with the nicotinamide-ribose rings, Phe-31
moves and has two alternate orientations, and Gln-35 and Thr-3X move to form hydrogen
bonds to Arg-70 in the absence of an inhibitor a-COOH (Fig. 2). This PTX orientation
also differs from those proposed from modeling studies which suggested that PTX could
occupy two different positions in which one of the methoxy groups could interact with
either Asn-21 or Asn-6411.

483
 Figure 2. Stereo companson of DHFR-PTX (solid) and MTXT (dashed).

Unusual binding interactiOns were also observed in the ternary complex of the F31A
mutant with TMQ. In th1s structure, preliminary results show the cofactor nicotinamide
ring is nearly perpendicular to the methoxy benzoyl ring of TMQ (Fig. 3). The binding
of TMQ also differs from that of MTX and PTX as the pteridine ring is displaced further
away from from the cofactor stte than the MTX ternary complexes and is in the opposite
direction observed for PTX. While the absence of the bulk of the phenyl ring of F31 in
this F31 A mutant may accommodate this movement, at this early stage of refinement,
there are no significant dtfferences between the two structures.

Figure 3. Stereo companson of DHFR-PTX (dashed) and F31A-TMQ (solid).

Interpretation of the two CB3717 folate inhibitor complexes shows the pterin ring
binds in a folate onentatwn with N3 and N2 interacting with Glu-30 and the NlO-
propargyl group occupies two different orientations in the wild type and F31 A mutant.
The propargyl group is pointed toward Ser-59 in the wild type complex because of the
close interaction wtth a partially occupied cofactor site, probably 10% (Fig. 4). There is
also density for the trigluatmate in more than one orientation. However, because of
ambiguities in interpretatiOn of weak density for the cofactor and triglutamate side chain,
a final model will have to await completion of the refinement. In the case of the F31A
mutant, the propargyl group is oriented toward Leu-22 and there is no interpretable
density for the cofactor. However, as observed in the folate binary complex7 , sulfate ions
from the buffer are bound in the phosphate positions of the NADPH site. There was also
no interpretable density for the triglutamate side chain. There are no sigmficant changes
in the active site because of the F31 A mutant and a structural water fills the cavity made
by the replacement of Phe with Ala at position 31.

484
 Data for the F31 G mutant bound with 10-EDAM and wild type MTXT (Fig. 5) show
that the orientation of the 10-ethyl differs from that of the NlO-methyl substituent. In this
case, the geometry and stereochemistry about the 10-position can vary compared to the
N 10 substituent in the normal folates. Again, these data are still in an unrefined state and
definitive details needs to await completion of the structure refmement.

Figure 4. Stereo comparison ofDHFR-F31A-CB3717 (solid) and wild type DHFR-CB3717TG (dashed).

Figure 5. Stereo comparison of wild type DHFR-MTXT (solid) and F31G-10-EDAM (dashed).

The interactions of MTXT and AMP in the ternary complex are similar to those of
MTX observed in bacterial ternary complexes in that Glu-30 hydrogen bonds to Nl and
N2 of MTXT and to Thr-136 which in turn interacts with a conserved water that also
hydrogen bonds to N2; N4 hydrogen bonds to the oxygens of Ile-7 and Tyr-121. While
the a-COOH of MTXT hydrogen bonds to the conserved Arg-70, there are no enzyme
contacts to the y-tetrazole ring which hydrogen bonds to a water. The cofactor NADPH
has an extended conformation, similar to other cofactor complexes and makes a close
contact to the MTXT N5 position 8. The conformation of AMP in its ternary complex is
similar to that of the MTXT.
The cofactor NADPH in all structures is bound in an extended conformation and
occupies a long, shallow cleft that covers the carboxy terminal ends of five strands of~­
sheet. The closest enzyme-cofactor contacts, with the exception of the F31 A-TMQ
structure, are to the nicotinamide ring.

SUMMARY

These structural studies reveal unusual intermolecular interactions for the binding of
inhibitors and cofactor in ternary complexes with both wild type and F31 mutant

485
recombinant human DHFR and show that these inhibitors have flexibility in occupying
the active site. These studies also possibly indicate the first structural data for a ternary
complex with a folate inhibitor and a polyglutamate side chain. However, further
refinement of this data is necessary before this can be confirmed. In contrast to the
ternary complexes of folate and MTX, the lipophilic antifolate PTX binds with its
methoxybenzoyl ring oriented toward the cofactor nicotinamide ring, while that of TMQ
it is bound closer to the Phe-31 position. Furthermore, the nicotinamide ring makes a
close contact to the NlO amine of TMQ, significantly different from its binding site
interactions in MTX complexes. These data also reveal that the conserved contacts
between the cofactor carboxyamide with the enzyme backbone residues Ala-9 and Ile-16
are dictated by the enzyme and that changes in the orientation of the structural elements
requires only subtle changes in the secondary structural units in which they are contained.
Therefore, only by careful analysis of a series of enzyme complexes can the mechanisms
of binding action be delineated.

ACKNOWLEGDEMENTS

This research was supported in part by a grant from the Buffalo Foundation (VC)
and NIH CA34714 (VC), CA41461 (JHF), CA31922 (RLB) and CA35212 (TIK).

REFERENCES

1. Bertino, J. R., Sobrero, A., Mini, E., Moroson, B. A. & Cashmore,A., Design and Rationale for Novel
Antifolates, NCI Monogr 5:87-91 (1987).
2. Li, W-W., Lin, J.T., Tong, W.P., Trippett, T.M., Breunan, M.F., Bertino, JR., Mechanisms of Natural
Resistance to Antifolates in Human Soft Tissue Sarcomas, Can. Res. 52, 1434-1438 (1992).
3. Srimatkandada, S., Schweitzer, B., Moroson, B., Dube, S., Bertino, J., Amplification of a
Polymorphic Dihydrofolate Reductase Gene Expressing an Enzyme with Decreased Binding to
Methotrexate in a Human Colon Carcinoma Line, HCT8R4, Resistant to this Drug. J. Bioi.
Chern. 264, 3524-3528 (1989).
4. Schweitzer, B.l., Srimatkandada, S., Gritsman, H., Sheridan, R., Venkatarajhavan, R., Bertino, J.R.,
Probing the Role of Two Hydrophobic Active Site Residues in the Human Dihydrofolate
Reductase by Site-directed Mutagenesis, J. Bioi. Chern. 264,20786-20798 (1989).
5. Tsay, J.T., Appleman, J.R., Beard, W.A., Prendergast, N.J., Delcamp, T.J., Freisheim, J.H. &
Blakley, R.L., Kinetic Investigation of the Functional Role of Phe-31 of Recombinant Human
DHFR, Biochem. 29:6428-6436 (1990).
6. McGuire, J.J., Russell, C.A., Bolanowska, W. E., Freitag, C.M., Jonas, C.S. & Kalman, T.I.,
Biochemical and Growth Inhibition Studies of Methotrexate and Aminopterin Analogues
Containing a Tetrazole Ring in Place of they-Carboxy Group, Can. Res. 50: 1726-1731 (1990).
7. Oefner, C., D' Arcy, A., Winkler, F.K., Crystal Structure of Human Dihydrofolate Reductase
complexed with Folate, Eur. J. Biochem., 174:377-387 (1988).
8. Cody, V., Ciszak, E., Luft, J., Kalman, T.I. & Freisheim, J.H., Crystal Structure of Human
Dihydrofolate Reductase-NADPH-Methotrexate Tetrazole Ternary Complex, Anticancer Drug
Design, 7, 483-491 (1992).
9. Tan, X, Huang, S, Ratnam, M, Thompson, PD & Freisheim, JH, The Importance of Loop Region
Residues 40-46 in Human Dihydrofolate Reductase as Revealed by Site-directed Mutagensis, J.
Bioi. Chern., 265, 8027-8032 (1990).
10. Davis, J.A., Delcamp, T.S., Prendergrast, N.J., Asford, V.A., Freisheim, J.H., Kraut, J., Crystal
Structures of Recombinant Human Dihydrofolate Reductase Complexed with Folate and 5-
Deazafolate, Biochem., 29:9467 (1990).
11. Sutton, P.A., Cody, V., Conformational Analysis of Lipophilic Antifolates: Crystal Structure of
Piritrexim and a Theoretical Evaluation of Its Binding to Dihydrofolate Reductase, J. Amer.
Chern. Soc, 110:6219 (1988).

486
COMPUTER-AIDED DESIGN OF MECHANISM-BASED
PTERIN ANALOGUES AND MD/FEP SIMULATIONS OF
THEIR BINDING TO DIHYDROFOLATE REDUCTASE

Jill E. Gready, 1 Peter L. Cummins 1 and Paul Wormeii2

1Department of Biochemistry, University of Sydney NSW 2006

2Department of Science, University of Western Sydney (Hawkesbury)
Richmond NSW 2753 Australia

INTRODUCTION

As part of our interest in the development of new inhibitors of dihydrofolate reductase

(DHFR), we have been using computer-based methods to predict likely compound classes
and their chemical properties and strength of binding with the enzyme, and also to analyse
data from a concurrent experimental program. The focus has been on mechanism-based
compounds which mimic the presumed protonated-activated form of folate in the enzymic
reduction by DHFR. 1·2 Analogues of two classes, the 8-substituted pterins (8-R-Pts) and 8-
substituted N5-deazapterins (8-R-N5d-Pts) [see Figure 1], have been synthesized and tested
and shown to be substrates and inhibitors, respectively, of DHFR.3-8 The activity data are
consistent with their binding in a substrate-like orientation9 in the enzyme active site as
predicted. Substrate,3,4 inhibitionS and binding data6,8,10 for analogues differently
substituted in the 8-position, and with variable methylation patterns in the 6-, 7- and 5- (8-
R-N5d-Pts) positions show substantial variation in DHFR activity and binding strengths with
dependence on pH and enzyme (chicken or human). Both the 8-R-Pts and 8-R-N5d-Pts are
tautomerically-activated formsll of the parent pterins and NS-deazapterins and are expected
to be more basic, as is found.6,12-14 The UV/vis and fluorescence spectra of these classes
are also interesting with both showing large red shifts in the long wavelength band compared
with the parent compounds, and also large differences between cation and neutral forms
arising from formation of an extended-guanidinium resonancell in the cation.6,12,14,15 We
report results illustrating the use of theoretical methods for studying some chemical and
enzyme-binding properties of these compounds.

COMPUTATIONAL METHODS

Structures, and protonation and tautomer energies were calculated by ab initio

SCF/3-21 G and SCF/6-31 G** methods with full geometry optimization using the
SPARTAN program16 running on a Silicon Graphics IRIS 4D/35 workstation. Spectral

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 487
modelling was done with the CNDO/S-CI program of Del Bene et a[.17 using SCF/3-21G
optimized geometries. The AMBER programs18 running on IBM RS6000 workstations
were used to carry out the molecular dynamics simulation and free energy perturbation
calculations, with force-fields for the DHFR molecule and NADPH and ligand as described
elsewhere.l9-21 The protein and cofactor coordinates were taken from the chicken liver
DHFR (clDHFR) X-ray crystal structure in which both cofactor (NADP+) and biopterin are
bound to the clDHFR molecule.22 The chemical mutations are achieved by coupling the
force fields for the initial and final state ligands, and the free energy changes are calculated
using a perturbation method: full details of the method and simulation conditions can be
found elsewhere.21,23,24 The mutations were performed both for ligand cation in water to
calculate the relative solvation term .6.Gsolv, and when bound to the clDHFR.NADPH
complex to calculate the relative binding term .6.Gbind· The net relative free energy for
binding M~ind is then calculated from the thermodynamic cycle and is related to the ratio of
experimental binding constants for the initial (K) and final (K') state ligands by:

MGbind =.6.Gbind- .6.Gsolv = -RTln(K'/K)

where R = gas constant and T = absolute temperature. Thus a negative MG indicates that
the mutation favours ligand binding. Two main types of mutation were performed, for
transformation of an 8-Me-N5d-Pt to an 8-Me-Pt involving mutation of a CH group toN in
the pyridine ring, and transformation of a CH3 group to H in the 6-, 7- or 5- positions. For
example:

DHFR(Giu-).NADPH.8-Me-N5d-Pt+ -7 DHFR(Glu-).NADPH.8-Me-Pt+
DHFR(Glu-).NADPH.6,8-diMe-Pt+ -7 DHFR(Glu-).NADPH.8-Me-Pt+

RESULTS AND DISCUSSION

Protonation and Tautomer Behaviour

A summary of theoretical SCF/6-31G** results for pterin, N5-deazapterin and their

N8(H)-tautomers and N8-protonated forms is given in Figure 1. As reported previously for
pterin,3,11 the N8(H) tautomer of N5-deazapterin is also less stable than the preferred
N3(H) form by 12 kcal/mol, somewhat less than for pterin (19 kcal/mol). Significant
differences are predicted and found for the protonation site and basicity of the parent
compounds. Experiment indicates pterin preferentially protonates on N1 with a pKa of 2.212
but with an accessible protonation of -1.7 for N8-protonation (see refs 2 and 11), in general
agreement with theory which, at the SCF/6-31G** level, predicts very similar (gas-phase)
protonation energies for the two sites (-238 kcal/mol). In contrast, theory predicts N5-
deazapterin should protonate on N8 (-251 kcal/mol) rather than N1 (-243 kcal/mol) and this
is consistent with the experimental UV/vis spectrum for the N5-deazapterin cation13 which
resembles that for 8-R-N5d-Pts,6,7 and the measured pKa of 3.713 which is consistent with
the higher predicted basicity. Both N8(H) tautomers preferentially protonate on N3 to form
the cations shown in Figure 1 but, as for its parent, the absolute basicity of the N5-deaza
compound is predicted to be higher (-263 compared with -257 kcallmol), a result consistent
with the measured pKa's of 8-Me-Pt and 8-Me-N5d-Pt of 5.212 and 6.4,6 respectively. The
combination of the two protonation energies and tautomer energy in the cycles shown in
Figure 1 necessarily results in a predicted smaller difference in basicity between the parent
and quinonoid forms of the N5-deazapterin than for the pterin: this is consistent with the
experimental differences in pKa of -2.7 and -3.5, respectively. As shown in Figure 2, a
plot of the theoretical protonation energies versus experimental pKa's yields a linear

488
 HN:.xo
I
x) 1. ·238 H.7)
)
1. X=N (PI)
2. X::C (N5d-Pt)
A .& ~·
-251 (-3.7
0

t:X12
H2N N N
N3(H)-tautomer + H+ HN~x)

+J:.x.:
19

7
••• ~ H,N;::.::.,)
.)i.:.,k. J
)l
N )
1. ·257 (-5.2)
extended-guanidinium
resonance substructure
H N N N 2. ·263 (-6.4)
2 H
NS(H )-tautomer

Figure 1. Relationship of SCF/6-31G** protonation and tautomer energies (kcal/mol) for pterin (1), N5-
deazapterin (2), and their N8(H) tautomers and N8-protonated forms. Experimental pKa's for compounds or
model compounds are given in brackets (see text).

2+-----.------.-----r----~----~~~
-265 -260 -255 -250 -245 -240 -235
Protonation Energies (kcal/mol)
Figure 2. Plot of SCF/6-31G** protonation energies versus experimental pKa's (see text) for pterin (0),
8-methyl-pterin<•).N5-deaza-pterin (0) and 8-methyl-N5-deazapterin (e).

375

!-5
X
• 0
350
•• 0
0

f 325
+ 0
0
0 • 0

~
[JO

~-
••
Ol
·il

~
~ 300
•/),.
A
275
300
t'7

325
* 350 375 400 425
Experimental Wavelength (nm)

Figure 3. Plot of experimental and theoretical results for the long-wavelength band of pterin (.L\), Nl+-
pterin (.&.), N5-deazapterin (V), N8+-N5-deazapterin (T), 8-methyl-pterins (0), N3+-8-methyl-pterins (e), 8-
methyl-NS-deazapterins (0), and N3+-8-methyl-N5-deazapterins <•).
Also shown are results for N5+-pterin
(x), N8+-pterin (+),and Nl+-N5-deazapterin (*):see text.

489
correlation 11 which may have value for predicting pKa's for similar compounds from
theoretical calculations.

Modelling of UV/visible Spectra

Experimental UV/vis spectral data6·7·12-15 show characteristic differences between the

parent and 8-substituted forms and between the pterins and NS-deazapterins which are
particularly evident in the maxima for the long-wavelength band: for the neutral forms these
are -340 and 400 nm for the pterins and -330 and 360 nm for the NS-deazapterins, with
characteristic blueshifts of 10 - 20nm for the 8-substituted cations. Calculated electronic
spectra showed a number of 1t* ~ 1t transitions whose band patterns and relative intensities
agree well with experimental data, although the absolute transition energies are too high. 25
The level of correlation is illustrated in the plot in Figure 3 of the experimental and theoretical
results for the long wavelength band for the parent compounds, their preferred protonated
forms, and for the neutral and cationic forms of a number of 8-methyl- pterins and NS-
deazapterins. Also shown are results of plotting spectral predictions for non-preferred
protonated forms for pterin (NS+ and N8+) and NS-deazapterin (Nl +) against experimental
values: the deviations from the correlation are obvious and provide some confidence that
theory could be used to discriminate preferred protonation sites in similar heterocycles for
which experimental data are available.

,J:
1.3 1.3 1.4
N HN
1.40 ,1.31 1.35 ,1.36 1.36

1.~ 1.~
H2 N 1.28 N 1.37 1.31 H2 N 1.37 N 1.30 1.36 H2 N 1.31 N 1.33
(a) (b) (c)

Figure 4. SCF/6-31G** geometries for: (a) N5-deazapterin, (b) N8(H)-N5-deazapterin, and (c) N8-
protonated-N5-deazapterin.

Molecular Geometries

As suggested by these spectral results, the two heterocyclic ring structures for pterins
and NS-deazapterins have very different 1t-electron distributions both between the parent and
8-substituted forms, between the pterin and N5-deazapterin forms, and between the neutral
and cationic forms. These differences are reflected in the molecular geometries, particularly
in the pattern of ring bond lengths which provide an approximate measure of the aromaticity
of the ring system26 and structural evidence for predicted 1t-electron delocalization in the N l-
and N8-protonated cations. Such differences in 1t-properties can be expected to contribute to
differences in ring-stacking interactions with the conserved aromatic residue in the DHFR
active site, although the relative magnitude of this effect compared with other active site
interactions is unknown.24 Results for pterin, its N8(H) tautomer and protonated forms at
the SCF/ST0-3G and SCF/3-21G levels have been reported previously:4,11,27 results at the
SCF/6-31G** level and at the SCF/3-21G level for the various methylated forms shown in
Figure 3 are similar (J.E. Gready, unpublished results). SCF/6-31G** geometries for NS-
deaza-pterin, its N8(H) tautomer and N8-protonated form are shown in Figure 4: SCF/3-
21G geometries for these and for the various methylated forms shown in Figure 3 are similar
(J.E. Gready, unpublished results). The main features to note in these structures are that the
mostly aromatic pyridine ring in the parent compound becomes much less aromatic
(alternating bond lengths) in the quinonid tautomer, and that changes in the N3-N8 grouping

490
in the cation are consistent with delocalization as shown in Figure 1. Our efforts to obtain
crystals for the 8-R-Pts and 8-R-N5d-Pts suitable for x-ray analysis to test these structural
predictions have been unsuccessful so far except for 6,8-dimethyl-NS-deazapterin
hydrochloride (M.J. Koen, T.W. Hambley and J.E. Gready, unpublished results) the
structure of which compares well with both the SCF/6-31G** geometry for the cation shown
in Figure 4 and the SCF/3-21G geometry for N3-protonated-6,8-dimethyl-N5-deazapterin.

Free Energy Perturbation/Molecular Dynamics (FEP/MD) Simulations

To illustrate the effect of replacing a ring-carbon with a nitrogen, results for the net
relative binding free energy and component solvation and enzyme binding terms for 8-Me-Pt
and 8-Me-N5d-Pt and their 6-methyl derivatives are shown in the first two lines of Table 1.
The solvation terms ilGsolv indicate the N5-deaza compounds are -4 kcal/molless stable in
solution and this hydrophobic effect could be expected to enhance their binding in the DHFR
active site. (Reference to other results in Table 1 for the 7,8-dimethyl and 6,7,8-trimethyl
analogues shows a very similar effect.) Consideration of the terms for the free energies for
interactions with the DHFR active site ilGbind shows they are of similar magnitude (-4
kcal/mol) and favour binding the pterins. When the solvation terms are subtracted from the
binding terms the net free energy change for binding is quite small, i.e neither compound
class is strongly favoured, a result roughly in agreement with available experimental kinetic
and binding data.4,6,8 Other results in Table 1 are for a number of transformations among 8-
Me-Pts and 8-Me-N5d-Pts involving ring methyl substituents. These results and
examination of the MD structures using molecular graphics suggest a quite complex interplay
of protein-ligand interactions, including steric effects and favourable hydrophobic
interactions, and solvation effects depending on the nature of the ring and substitution in
other ring positions. Although the trends correlate reasonably well with available
experimental binding data, it has become clear that in order to obtain more quantitative
predictions the theoretical model needs to be developed to allow better relaxation of the
enzyme with the different bound ligands.

Table 1. FEP/MD solvation and enzyme-binding contributions to the relative binding of 8-

Me-Pts and 8-Me-N5d-Pts to clDHFR.
Simulation ~Gsolv ~Gbinl ~~Gbind

8dp ~ 8p -4.03 ± 0.03 -3.83 ± 0.16b 0.20 ± 0.19

68dp ~ 68p -3.78 ± 0.03 -4.12 ± 0.18 -0.34 ± 0.21
78d ~ 78p -4.35 ± 0.06 -4.65 ± 0.20 -0.30 ± 0.26
678dp ~ 678p -4.12 ± 0.03 -5.44 ± 0.57 -1.32 ± 0.60
678dp ~ 78dp -0.97 ± 0.04 -0.25 ± 0.10 0.72 ± 0.14
~ 68dp -1.13 ± 0.09 -0.83 ± 0.32 0.30 ± 0.41
78dp ~ 8dp -1.07 ± 0.03 -1.95 ± 1.oob -0.88 ± 1.03
68dp ~ 8dp -1.49 ± 0.06 -0.01 ± 0.13 1.48 ± 0.19
58dp ~ 8dp -2.29 ± 0.04 -1.43 ± 0.12 0.86 ± 0.16
5678dp ~ 568dp -0.44 ± 0.00 -0.14 ± 0.44 0.30 ± 0.44
568dp ~ 68dp -1.50 ± 0.05 -1.38 ± 0.31 0.12 ± 0.36
~ 58dp -1.13 ± 0.03 0.14 ± 0.32 1.27 ± 0.35
678p ~ 78p -0.97 ± 0.08 0.11 ± 0.07 1.08 ± 0.15
~ 68p -0.27 ± 0.05 -0.23 ± 0.41 0.04 ± 0.46
68p ~ 8p -2.07 ± 0.08 0.37 ± 0.09c 2.44 ± 0.17
a) ~Gbind from average of forward and backward mutations with double-wide sampling
b) Geometry change m 'A= 1~ 0 mutation c) Geometry change in 'A= 0~ 1 mutation

491
SUMMARY

Using a combined theoretical and experimental approach we have been able to predict
several chemical properties and the contributions of the many factors which determine the
macroscopic binding behaviour of these new mechanism-based compounds with DHFR, and
also analyse experimental data to develop structure-activity relationships.

ACKNOWLEDGEMENT

We thank Dr Jeff Reimers for access to the VAX version of the CNDO/S-CI program
and Prof Wolfgang Pfleiderer, Dr Mark Koen and Michael Ivery and Soon-Seog Jeong for
compounds and experimental data. Funding from the National Health and Medical Research
Council is gratefully acknowledged.

REFERENCES

1. F.M. Huennekens and K.G. Scrimgeour, in: "Pteridine Chemistry," W. Pfleiderer and E.C. Taylor,
eds., Pergamon, Oxford (1964), pp. 355-76.
2. J.E. Gready, Biochemistry 24: 4761 (1985).
3. V. Thibault, M.J. Koen and J.E. Gready, Biochemistry 28:6042 (1989).
4. J.E. Gready, in: "Chemistry and Biology ofPteridines 1989," H.-Ch. Curtius, S. Ghisla and N. Blau,
eds., de Gruyter, Berlin (1990), pp. 23-30.
5. M.T.G. Ivery and J.E. Gready, Bioi. Chern. Hoppe-Seyler 373:1125 (1992).
6. J.E. Gready, M.T.G. Ivery, M.J. Koen and H.J. Yang, in: "Pteridines and Related Biogenic Amines and
Folates 1992," N. Blau, H.-Ch. Curtius, R. Levine and J. Yim, eds., Hanrim, Seoul (1992), pp.265-76.
7. M.J. Koen and J.E. Gready, J. Org. Chern. 58:1104 (1993).
8. M.T.G. Ivery and J.E. Gready, other paper this volume.
9. C. Oefner, A. D'Arcy and F.K. Winkler, Eur. J. Biochem. 174:377 (1988).
10. S.-S. Jeong and J.E. Gready, other paper this volume.
11. J.E. Gready, J. Comput. Chern. 6: 37 (1985).
12. W. Pfleiderer, in: "Comprehensive Heterocyclic Chemistry," A.J. Boulton and A. McKillop,
eds., Pergamon, New York (1984), pp. 263-327.
13. M. Pfleiderer, Masters Thesis, University of Konstanz (1986), p.19.
14. S.-S. Jeong, P. Wormell and J.E. Gready, Pteridines (1993) in press.
15. S.-S. Jeong and J.E. Gready, Bioi. Chern. Hoppe-Seyler 373:1139 (1992).
16. W.J. Hehre, SPARTAN (Version 2.0), Wavefunction Inc., Irvine CA, USA.
17. J. Del Bene, H.H. Jaffe, R.L. Ellis and G. Kuehnlenz, CNDO/S-CI, QCPE #174, Dept of Chemistry,
Indiana University.
18. U.C. Singh, P.K. Weiner, J.W. Caldwell and P.A. Kollman, AMBER (Version 3.2), Dept. of
Pharmaceutical Chemistry, University of California, San Francisco (1988).
19. P.L. Cummins, K. Ramnarayan, U.C. Singh and J.E. Gready, J. Am. Chern. Soc. 113:8247 (1991).
20. P.L. Cummins and J.E. Gready, Proteins: Struct. Funct. Genet. (1993) in press.
21. P.L. Cummins and J.E. Gready, J. Comput.-Aided Mol. Des. (1993) in press.
22. M.A. McTigue, J.F. Davies, B.T. Kaufman and J. Kraut, Biochemistry 31:7264 (1992).
23. U.C. Singh, F.K. Brown, P.A. Bash and P.A. Kollman, J. Am. Chern. Soc. 109:1607 (1987).
24. P.L. Cummins and J.E. Gready, other paper this volume.
25. P. Wormell and J.E. Gready, Chern. Phys., submitted.
26. J.E. Gready, Int. J. Quant. Chern. 91:369 (1987).
27. J.E. Gready, J. Mol. Struct.: THEOCHEM 124:1 (1985).

492
DOES R67 DffiYDROFOLATE REDUCTASE POSSESS A PROTON DONOR?

Janel C. Holland, Charles E. Linn, Enrico DiGiammarino, Robert Nichols and

Elizabeth E. Howell

Department of Biochemistry
University of Tennessee
Knoxville, TN 37996-0840

Dihydrofolate reductase (DHFR, EC 1.5.1.3) catalyzes the reduction of dihydrofolate to

tetrahydrofolate using NADPH as a cofactor. DHFR is important in folate metabolism as
generation of tetrahydrofolate is required for the synthesis of thymidylate, purine nucleosides,
methionine and other metabolic intermediates. Efficient inhibition of DHFR results in blockage
of DNA synthesis and consequent cell death. Therefore specific inhibition of bacterial DHFR
by trimethoprim is the basis for clinical treatment of numerous bacterial infections.
Clinical resistance to trimethoprim has been observed and correlated with the production
of novel DHFRs encoded by R-plasmids. At least 10 different types of plasmid specified DHFR
genes have been identified 1• Type II DHFR is of particular interest as it is unrelated genetically
and structurally to chromosomal DHFR. Some pertinent information comparing E. coli
chromosomal DHFR and R67 DHFR, a prototypic type II DHFR, is summarized in Table I.
The origin of type II DHFR genes is not known. Stone and Smith2 hypothesized that a
NADP-linked oxidoreductase originally specific for another substrate may have been recruited
to catalyze the reduction of dihydrofolate and to provide resistance to TMP.
A crystal structure of a dimeric form of R67 DHFR was reported by Matthews et al. in
19863 • More recently, the active tetrameric species has been crystallized as an apoenzyme and
in a binary complex with thio-NADP+ (Matthews, Xuong and Narayana, personal
communication). From the refined structures, it is clear that the overall structure is entirely
different from that of E. coli chromosomal DHFR. Difference Fourier maps describing binding
of thio-NADP+ indicate the active site is a pore traversing the length of the molecule. Of
especial interest is the implication from the crystal structure that one active site exists per
tetramer and that residues from each monomer contribute to binding in the single active site.
While a shared active site between protomers is not surprising, only one active site per multimer
is quite unusual.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling eta!., Plenum Press, New York, 1993 493
 Table I. A Comparison of E. coli Chromosomal and R67 DHFRs

E. coli DHFR R67DHFR

Enzyme Form Monomer; 18,000 daltons Tetramer; 34,000 daltons

Crystal Structure 8-stranded P-sheet with 4 4 P-barrels; single active site

a-helical connecting strands pore composed of residues from
4 subunits
Trimethoprim K; 20pM4 0.15 mW
Dihydrofolate K, 1.2 p.M6 5.8 p.M7
NADPH Km 0.94 p.M6 3.0 p.M7
kcal (pH 7.0) 29 sec· 1 1.3 sec· 1

The difference Fourier map describing thio-NADP+ binding to tetrameric R67 DHFR
shows electron density in the active site pore describing at least 2 symmetry related binding sites
(Matthews, Xuong, Narayana, personal communication). Therefore a productive model for
catalysis may describe binding of dihydrofolate in half the pore and binding of cofactor in the
other half. The pteridine ring of dihydrofolate and the nicotinamide ring of NADPH would
encounter each other at the center of the pore. This model is consistent with our preliminary
kinetic studies. Surprisingly, a proton donor has not been identified in the active site pore of
R67 DHFR although a proton donor greatly facilitates catalysis in E. coli chromosomal DHFR.
In a D27S mutant of E. coli chromosomal DHFR, the proton donor, Asp27, was removed by
site directed mutagenesis8 • The D27S DHFR partially compensated by binding pre-protonated
substrate (pKa=2.59~ and k..t increased at low pH where protonated dihydrofolate is the
predominate species.
To confirm our interpretation of the crystal structure indicating tetrameric R67 DHFR
does not possess a proton donor, we examined a pH profile of R67 DHFR activity expecting to
see a similar profile to D27S E. coli DHFR, ie. increased activity at low pH as the pKa of
protonated dihydrofolate (- 2.59)9 is approached. Instead a pH profile of percent activity in
R67 DHFR displays a bell shape with pKas of -6.2 and -7.3. The lower pKa value suggests
histidine may be ionizing and affecting enzyme activity. A single histidine (H62) occurs per
monomer in R67 DHFR and is found at the dimer-dimer interface between tetramers. From the
222 symmetry of the crystal structure, 2 histidines appear per interface (ie. H62,H362 at one
interface and Hl62,262 at the second). Protonation of H62 could potentially destabilize
tetrameric R67 DHFR and favor dimer formation. As the putative active site pore would be lost
in dimeric R67 DHFR, decreased activity should result.
To determine if the lower pKa in kinetic pH profiles is related to ionization of H62, the
stability of tetrameric R67 DHFR was monitored as a function of pH10• For the pH range 5-8,
tetrameric R67 DHFR reversibly dissociates into dimers as monitored by sedimentation
equilibrium and molecular sieving techniques. Ionization of histidine was confirmed by
monitoring the chemical shifts of the C2 proton in NMR experiments and best fits of an
incomplete titration curve yield a pKa of 6. 77. Since W38, 138,238,338 also occur at the dimer-
dimer interfaces, fluorescence can monitor the tetramer <:::% 2 dimer equilibrium. When
fluorescence was monitored over the pH range 5-8, a protein concentration dependence of
fluorescence was observed (since the reaction is bimolecular) and global fitting of three titration
curves yields Kd = 9. 72 nM, pK. = 6. 84 and n =2 for the linked reactions:

494
 K.t K.2n
tetramer ~ 2 dimers + 2n H+ ~ 2n protonated dimers.

Additionally, modification of H62 by diethylpyrocarbonate (DEPC) stabilizes dimeric

R67 DHFR and causes a 200-600 fold decrease in catalytic efficiency (k.,.1 =0.052 sec- 1, Krn <DHFl
= 72 I'M, Kro (NADPH) = 40 I'M 1 ~. These values are upper limits for dimeric enzyme activity
as our 14C-DEPC labeling studies indicate only 1.35 histidines per dimer are labeled after
separation of any unmodified tetramer by affmity chromatography. At increased DEPC
concentrations, modification is only partially reversible by hydroxylamine treatment, suggesting
either modification of other groups essential for enzyme activity or disubstitution at histidine11 •
The decreased activity of modified, dimeric R67 DHFR is consistent with loss of the active site
pore.

ENGINEERING A STABLE TETRAMER

Since the ionization of H62 is linked to dissociation of active tetramer into inactive or
partially active dimers, any potential activity increase at low pH associated with protonation of
dihydrofolate in solution (pKa=2.59) will be masked. To investigate whether there is a proton
donor in R67 DHFR using kinetic methods, a stable tetramer needed to be constructed.
From our analysis of the tetrameric x-ray structure, a H62C mutation should allow
formation of disulfide bonds between H62C and H362C (and H162C & H262C) at the dimer-
dimer interfaces and results in covalently stabilized tetrameric R67 DHFR. The H62C mutation
was constructed by standard site directed mutagenesis techniques using a synthetic R67 DHFR
gene7 • The H62C mutant gene was sequenced to confirm that no additional mutations were
present.
H62C R67 DHFR was purified according to the protocol for purification of native R67
DHFR7 except that E. coli cells were lysed by sonication and no reducing agents were added to
buffers. Purification steps included PBE chromatofocusing, 075 molecular sieving and DEAE
ion-exchange chromatography.
Purified H62C R67 DHFR is predominately reduced and dimeric as determined by
molecular sieving studies and Ellmans titrations. Disulfide bond formation does occur
spontaneously, albeit slowly. Numerous procedures, for example disulfide exchange, were
investigated to induce disulfide bond formation, however the easiest and most efficient method
was to incubate H62C R67 DHFR at high protein concentrations ( ~ 15 mg/ml) at pH 8. 8, 4' C
for > 1 week. Formation of active tetramer was monitored by either increased enzyme activity
and/or by molecular sieving chromatography. For the latter, two peaks were observed during
elution from a Superose 12 HR10-30 column (Pharmacia FPLC), indicating tetrameric and
dimeric species are not in rapid equilibrium in H62C R67 DHFR. After 1 week, stable (S-S
crosslinked) tetramer comprises ~ 20% of the total H62C R67 DHFR population. To separate
active tetramer from dimer, a 0.03-0.05M KCl gradient on a monoQ column equilibrated in 10
mM Tris pH 8 was utilized (Pharmacia FPLC).
As shown in Figure 1, elution of native R67 DHFR from a Superose 12 HR 10-30
column at pH 7 and 5 shows a large change in elution position, consistent with dissociation of
tetramer to 2 dimers 10 • In contrast, molecular sieving studies of active H62C R67 DHFR at pHs
5 and 7 show only minimal changes in elution position, indicating perhaps a minor
conformational change. From a plot of K.. (= (elution volume - void volume) I (total bed
volume- void volume)) versus MW standards, the molecular weight of H62C R67 DHFR at pH
7 (and 5) is 42,000. This value is consistent with active H62C R67 DHFR being tetrameric.

495
 (A)

000

~ 1----..J

A
100
(B)

-
";"

-...
(,)
Q)
0

0 10 15 20 25 >-
·s;
:;::
(,)

(C) <C
Q)
1G-1
>
:;::
ftl
Q)
a: 0
(D)

1~~~~~~~-L~~~~~~

U M U M U M n M M
Elutton Volume (mL) pH
Figure 1. Elution profiles from a Superose 12 Figure 2. pH profiles for native ( o) R67 DHFR
HR10-30 column. Native R67 DHFR at pH 7 and & the oxidiz.dd, tetrameric, H62C mutant ("') R67
5, panels A & B. H62C R67 DHFR at pH 7 and 5, DHFR.
panels C & D.

Ellmans titrations were performed to verify the formation of disulfide bonds in tetrameric
H62C R67 DHFR. Non-reducing Ellmans titrations yield 0.94 and 0.90 free sulfuydryls per
monomer in native and H62C R67 DHFRs respectively. In contrast, reducing Ellmans titrations
yield 1.36 and 1.96 sulfuydryls per monomer for native and H62C R67 DHFR respectively.
These studies clearly confirm a stable tetrameric species has been constructed by disulfide
crosslinking.
Oxidized, tetrameric H62C R67 DHFR is quite active with k.,.1 = 0.64 s· 1, K.n <DHFJ =
3.8~M and K.n <NADPHl = 6.2~M at pH 7.0. In comparison to native R67 DHFR values (see
Table I), K.n<NADPHl increases 2 fold while kcat and K.n<DHF) decrease 2 and 1.5 fold respectively.
These minimal changes indicate the conformation of the active site pore in H62C R67 DHFR
has changed only slightly.
As shown in Figure 2, a pH profile describing H62C R67 DHFR catalysis shows
increasing activity at low pH. This behavior is reminiscent of the pH profile for the mutant
D27S E. coli chromosomal DHFR8 and supports our model that R67 DHFR uses protonated
dihydrofolate as substrate. Catalytic activity increases at low pH as the pKa for N5 in
dihydrofolate ( =2.59~ is approached.
Based on the above results, our current model describing the mechanism of R67 DHFR
is shown in Scheme I below where T is tetramer and D, DH,. are unprotonated & protonated
dimers respectively. In this model, R67 DHFR uses protonated dihydrofolate (HDHF) as
substrate; binding of unprotonated dihydrofolate is unproductive. We note the binding
stoichiometry of ligands has not yet been determined.

496
 2DH,.
+2nH+ 1 ~ p:£<.-6.77-6.84
Scheme I
2D
1~ ~
T + DHF + NADPH .;::: T • DHF •NADPH
p:£<.=2.591 ~ +H+ 1 ~ +H+
T + HDHF + NADPH :;::::: T • HDHF • NADPH ~ products

SUBSTRATE TOLERANCE STUDIES

A broad cofactor tolerance has previously been noted in R67 DHFR as it can utilize
{3-NADPH, a-NADPH and NADH as cofactors; the latter two cofactors display 4 and 40
fold increases in K, and 1.5 and 17 fold decreases in ).(.,,1 respectively 12 • To investigate the
substrate tolerance of this enzyme, we have examined the ability of a quinonoid tautomer of
dihydropterin to serve as a substrate (Figure 3B). This tautomer is not reduced by
chromosomal DHFRs and a separate, nonhomologous eukaryotic enzyme, dihydropteridine
reductase (DHPR), reduces quinonoid-dihydropterin to tetrahydropterin 13 • We find R67
DHFR reduces quinonoid 6,6-dimethyl-dihydropterin (q-DMP) with ).(.,.1 = 0.40 sec· 1, K, (q-
DMPJ = 185 ~-tM and K, (IIIADPH) = 11 ~-tM (pH 7.0). Since the pKa of N3 m q-DMP is 5.15 14,
the ability of R67 DHFR to utilize this substrate shou!d be facilitated by the availability of
protonated q-DMP in solution. From a mechanistic perspective, reduction of q-DMP by R67
DHFR is surprising as reduction of dihydrofolate is proposed to occur via hydride transfer
from NADPH to C6 of the pteridine ring in chromosomal DHFR whereas reduction of q-
DMP is proposed to involve hydride transfer to N5 in DHPR15 •

Figure 3. Alternate substrates for R67 DHFR- A. DHF; B. quinonoid 6,6-dimethyldihydropterin; C. MTHF;
D. 5-iminium cation of MTHF. R = 4-aminobenzoyl[a~](L)-glutamic acid.

Another substrate we have recently determined to be reduced by R67 DHFR is

N5,N10-methylene-tetrahydrofolate (MTHF; see Figure 3C) with }(..1 = 0.84 sec· 1, K, <MTHF)
= 78.1 ~-tM, and K, <NADPH) = 7.1~-tM (pH 7.0; anaerobic conditions). MTHF is not a
substrate for chromosomal DHFRs and different methylene-tetrahydrofolate reductases
(MTHFRs) are present in pro- and eukaryotes to reduce MTHF to 5-methyl-THF16 • In

497
solution, ring opening of MTHF is facilitated by acid catalysis to yield the 5-iminium cation
(Figure 3D 17). Interestingly, eukaryotic MTHFR, a flavin enzyme, also possesses a DHPR
activity. The 5-iminium cation has been proposed to tautomerize to a quinonoid 5-CHrDHF
intermediate which could serve as a substrate for the DHPR activity of MTHFR18 • From
these model folate and eukaryotic MTHFR studies, we anticipate the activated MTHF species
being reduced by R67 DHFR is either the 5-iminium cation or the quinonoid 5-CH3-DHF
tautomer.
We note that folate is not a substrate for R67 DHFR, most likely because its pKa is
< -1.5 19 and the concentration of activated substrate (protonated folate) would be minimal in
our assays.

CONCLUSIONS The altered pH profile for the mutant H62C R67 DHFR as well as the
substrate tolerance studies for native R67 DHFR are both consistent with a model for
catalysis such that a proton is obtained from solution, not directly from the enzyme. This
contrasts with mechanisms proposed for chromosomally encoded DHFRs, which possess a
proton donor to facilitate catalysis.

REFERENCES

I. Amyes, S.G.B., Towner, K.J. and Young, H.-K., J. Med. Microbial. 36:1-3 (1992).
2. Stone, D. and Smith, S., J. Bioi. Chem. 254:10857-10861 (1979).
3. Matthews, D.A., Smith, S.L., Baccanari, D.P., Burchall, J.J., Oatley, S.J. and Kraut,
I., Biochemistry 25:4194-4204 (1986).
4. Stone, S.R. and Morrison, J.F., Biochim. Biophys. Acta 869:275-285 (1986).
5. Amyes, S.G.B. and Smith, J.T., Eur. J. Biochem. 61:597-603 (1976).
6. Howell, E.E., Booth, C.B., Farnum, M., Kraut, J. and Warren, M.S., Biochemistry
29:8561-8569 (1990).
7. Reece, L.J., Nichols, R., Ogden, R.C. and Howell, E.E., Biochemistry 30:10895-
10904 (1991).
8. Howell, E.E., Villafranca, I.E., Warren, M.S., Oatley, S.J. and Kraut, J., Science
231:1123-1128 (1986).
9. Maharaj, G., Selinsky, B.S., Appleman, J.R., Perlman, M., London, R.E. and Blakley,
R.L., Biochemistry 29:4554-4560 (1990).
10. Nichols, R., Weaver, C.D., Eisenstein, E., Blakley, R.L., Appleman, J., Huang, T.H.,
Huang, F.Y. and Howell, E.E., Biochemistry 32:1695-1706 (1993).
11. Miles, E.W., Methods Enzymol. 47:431-442 (1977).
12. Smith, S.L. and Burchall, J.J., Proc. Nat/. Acad. Sci. 80:4619-4623 (1983).
13. Kaufman, S., Neurochemical Research 16:1031-1036 (1991).
14. Bailey, S.W. and Ayling, I.E., Biochemistry 22:1790-1798 (1983).
15. Armarego, W.L.F. and Vasudevan, S.G. (1989) in: "Chemistry & Biology of
Pteridines, 1989," Curtius, H. -Ch., Ghisla, S. and Blau, N., Eds., Walter de
Gruyter, Berlin (1990).
16. Vanoni, M.A., Ballou, D.P. and Matthews, R.G., J. Bioi. Chem. 258:11510-11514
(1983).
17. Kallen, R.G. & Jencks, W.P., J. Bioi. Chem. 241:5851-5863 (1966).
18. Matthews, R.G. and Kaufman, S., J. Bioi. Chem. 255:6014-6017 (1980).
19. Poe, M., J. Bioi. Chem. 252:3724-3728 (1977).

498
LASER-SENSITIZED TAUTOMERS IN DIHYDROFOLATE REDUCTASE

John W. Ledbetter, I Wolfgang Pfleiderer,2 and James H.

Freisheim3

1Department of Biochemistry and Molecular Biology

Medical University of South Carolina
Charleston, SC 29425-2211
2Fakultat fiir Chemie
Universitat Konstanz
D-7750 Konstanz, Germany
3Department of Biochemistry and Molecular Biology
Medical College of Ohio
Toledo, Ohio 43699-008

INTRODUCTION

The mechanism of protonation of the dihydropterin ring prior to hydride

transfer in the catalytic mechanism of dihydrofolate reductase (DHFR) (5,6,7,8-
tetrahydrofolate: NADP oxidoreductase, E.C.l.5.1.3) remains controversial.
Several proposals have emerged. One follows protonation of 04 followed by
tautomerism 1 which may be accompanied by required conformational
changes. 2 Another3 proposes a proton transfer from glu30 in chicken liver
DHFR to N5 via 04 and the intervening water molecule. A different scheme4,5
for protonation involves the formation of the enol structure of 04.
The experiments reported herein were found to offer new data on the
lactam-lactim tautomers of pterins. Two laser-induced triplet states were
observed in this work and previously6 for pterin and for biopterin. With
substituted pterins which inhibit tautomerization we now assign these two
states to the lactam and lactim tautomers. The very interesting observation
that the lactim triplet was prominent in the DHFR-bound biopterin may signal a
preference for a tautomer.

EXPERIMENTAL METHODS

L-Biopterin was purchased from Dr. B. Schircks Laboratories, Jona,

Switzerland. Pterin was obtained from Fluka Chemika-Biochemika, Buchs,
Switzerland. The 3-methylpterin (3MP) and 2-amino-4-methoxypteridine (4MP)
were prepared as previously described. 7 Bovine serum albumin (BSA) was
purchased from Sigma Chemical Co. The recombinant human DHFR (rHDHFR)
was prepared according to Prendergast et aLB The laser excitation
experiments9-ll utilized the 337-nm ultraviolet light from a laboratory-
constructed nitrogen laser to populate the triplet state whose decay was
observed by a Spectra-Physics Model 165 Ar+ laser. The data for the decay
plots were collected with a Tektronix 7912 AD programmable digitizer. The

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 499
decay curves were analyzed by a nonlinear regression subroutine based on a
Levenberg-Marquardt algorithm supplied by IMSL, Inc.

RESULTS AND DISCUSSION

The upper absorption decay curve of Figure 1 shows for an Ar-saturated

0.4 mM solution of pterin at pH 8.4 in phosphate a biexponential decay. The
fast decay with k = 3.4 ± .13 x 106 s-1 and the slow decay with k = 0.45 ± .01 x
106 s-1 agree well with earlier results of 3.3 x 106 s-1 and 0.43 x 106 s-1 by
Chahidi et ai.6 They suggested that the biexponential decay results from two
triplet states which could arise from the lactam P lactim tautomers. Pursuing
this possibility we have investigated 3-methylpterin (3MP) and 2-amino-4-
methoxypteridine (4MP) which cannot tautomerize because the methyl group
has replaced the required hydrogen. The absorption decay of 4MP at pH 6.5 is
also shown in Figure 1 as the middle curve. Clearly, the absorption decays as a
single exponential with k = 2.2 ± .04 x 106 s-1. The much weaker absorption
decay of 0.4 mM 3MP, the lower curve, also occurs by a single exponential with
k = 1.57 ± .06 x 106 s-1. From these decay curves it is easy to draw the
conclusion that the 3MP and 4MP each have a single triplet state absorption
and decay. Since 3MP represents the lactam structure with a 4-ketoid
function and 4MP represents the lactim structure it would appear that the
solution of pterin (and biopterin9) possesses both tautomers in the triplet
excited state. Excitation makes OH and NH groups many times more acidic
while C=O and C=N groups become more basic.l2 From a pterin solution in the
ground state with the lactam tautomer present7, excitation then yields an
observable mixture of tautomers. The higher rate constant of 4MP suggests

PTER I N~ DECAY COMPONENTS WITH oH

FRACTION DECAY COMPONENT
o. 034 I 1.1313 r - -- - - - -- -- ----,

0. 026 t
I

0
G. 0!71
0

....<_J

II
"'0
G. GG9

ru ~ !"J !" p
0 0 0 0
0 0 0 0 0
0
MiCROSECONDS

Figure 1. Triplet absorption decay at Figure 2. Fraction of preexponential

4765 A for 0.4 mM pterin (upper) at factors of the biexponential decay for
pH 8.4, 0.2 mM 4MP (middle) at pterin with pH.
pH 6.5 and 0.4 mM 3MP (lower) at
pH 6 .5 in phosphate buffer.

500
that the faster decay of pterin belongs to the lactim tautomer. However, the
rate constant is 1.4 times that of 3MP and not the 7.6 ratio for the pterin
decays. The methyl groups may account for this difference in the ratios.
The most obvious difference in the absorption decays of the derivatives is
the intensity of the triplet absorbance. This is clearly evident in Figure 1. We
have been able to use this difference to assign the triplet state tautomers of
pterin and L-biopterin in the following experiments. In Figure 2 we have plotted
the relative magnitudes of the two decay components of pterin as a function of
pH. The preexponential factors were determined by holding constant the decay
rate constants at 3.3 x 106 s-1 and 0.7 x 1o6 s-1 which represent an average
below pH 9.5. Above pH 9.5 the anionic pterin decayed with k = 1.5 x 1Q6 s-1,
so that number was used. The regression analysis then produced the
preexponential factors required to fit the curve. Residual absorption was
included. The factors as a fraction of 1 are plotted in Figure 2 for each decay.
This is an interesting plot in that it shows not only the pKa -9.8 for the triplet
state (Chahidi et al.6 reported 9.5-10) but also the formation at lower pH's of
one tautomer at the expense of the other. On excitation the tautomeric
equilibrium shifts from a predominately lactam structure to more of the lactim
structure depending on the pH. This process maximizes at pH -8.5. To prove
this point we now plot in Figure 3 the top absorbance change at t=O for the
triplet states against pH. At pH 6.5 the top 600's for equal concentrations of
pterin and 3MP coincide. At this pH the lactam tautomer clearly dominates in
the excited pterin solution. As the pH approaches 9.5 the top 600 increases
rapidly for pterin while that for 3MP decreases. The increase in 600 for pterin
approaches that for 4MP shown here at half the concentration, 0.2 mM. The
preexponential factors approach 40% at pH -8.5 in Figure 2 and, therefore, the
comparison of 4MP at one-half the pterin concentration may be
appropriate. So, while the decay rates of the derivatives suggest an assignment

PT£RINw T~UTOM£R TOP OD WITH PH

TOP D£LT~ OD
e.0seee r - -- -- -- - - ---, 0.045

0 . 034

0.023
0
0

...<
.J
U1
0

0 . 011

!'l ,b. ~ !"

0 0 0 0
p
0 0 0 0 0
0
pH MICROSECONDS

Figure 3. Top change in OD at 4765 Figure 4. Triplet absorption decay

A with pH for 0.4 mM pterin at 4579 A for 0.2 mM L-biopterin
(star), 0 .2 mM 4MP (box) and 0.4 mM (upper) and for 0.4 mM rHDHFR
3MP (cross) in phosphate buffer. added (lower) at pH 9.35 in 0.05 M
Tris and 0.5 M KCI buffer.

501
of tautomeric structures to the biexponential decay of pterin, the intensities
serve to substantiate that assignment. The faster triplet state decay of pterin
and L-biopterin results from the excited lactim tautomer. The slower decay
belongs to the lactam tautomer.
The assignment now allows a determination of the effect of the rHDHFR
binding site on the tautomeric equilibrium. For this comparison between
substrate in free solution and in the enzyme we used L-biopterin for which we
have both binding data and decay data. L-Biopterin binds to rHDHFR with a
KD = 202 J.1.M9. At the concentrations used, 60% of the L-biopterin was bound
to rHDHFR, so the results should be interpreted with caution. The lower plot of
Figure 4 represents the remarkable effect of rHDHFR on the triplet state
behavior of L-biopterin. Because an equivalent amount of BSA had a minor
effect (10% increase in the ratio oflactim/lactam preexponential factors) on the
biexponential decay. the effects of rHDHFR on L-biopterin probably occur at the
active site. The remaining exponential with k = 3.4 x106 s-1 could be the result
of active-site quenching of the slower decay of Figure 4 which is observed at pH
9.35 for free L-biopterin. However, the single exponential decay in rHDHFR
which also occurs at pH's 8.0 and 6.9 with a rate constant equal to that for the
lactim tautomer suggests a shift in the tautomeric equilibrium rather than
protein quenching. The lactim preexponential factor in fact increases from
-20% for free solution to -50% for the bound substrate. This picture could
mean that in rHDHFR the lactim tautomer predominates unless possibly the
triplet state of the lactam tautomer quenches so rapidly at the site that its
decay isn't observed.

REFERENCES
1. C. Oefner, A. D'Arcy, and F.K. Winkler, Eur. J. Biochem. 174:377-385
(1988).
2. W.A. Beard, J.R. Appleman, T.J. Delcamp, J.H. Freisheim, and R.L.
Blakley. J. Biol. Chern. 264: 9391-9399 (1989).
3. M.A. McTigue, J.F. Davies. B.T. Kaufmann, and J. Kraut, Biochemistry
31:7264-7273 (1992).
4. K. Taira, J.-T. Chen, C.A. Fierke, and S.J. Benkovic, Bull. Chern. Soc.
Japan 60:3025-3030 (1987).
5. B.S. Selinsky. M.E. Perlman, R.E. London, L.J. Unkefer, J. Mitchell, and
R.L. Blakley, Biochemistry 29:1290-1296 (1990).
6. L. Chahidi, M. Aubailly. A. Momzikoff, M. Bazin, and R. Santus,
Photochem. Photobiol. 33:641-649 (1981).
7. W. Pfleiderer, E. Liedek, R. Lohrmann, and M. Rukwied, Chern. Ber.
93:2015-2024 (1960).
8. N.J. Prendergast, T.J. Delcamp, P.L. Smith, and J.H. Freisheim,
Biochemistry 27:3664-3671 (1988).
9. J.W. Ledbetter and J.H. Freisheim, submitted for publication.
10. J.R. Lindroth, S.M. Martin, and J.W. Ledbetter, Comput. Biol. Med.
17:369-381 (1987).
11. L.S. Walters, T.J. Cornish. H.W. Askins, and J.W. Ledbetter, Anal.
Biochem. 127:361-367 (1982).
12. J.F. Ireland and P.A.H. Wyatt. Adv. Phys. Org. Chern. 12:131-221 (1976).

502
METHOTREXATE-INSENSITIVE MUTANTS OF HUMAN
DIHYDROFOLATE REDUCTASE (hDHFR) CONSTRUCTED BY SITE-
DIRECTED MUTAGENESIS AT PHENYLALANINE 34

Takayuki Nakano, James R. Appleman and Raymond L. Blakley

Department of Molecular Phannacology

St. Jude Children's Research Hospital
Memphis, TN 38101

INTRODUCTION
Dihydrofolate reductase (DHFR) catalyzes NADPH-dependent reduction of 7,8-
dihydrofolate (H2folate) into 5,6,7 ,8-tetrahydrofolate (H4folate), which is essential for the
synthesis of thymidylate and hence the synthesis of DNA Since rapidly growing cells can be
killed by inhibition of hDHFR by methotrexate (MTX), this and other inhibitory drugs are
used in the chemotherapy of cancer. A serious problem in such chemotherapy with MTX is
development of MTX-resistance in tumor cells. Spontaneous mutation in the DHFR gene is
suspected to be one of the causes for such resistance.
Phe 34 of hDHFR is a strictly conserved active site residue and interacts
hydrophobically with, and is in van der Waals contact with, the pteridine ring of bound
substrate or bound MTXl. Thus exchanging this residue for others should lead to variants of
hDHFR having greatly changed properties. The Phe 34 ~ Ser variant of hDHFR has been
partially characterized previously by Schweitzer et al.2. and shown to possess very low
affinity for MTX. We have produced variants ofhDHFR in which Phe 34 is replaced by Ala,
Ser or Thr by oligonucleotide-directed mutagenesis. The effects of the residue changes at
position 34 of hDHFR on the catalytic properties and on the interaction with MTX have been
studied.

MATERIALS AND METHODS

Mutagenesis, Expression and Purification

An expression vector carrying eDNA for wild type (WT) hDHFR in pDS5/RBSII3
was generously provided by Dr. D. Stueber. Oligonucleotide-directed mutagenesis was
carried out with a MutaGene In Vitro Mutagenesis Kit (BioRad). Escherichia coli
Ml5(pREP4) was transformed by expression plasmids. Since the variants of hDHFR
produced have very low affinity for MTX, they could not be purified by affinity
chromatography on a MTX-Sepharose as routinely used for the purification of WT
hDHFR4. The methods used for purification of these variant hDHFR's included anion
exchange and gel-flltration columns.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling eta!., Plenum Press, New York, 1993 503
Steady State Kinetic Experiments

The enzymic reaction rate was determined at 20°C at pH 7.65, by measuring the
decrease in absorbance at 340 nm with a Beckman DU-70 spectrophotometer. The reaction
was initiated by addition of H2folate solution to a mixture containing enzyme and 100 j.tM
NADPH in MATS buffer. Initial reaction rates at various concentrations of H2folate were
fitted to the appropriate rate equation.

Determination of Thermodynamic Dissociation Constants, Kd

In most cases the quenching of protein fluorescence emission at 330 nm upon addition
of ligand was determined with an SLM AMINCO fluorimeter. The excitation wavelength was
either 280 nm or 295 nm. For the measurement of Kd for MTX dissociation from the
E.NADPH.MTX complex, the decrease in fluorescence at 440 nm was monitored. The
inner-filter effects were corrected by use of the data from parallel measurements of the
quenching of the fluorescence of tryptophan.

Transient State Kinetic Experiments

All these experiments were carried out with a Hi-Tech PQ/SF53 stopped-flow
spectrophotometer/fluorimeter.
To determine association (k0 n) and dissociation (koff) rate constants for MTX the
enzyme.NADPH binary complex was mixed with MTX, and the fluorescence above 310 nm
(which includes both the protein intrinsic fluorescence and energy transfer fluorescence from
NADPH) was recorded over a period of several seconds. These data were fitted to a single
exponential decay curve. The excitation wave length in this experiment was 280 nm. kon was
determined from the dependence of the observed rate constant kobs on the concentration of
H 2folate. The apparent dissociation rate constant (koff,app) was determined by the
competition method, in which a solution of the enzyme.NADPH.MTX complex is mixed
with edatrexate, used as trapping ligand, in the stopped-flow spectrophotometer and the
absorbance change due to MTX deprotonation followed as MTX dissociated.
The rate constant (kchem) describing the chemical transformation of
enzyme.NADPH.H2folate to enzyme.NADP.H4folate, and the thermodynamic dissociation
constant Kd for H2folate dissociation from enzyme.NADPH.H2folate were determined in
single turnover stopped-flow experiments, in which a concentration of NADPH
substoichiometric with respect to enzyme was used, and absorbance change due to
conversion of bound substrates to bound products was measured ..

RESULTS AND DISCUSSION

Preparation of Phe 34 variant enzymes

Satisfactory purification of the variants of hDHFR has been achieved with good yields.
The purity of the final preparation was generally above 90 % based on analysis by SDS-
PAGE on a 12 % gel followed by densitometer scanning.

Effects of Mutations on Catalysis

WT hDHFR turns over primarily through the pathway E.NADPH ~

E.NADPH.H2folate ~ E.NADP.H4folate ~ E.H4folate ~ E.NADPH.H4folate ~

E.NADPH. The dependence of the catalytic rate of the WT enzyme as a function of Hzfolate
concentration is described by a rectangular hyperbola as predicted by the Michaelis-Menten
equation. However, dependence of the catalytic rates for these variants do not obey the
simple Michaelis-Menten equation, but exhibit marked substrate inhibition, which is
explicable by a variation of the branched catalytic pathway used by WT enzyme.
kchem for WT hDHFR is much greater than kcat. and the overall catalytic rate for WT is
limited by dissociation of NADP and J4folate. Although the mutations either increase kcat. or

504
do not reduce it greatly, the values of kchem are reduced to the point that the chemical
transformation step becomes the rate-limiting step of the catalytic cycle.
kcatfKm values for all the variant enzymes are much lower than for WT indicating
greatly decreased efficiency.

Effects of mutation on H2folate and MTX binding

Kct for H2folate dissociation from the enzyme.NADPH.H2folate ternary complex is

greatly increased compared with WT. This large decrease in affinity for H2folate is not
surprising considering that the Phe 34 residue is in van der Waals contact with the pteridine
ring of the bound substrate. Substitution of smaller residues for Phe 34 would probably
eliminate this interaction.
Kct values for MTX dissociation from enzyme.NADPH.MTX are much greater for Phe
34 variants of hDHFR than for WT. Furthermore, the decrease in affinity for MTX is in all
cases substantially larger than the decrease in affinity for H2folate. This differential effect is
perhaps to be expected because the pteridine ring of bound MTX is in the inverse orientation
to that of the pteridine ring of bound H2folate.

CONCLUSIONS

Variants of hDHFR having good catalytic efficiency as well as low affinity for MTX
are of particular interest in this study. Although the mutations described here cause very large
increases in Ki for MTX, without large decreases in kcat. there are sufficiently large increases
in Km for H2folate that catalytic efficiency, as measured by kcatfKm. is decreased by a factor
of 340 or more. Catalytic activity by the variants would therefore be very weak at the low
concentration of H2folate present in cells. It is therefore unlikely that these mutations could
cause clinical resistance to MTX. Results of the ongoing crystallography for these variant
enzymes are awaited for more detailed interpretation of the effects of the structural changes.

ACKNOWLEDGEMENT

This work was supported in part by USPHS Grant CA 31922 from the National
Cancer Institute, by a Post Doctoral Fellowship from St. Jude Children's Research Hospital
(toT. N)., and by American Lebanese Syrian Associated Charities.

REFERENCES
1. J. F. Davies, II, T. J. Delcamp, N.J. Prendergast, V. A. Ashford, J. H. Freisheim, and J. Kraut, Crystal
structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate,
Biochemistry 29:9467, (1990).
2. B. I. Schweitzer, S. Srimatkandada, H. Gritsman, R. Sheridan, R. Venkataraghavan and J. R. Bertino,
Probing the role of two hydrophobic active site residue in the human dihydrofolate reductase by
site-directed mutagenesis, J. Biol. Chem. 264:20786, (1989).
3. D. Stueber, I. Ibrahimi, D. Cutler, B. Dobberstein and H. Bujard, A novel in vitro transcription-
translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences,
EMBO J. 3:3143, (1984).
4. N.J. Predergast, T. J. Delcamp, P. L. Smith and J. H. Freisheim, Expression and site-directed
mutagenesis of human dihydrofolate reductase. Biochemistry 27:3664, (1988).

505
KINETIC INVESTIGATION OF METHOTREXATE RESISTANT HUMAN
DIHYDROFOLATE REDUCTASE (hDHFR) MUTANTS AT PHE31

Srinivas K. Chunduru, James R. Appleman and

Raymond L. Blakley

Department of Molecular Pharmacology

St. Jude Children's Research Hospital
Memphis, TN 38101

INTRODUCTION

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of

dihydrofolate (Hlolate) to tetrahydrofolate (Hlolate). This enzyme is necessary for
maintaining adequate levels of H4folate and its derivatives, which are essential for the
biosynthesis of purines, thymidylate and several amino acids. This enzyme is therefore
a target for anticancer drugs such as methotrexate (MTX). Cancer patients receiving high
dose MTX as part of their therapy frequently develop clinical resistance and molecular
mechanisms of such resistance are of interest.
Schweitzer et al. 1 have shown that hDHFR in a human cell line selected for resistance
to MTX has the Phe at position 31 altered to Ser. In the crystal structure of hDHFR,2
Phe31 is present near the active site cleft and interacts hydrophobically with the pteridine
and benzene of ring of the bound substrate Hlolate. We have therefore used
oligonucleotide-directed mutagenesis to construct eDNA for a series of Phe~ 1 mutants
(F31V, F31T, F31S, F31A and F31G) and have investigated these substitutions by
measuring transient- and steady-state kinetics of catalysis, and kinetics of MTX and
substrate and product binding.

MATERIALS AND METHODS

Construction and Purification of Phe31 Variants

Single stranded DNA for the mutagenesis reactions was prepared by cloning a 705 bp
fragment containing the full length eDNA for hDHFR (derived from pDSS/RBSII,
Sphi[Sphl-Pstl]/hDHFR), kindly provided by Dr. Sttiber, Hoffman-LaRoche, Basel,
Switzerland), into Ml3mpl8. Phe11 mutants were generated using the Mutagene Ml3 kit

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 507
supplied by Bio-Rad. Colonies harboring Phe31 mutations were grown in LB media and
purified by MTX-Sepharose affinity chromatography.

Detennination of Ki for MTX

The inhibition constant K; for MTX was obtained from the dependence of steady state
enzyme activity on inhibitor concentration as described previously. 3 Enzymes were
preincubated with 100 JLM NADPH and various concentrations of MTX and the reaction
was initiated by the addition of Hlolate to a final concentration of 50 J!M. After an initial
Jag, the reaction velocity increased until a constant reaction rate, i.e., steady-state rate is
reached. The steady-state rates thus obtained were then fitted by nonlinear least squares
techniques to the equation for tight binding inhibitors as described previously. 3

Determination of Steady-State Parameters

The steady-state kinetic parameters kcat• K"' for Hlolate substrate were determined by
following the disappearance of NADPH at 340 nm and fitting the data to the Michaelis-
Menten equation using the Kaleidagraph program.

Detennination of Equilibrium Dissociation Constants

The equilibrium dissociation constants (Kd) for the interaction of H2folate and folate
with all five Phe31 variants were determined by measuring the quenching of protein
fluorescence upon addition of ligand. The sample was excited at 280 nm and the emission
monitored at 330 nm with an SLM 8000 fluorimeter. The fluorescence values at various
ligand concentrations were fitted to the standard equation as described by Dunn et al.4 in
order to determine Kd values.

Detennination of Association and Dissociation Rate Constants

The association (k,J and dissociation (k.,rr) rate constants were determined by
measuring the exponential decrease in protein fluorescence following mixing of enzyme
and ligand in a Hi-Tech PQ/SF53 stopped-flow fluorimeter. Enzyme was excited at 280
nm, or in some cases at 295 nm to reduce ligand inner filter effects and/or to reduce ligand
fluorescence. Data from 5 to 10 replicate binding reactions were averaged and the mean
set analyzed. The apparent rate constant, k.x,, was obtained from each mean data set by
fitting to the equation F, = Feq + ilFe·kobs', where F, and Feq are the fluorescence values
at time t and after attainment of equilibrium, respectively, and ilF is the difference
between the initial and the equilibrium fluorescence. ko1,. values obtained at different ligand
concentrations were then fitted by Kaleidagraph to the equation k.,b, = k"" [L] + korr run
on a Macintosh computer system.

RESULTS AND DISCUSSION

Inhibition of Variant DHFRs by MTX

Three out of 5 variants were found to have significantly decreased affinity for MTX,
up to 100-fold higher K; than wild type hDHFR (wt). The other two substitutions resulted
in little change in K; to MTX (0. 73 and 1.47 times that of wt, respectively).

508
MTX Binding by Stopped Flow

Detailed studies of the interaction of MTX with one of the variants were carried out
using stopped-flow spectrophotometry/fluorimetry. The association rate constant (k,J for
MTX binding to E.NADPH was found to be 0.2 that for wt, and the apparent dissociation
rate constant (k.,rr,arr> for MTX from this complex was increased 94-fold. The latter
increase could be due either to an increase in the true k.,rr, or to decreased isomerization
of the initial MTX complex. Since no evidence could be obtained for the isomerization
of the MTX complex of the variant hDHFR, whereas such an isomerization increases MTX
binding by a factor of 60 for wt hDHFR, the Joss of this isomerization appears to be the
major factor in decreasing affinity for MTX, and korr actually decreases by a factor of 3.

Steady State Behavior and Michaelis Constants

The steady state kinetics of the variant DHFRs were examined in order to make an
assessment of the effects of mutations on the catalytic behavior of the enzyme. The K.n
values for Hlolate for the five variant enzymes were increased above that of wt several
fold, and the lowest 1<.:.1 (i.e., V01,./[E]) was 0.3 that for wt. Some variants had a higher
1<.,,1 than wt. 1<.,,/K"" the enzyme's catalytic efficiency for all five variant enzymes was
found to be slightly lower than for wt.

Equilibrium Dissociation Constants from Fluorescence Titration

Kd for Hlolate in binary complexes were found to be considerably lower than for wt,
and the Kd for folate in binary complexes is slightly lower. Thus, although these mutations
greatly decreased affinity for MTX, they increased affinity for folate and Hlolate.

Effect of Mutation on k.n and korr for Substrate and Product Complexes

In order to understand the changes in steady-state kinetics induced by the mutations,

we have determined the effect of one of the mutations on the various association and
dissociation rate constants for various complexes of substrates and ligand with the enzyme.
k.,. values for formation of binary complexes of folate, Hlolate and Hlolate decreased few
fold. k.,. values for NADP binding to E.H 2folate remained virtually unchanged. k.,rr values
for folate and H 2 folate from binary complexes markedly decreased.

Effect of Mutation on the Rate of Chemical Transformation Step (kchem)

The conversion of enzyme-bound substrates to enzyme bound products is most

conveniently measured by a single turnover experiment in a stopped-flow
spectrophotometer. Data for the decrease in absorbance with time fitted to a rate equation
with a single exponential term. k.:hcm measured this way is markedly decreased from the
value of 1360 s·• for wt hDHFR.

CONCLUSIONS

The steady-state velocity under intracellular conditions at 0.1 ~-tM H2folate and
saturating NADPH for these variants is 1-2 s·• (6.9 s·• for wt). Thus, the catalytic
efficiency and affinity for MTX would probably permit cell survival in the presence of
high concentrations of intracellular MTX. The fact that only the F31S variant has been

509
found by selection with MTX in tissue culture, is probably because mutation to other
variants requires 2 or 3 base changes.

Acknowledgements

This work was supported in part by USPHS research grant CA 31922 from the
National Cancer Institute, by a John H. Sununu postdoctoral fellowship (S.K.C.), and by
the American Lebanese Syrian Associated Charities.

REFERENCES

l. B.I. Schweitzer, S. Srimatkandada, H. Gritsman, R. Sheridan, R. Venkataraghavan

and J.R. Bertino, Probing the role of two hydrophobic active site residues
in the human dihydrofolate reductase by site-directed mutagenesis, J. Bioi.
Chem. 264:20786 (1989).
2. J.F. Davies, II, T.J. Delcamp, N.J. Prendergast, V.A. Ashford, J.H. Freishim and
J. Kraut, Crystal structures of recombinant human dihydrofolate reductase
complexed with folate and 5-deazafo\ate, Biochemistry 27:3664 (1988).
3. J.R. Appleman, N. Pendergast, T.J. Delcamp, J.H. Freisheim and R.L. Blakley,
Kinetics of the formation and isomerization of methotrexate complexes of
recombinant human dihydrofolate reductase, J. Bioi. Chem. 263:10304
(1988).
4. S.M.J. Dunn, J.G. Batchelor and R.W. King, Kinetics of ligand binding to
dihydrofolate reductase. Binary complex formation with NADPH and
coenzyme analogues, Biochemistry 17:2356 (2978).

510
THE EFFECT OF CODON 31 ON THE RELATIVE AFFINITIES FOR THE
BINDING OF DESIGNED 8-ALKYL-PTERINS TO DIHYDROFOLATE
REDUCTASE: A STATISTICAL PERTURBATION THEORY AND
MOLECULAR DYNAMICS SIMULATION STUDY

Peter L. Cummins and Jill E. Gready

Department of Biochemistry
University of Sydney
N.S.W. 2006 Australia

INTRODUCTION

Although a high degree of primary sequence and structural homology exists between
rhDHFR (recombinant human dihydrofolate reductase) and clDHFR (chicken liver DHFR),
particularly in the active-site region,l,2 kinetic studies of the designed 8-alkyl-pterin
substrates appear to have revealed small but consistent differences in the Km's for these two
enzymes,3 with those for clDHFR being the lower. In contrast, the dissociation constants
(~<d's) for the binding of a number of 8-alkyl-NS-deazapterin inhibitors indicate a preference
for rhDHFR.4 The elucidation of the origin of these small differences in terms of specific
structural differences between the enzymes and resulting differences in molecular interactions
represents an interesting and challenging problem which lends itself to computational study
using modem-day molecular simulation techniques.S The structurally-aligned primary
sequences of clDHFR and rhDHFR are compared below for sections that contain most of the
important active-site residues (within ca. 8A of the pterin center):

8 17 23 25 31 34 39 57 68 116 122 137

clDHFR VAV-G-P-P-EYK-FQR-S-W -K-GG-K-R
rhDHFR VAV-G-P-P-EFR-FQR-T-W -K-GG-K-R

As may be seen, the sequences differ in codons 31, 32 and 39 (Y = tyrosine, F =

phenylalanine, K = lysine, R = arginine, S = serine, T = threonine). Codon 31 is of
particular interest as the side chain closes over the active site in both clDHFR and rhDHFR,
thereby helping to eliminate solvent from the reaction. We have used computer simulation

Chemistry and Biology of Pteridines and Palates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 511
methods to calculate the free energy change for the transformation Y ~ F in order to gain
some understanding of the species-dependent differences in enzyme activity.

METHODS

The protein and cofactor coordinates were taken from the clDHFR X-ray crystal
structure in which both cofactor (NADP+) and biopterin are bound to the DHFR molecule}
The AMBER programs6 were used to carry out the molecular dynamics simulation and free
energy perturbation calculations. The force-fields of Weiner et alJ,8 were used for the
DHFR molecules. The NADPH and ligand force fields have been described elesewhere.9-ll
The chemical mutations are achieved by coupling the force fields for residue 31, and the free
energy changes are calculated using a perturbation method: full details of this computational
method can be found elesewhere.12 The following three chemical mutations were carried out
with AMBER:

I DHFR(Glu-:Tyr).NADPH ~ DHFR(Glu-:Phe).NADPH
II DHFR(Glu-:Tyr).NADPH.8p+ ~ DHFR(Glu-:Phe).NADPH.8p+
III DHFR(Glu-:Tyr).NADPH.68p+ ~ DHFR(Glu-:Phe).NADPH.68p+

I gives the free energy change for mutation of Codon 31 for an ionized Glu (Codon 30)
active site without bound substrate, while II and III give the corresponding changes for the
bound protonated substrates 8-methyl-pterin (8p+) and 6,8-dimethyl-pterin (68p+). Each
simulation for the free energy calculation was carried out over a period of 120 ps of
molecular dynamics. The relative free energies can be obtained from the thermodynamic
cycle shown in Figure 1.

Simulation: "Chemical mutation"

E' (aq)
~Gr
Ci3
E
Q)
E
-~ K
a.
X
w
~Grr & ~Grn
E:L(aq) E':L(aq)
Figure 1 Thermodynamic cycle used to determine free energy change due to the mutation at Codon 31 in
clDHFR. E = clDHFR.NADPH, L = Sp+ or 68p+.

~G 1 , ~Gn and ~Gm are the calculated free energy changes for the mutations I, II and III
respectively. The net free energy change 66G, for example when 8p+ binds to DHFR, is
related to the ratio of binding constants by:

MG =~Gn - ~GI =-RTln(K'/K)

512
where R = gas constant and T = absolute temperature. Thus a negative ~~G indicates that
the mutation favours ligand binding.

RESULTS

The results of the free energy calculations for mutations I-III are given Table 1. The
free energies have been decomposed into electrostatic (~Geie) and van der Waals (~Gvdw)
contributions from the molecular dynamics force-field, i.e. the total ~Gtotal = ~Gele +
~Gvdw· The net total free energy changes MGtotal on the binding of each ligand due to the
mutation are also given.

Table 1. Computed free energy differences for the mutation Y ~ F at Codon 31 (clDHFR).

Simulation ~Ge!e ~Gvdw ~Gtotal ~~Gtotal

-2.00 ± 0.00 0.89 ± 0.02 -1.11 ± 0.02

II -1.57 ± 0.05 0.68 ± 0.00 -0.89 ± 0.05 0.22 ± 0.07
ill -2.13 + 0.02 0.82 + 0.00 -1.31 + 0.02 -0.20 + 0.04

First we note that the magnitude of the electrostatic contribution is appreciably larger
than the vdW term, which is not surprising as the structural, i.e. steric, changes would be
relatively small for OR mutated to H, whereas the dipole of OR group would be expected to
introduce a more significant electrostatic effect. It is interesting to compare these results with
the mutation of phenol to benzene molecule in aqueous solution. Rao and Singh13 obtain
2.52 ± 0.01 and 1.00 ± 0.00 kcal/mol respectively for ~Gele and ~Gvdw· While the vdW
term is close to the values given in Table 1, the electrostatic component is of the opposite
sign. This indicates that the local electrostatic environments in protein and solvent are quite
dissimilar. Although the individual free energy changes are substantial, the net free energy
change (MGtotai) is small, and hence we would predict small differences in the binding for
the mutant enzyme. The results suggest that binding of 8p+ is stronger for native clDHFR
consistent with experimental Km's, although stronger binding to the mutant is predicted for
68p+. However, it should be stressed that the differences are quite small and we have not
made an assessment of the errors in the simulation. Also we have considered only one
difference by chemical mutation, but other sequence differences may also contribute to the
free energy. In particular long-range electrostatic interactions may play a role since both the
active substrates and inhibitors are expected to be protonated when bound to DHFR:3 this has
now been shown to be the case for the inhibitors.14 The free energy due to interaction of two
charges q and q' separated by distance r in the protein may be calculated using the formula 15

Gion = qq'exp(Kr)/(E(r)r)

where exp(Kr) is an electrolyte screening factor with Debye length K-1 and e(r) is an
empirically-derived distance-dependent dielectric permittivity. The positions of the charges

513
are fixed by the X-ray coordinates for clDHFR 1 and rhDHFR,2 assuming these are close to
the average solution structures. Using this approach for calculating electrostatic free energies
at infinite dilution (exp(Kr) = 1), we estimate the interactions between the charged residues of
DHFR and a cation in the active site would lead to differences in the range 0.5-1.0 kcal/mol
with the rhDHFR complex the more stable.

CONCLUSION

We have used computer simulation methods (molecular dynamics combined with free
energy perturbation techniques) to calculate the free energy change for the transformation Y
--7 F (at codon 31) in an effort to gain some general understanding of the species-dependent

differences in enzyme activity with our new substrates and inhibitors. The magnitude of the
free energy change for Y --7 F is computed to be small, i.e. < 1 kcal/mol, consistent with the
experimental Km's for binding of 8-R-pterin substrates. Other amino acid replacements (e.g.
K --7 R at 32 or S --7 T at 39) may well produce similarly small free energy changes.
Differences in long-range electrostatic interactions with charged residues may also be a
factor. We have made an estimate of the total ion-pair contribution to the free energy
difference using a macroscopic model for the electrostatics, and found a greater tendency for
binding to rhDHFR. The observed differences in binding are thus likely to result from the
cumulative effects of many small structural/sequence variations. However, the simulation
methods were unable to resolve such small free energy changes with sufficient accuracy to
confidently predict that clDHFR rather than rhDHFR provides the stronger binding as
measured by Km's, simply on the basis of a single mutation at Codon 31.

ACKNOWLEDGEMENT

This work was supported by the National Health and Medical Research Council.

REFERENCES
1. M.A. McTigue, J.F. Davies, B.T. Kaufman and J. Kraut, Biochem. 31:7264 (1992).
2. C. Oefner, A. D'Arcy and F.K. Winkler, Eur. 1. Biochem. 174:377 (1988).
3. J.E. Gready, in: "Chemistry and Biology of Pteridines 1989," H.-Ch. Curti us, S. Ghisla and N. Blau,
eds., de Gruyter, Berlin (1990), pp. 23-30.
4. M.T.G. Ivery and J.E. Gready, this volume and to be published.
5. B.L. Tern be and J.A. McCammon, Comput. Chern. 8:281 (1984).
6. U.C. Singh, P.K. Weiner, J.W. Caldwell and P.A. Kollman, AMBER (Version 3.2), Dept.
Pharmaceutical Chern., Univ. of California, San Francisco, 1988.
7. S.J. Weiner, P.A. Kollman, D.A. Case, U.C. Singh, C. Ghio, G. Alagona, S. Profeta and P. Weiner,
1. Am. Chern. Soc. 106:765 (1984).
8. S.J. Weiner, P.A. Kollman, D.T. Nguyen and D.A. Case, 1. Comput. Chern. 7:230 (1986).
9. P.L. Cummins, K. Ramnarayan, U.C. Singh and J.E. Gready, 1. Am. Chern. Soc. 113:8247 (1991).
10. P.L. Cummins and J.E. Gready, Proteins: Struct. Funct. Genet., in press.
11. P.L. Cummins and J.E. Gready, 1. Comput.-Aided Mol. Des., in press.
12. U.C. Singh, F.K. Brown, P.A. Bash and P.A. Kollman, 1. Am. Chern. Soc. 109:1607 (1987).
13. B.G. Rao and U.C. Singh, 1. Am. Chern. Soc. 111:3125 (1989).
14. S.-S. Jeong and J.E. Gready, this volume.
15. E.L. Mehler and T. Solmajer, Protein Eng. 4:903 (1991).

514
EFFECT OF CODON 22 MUTATIONS ON SUBSTRATE AND INHIBITOR
BINDING FOR HUMAN DffiYDROFOLATE REDUCTASE

E. Ercikan, M. Waltham, A. Dicker, B. Schweitzer and J. R. Bertino.

Program of Molecular Pharmacology

Memorial Sloan-Kettering Cancer Center
New York, NY 10021

INTRODUCTION

Previous studies in this laboratory have identified an altered dihydrofolate reductase

(DHFR) enzyme in the methotrexate (MTX) resistant Chinese hamster ovary (CHO) cell
line Pro-3MtxRIII [1,2]. The alteration involved the replacement of the active site
residue Leu (L, position 22) by Phe as the result of a base transition at nucleotide 67 in
the DHFR gene [3]. Enzyme kinetic studies of the altered enzyme revealed that the Km
for both substrates (H2folate & NADPH) were similar when compared with wild-type
vertebrate enzyme, but the Ki for MTX was increased some 100-fold. Interestingly,
previous studies by other researchers have also identified a mutation at residue 22 in
MTX-resistant cell lines: a Leu to Arg mutation in the mouse line 3T6-R400 [4], and a
Leu to Phe mutation in yet another CHO cell line [5]. Combined, these results suggest
that codon 22 may be a "hot spot" which readily mutates to impart a MTX-resistant
phenotype to its host cell. While definitive proof still awaits, it seems plausible that
these or similar mutations in DHFR may be responsible for some instances of resistance
in tumors of patients who receive antifolates therapeutically.
Here we report the characterization of several residue 22 mutations of the human
DHFR. Earlier work by Thillet & coworkers [6] examined the kinetic properties of a
Leu to Arg mutation at this position for recombinant mouse enzyme. In this paper we
report Ile (I), Met (M), Phe (F) and Tyr (Y) substitutions (Trp and Arg mutagenesis in
progress) to determine the extent of change that can be tolerated before altering kinetic
and antifolate inhibitory properties of the human enzyme. This study also permits us to
test various predictions derived from molecular modeling regarding substrate and
antifolate affinity of the different mutant species, and forms part of a general goal of this
laboratory to identify mutant DHFRs which may serve as superior dominant selectable
markers in gene transfer technologies.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 515
EXPEREMENTALPROCEDURES

Mutagenesis was performed using a PCR technique [7] involving two separate sets
of primers; one set flanking the entire eDNA of DHFR and the second set being
complementary to one another and containing the desired mutation. The final PCR
product (incorporating a mutated full length eDNA) was digested and cloned into a
pBR322 vector then infected into JM109 E. coli.
Standard techniques [8] were used to purify recombinant mutant DHFRs from E.
coli lysates, however column dimensions, bed volume and washing & elution conditions
for the MTX-affinity absorption step were altered to maximize retardation of the mutant
enzyme during chromatography. Subsequent steps of dialysis and ion-exchange
chromatography resulted in homogeneous preparations by the criteria of SDS-PAGE.
Catalytic properties were determined using the spectrophotometric assay at 25°C in
MATS assay buffer (pH 7.4, [9]) unless otherwise stated.

RESULTS AND DISCUSSION

To better rationalize the mechanism for decreased MTX binding for the Phe 22
mutant, molecular modeling of enzyme, substrate and inhibitor were undertaken. It
appears that the Phe side chain protrudes towards the bridge area (C6-C9-N 10) of the
inhibitor, thus steric hindrance due to an overlap of the van der Waals forces of the
phenyl and MTX (especially the methyl group of N 10) would weaken the binding of
ligand. As substrate (H2folate) binds the active site in a flipped orientation (180° rotated
about the C6-C9 bond axis relative to the pteridine ring system of inhibitor, [10]), then
binding of this ligand should be less perturbed.
The substrate Michaelis constants for mutant enzymes (Table 1a) are all elevated
though surprisingly less so for those involving bulky side chain replacements of Phe and
Tyr. In general, the decreased affinity for H2folate in this series is less dramatic than
that evaluated for other codon 22 DHFR mutants identified from cell lines [1-5,11]. By
contrast, the catalytic turnover for the Phe and Tyr mutants are more affected than Ile
and Met which are near identical to wild-type. The overall catalytic efficiency (as
judged by kcat/Km) is reduced by 113 for L22M and L22F, and more so for L22I and
L22Y.
The pH rate profile for wild-type enzyme is relatively flat except below pH 5.0
(Fig. 2). All codon 22 mutants show some pH dependence across the entire pH range
tested. The Tyr mutant shows marked dependence with a catalytic rate 5.5-fold higher at
pH 5.5 as compared to neutral pH. Further experiments examining the deuterium
isotope effect (with NADPD) are planned to determine if hydride transfer is the sole rate
limiting process for this mutant. It is interesting to note that a similar pH rate profile
(and thus perhaps a similar change in the rate limiting step) for wild-type enzyme can be
induced by 4 M urea, presumably by elevating the rate constant for dissociation of
product from the slightly unfolded protein. L22Y may have an altered conformational
structure (more loose or open).
While substrate affinity for mutants is several fold reduced, the binding affinity for
MTX and APT is more dramatically affected particularly for the Phe and Tyr mutants;
MTX binding is reduced 88 and 1600-fold respectively (Table 1b). The more conserved
side chain replacements (Ile and Met) affect binding of antifolate only marginally. Thus,
Tyr and Phe mutations at this position for human DHFR may be useful as dominant

516
Table 1. Kinetic and inhibition parameters of wild-type and codon 22 mutants of
humanDHFR

wild-type L221 L22M L22F L22Y

(a) Kinetic parameter~

Km (H2folate) (tJ.M) 0.08 0.55 0.23 0.16 0.15

kcat (sec-1) 12.7 13.2 13.4 7.4 1.5

kcat1Km (sec-1J.lM-1) 159 24 58 46 10

(b) Inhibition constantsb

Methotrexate (pM) 1.2c 1.8 2.8 106 1,980

Aminopterin (pM) 1.8 7.6 9.2 212 ND

Piritrexim (pM) 13.2c ND ND 16 1,190

a Michaelis constants were obtained by fitting the hyperbolic form of the Michaelis-Menten equation (by
iterative analysis) to progress curve data. 10-cm path-length cuvettes were used for these experiments to
assist in monitoring reactions at low H2folate concentrations. Values of kcat were obtained by active site
titration experiments using MTX and/or from activity and total protein measurements of purified enzyme
species.
b Ki values for methotrexate (MTX), aminopterin (APT) and piritrexim (PTX) were evaluated by fitting
the equation for tight-binding competitive inhibition to inhibited steady-state data. ND = not determined.
c These values were estimated previously in this laboratory under similar experimental conditions.

350

2
·c:
300

"'
Ill 250
.::
iii
! 200
~
·;;
150
~
"'
.2 100
>-
iU
iii 50
0

0
5.50 6.50 7.50 8.50 9.50

pH

Figure 1. pH rate profiles for wild-type and codon 22 mutants of human DHFR. Assays were performed
in MTEN buffer [9} at 25°C and rates normalized for activity at pH 7.4 (i.e. rate at pH 7.4 set to 100).
Wild-type, +; L221, £; L22M, +; L22F, •; L22Y, e.

517
selectable markers. The human Phe 22 mutant may be superior to the rodent versions
(already in use by a number of laboratories) as the Km for substrate is less affected, yet it
displays a similar reduced affinity for MTX. One remarkable feature is that L22F is
more potently inhibited by piritrexim (by comparison with MTX & APT), presumably
due to the shortened bridge region between the pteridine and the benzoyl group. Thus,
piritrexim is predicted to be somewhat more cytotoxic towards established cell lines
which harbor this form of mutant DHFR [1,2,5].
Inspection of the inhibition progress curves also reveal that the mechanism of
inhibition or the pathway to formation of a final tight antifolate complex, as usually
observed for wild-type DHFR, is impaired for Phe and Tyr mutants. The Tyr mutant
displays an initial inhibited reaction rate without slow onset of inhibition or
"isomerization" (for MTX, APT & PTX, data not shown). Likewise, the Phe mutant
does not isomerize but only when in combination with APT. These results imply that
either an antifolate induced isomerization process does not occur for these particular
combinations, or that a naturally occuring isomerized form of the protein (also occuring
for the mutant enzyme) is not stabilized by the antifolate. It appears that the
isomerization must not be crucial for catalysis as both mutants display reasonable
catalytic activity.

Methotrexate Compound 1 Compound2 Compound 3

Figure 2. Putative inhibitors of Phe and Tyr mutants at position 22. Steric hinderance should be removed
if the bridge region between pteridine and the p-aminobenzoyl moiety is removed from C6 to C5. An
oxygen, nitrogen or double bond in the bridging region is introduced for rigidity, preventing rotation
about the C6-C9 axis.

As the rank order of inhibitor binding differs between wild-type and codon 22
mutants, particular antifolates may be more cytotoxic to cell lines that contain different
residue 22 mutants, and specific inhibitor compounds could be developed for these
altered DHFR forms. On this latter point, three plausible inhibitor compounds have
been designed for the Phe and Tyr mutants (Fig. 2). We plan to synthesize and trial
these compounds in the near future.

518
REFERENCES

1. Flintoff, W.F. and Essani, K. (1980) Biochemistry 19:4321-4327.

2. Flintoff, W.F., Davidson, S.V. and Siminovitch,L. (1976) Somatic Cell Genet.
2:245-261.

3. Dicker, A.P., Volkenandt, M., Schweitzer, B.I., Banerjee, D. and Bertino, J.R.
(1990) J. Biol. Chern. 265:8317-8321.

4. Simonsen, C.C., and Levinson, A.D. (1983) Proc. Natl. Acad. Sci. U.S.A.
80:2495-2499.

5. Melera, P.W., Davide, J.P., Hession, C.A. and Scotto, K.W. (1984) Mol. Cell.
Biol. 4:38-48.

6. Thillet, J., Absil, J., Stone, S.R. and Pictet, R. (1988) J. Biol. Chern. 263:12500-
12508.

7. Higuchi, R., Krummel, B. and Saiki, R.K. (1988) Nuc. Acid Res. 16:7351-7367.

8. Schweitzer, B., Srimatkandada, S., Gritsman, H., Sheridan, R., Venkataraghavan,

R. and Bertino, J.R. (1989) J. Biol. Chern. 264:20786-20795.

9. Ellis, K.J. and Morrison, J.P. (1982) Methods Enzymol. 87:405-426.

10. Bolin, J.T., Filman, D.J., Matthews, D.A., Hamlin, R.C. and Krraut, J. (1982) J.
Biol. Chern. 257:1360-13662.

11. Schweitzer, B.I., Gritsman, H., Dicker, A., Volkenandt, M. and Bertino, J.R. in
Chemistry and Biology of Pteridines: Pteridines & Folic acid derivatives (eds Curtis, H-
Ch., Ghilsa, S. & Blau, N.) 760-764 (Walter de Gruyter, Berlin, New York., 1990).

519
THERMODYNAMIC STUDY OF FOLATE ANALOGUE BINDING TO

DIHYDROFOLATE REDUCTASES FROM DIFFERENT SPECIES

Sophie Sasso, Robert Gilli, Colette Lopez,

Jean Claude Sari, and Claudette Briand

Groupe de Recherche sur les Interactions des

Proteines en Pharmacologie (G.R.I.P.P.)
Faculte de Pharmacie, 27 Bd Jean Moulin,
13005 Marseille, France

INTRODUCTION

Dihydrofolate reductase (DHFR) is the target of

numerous folate analogues including the antibacterial agent
Trimethoprim (TMP), the anticancer agent Methotrexate (MTX),
and a second generation antifolate compound, Trimetrexate
(TMQ).
TMP is known to present a remarkable species
selectivity, binding about 10 5 more tightly to bacterial
DHFRs than to vertebrate enzymes, whereas MTX has the same
affinity for both types of enzymes. A component of this
selectivity depends upon the cooperative binding to DHFR of
TMP and cofactor, NADPHl.
Our purpose was to evidence, at physiological
temperature, the thermodynamic origin of the role of NADPH
in this selectivity by using a microcalorimetric method.
Binary and ternary complexes have been directly studied in
order to develop a new thermodynamic approach of
selectivity, concurrently with enzymatic, cristallographic
and NMR studies. Few quantitative studies concerning the
interactions in binary system between folates and bacterial
DHFRs have been presented, however, and the experiments were
never done at 37"c. The velocity of our flow isothermal
microcalorimetric experiments made it possible to conduct
the study at 37"c, in spite of the poor thermal stability of
the protein.

RESULTS and DISCUSSION

I) study of folate analogue binding in binary system

We measured, at physiological temperature and pH 6. 8,

the thermodynamic parameters (enthalpy variation ~H,

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 521
association constant Ka, and entropy variation L1S) of the
interactions of TMP, MTX and TMQ with bovine liver enzyme
(DHFRb) and with Escherichia coli DHFR (DHFRc). Both enzymes
were purified by affinity chromatography. By using
microcalorimetry 2 , one can determine whether the interaction
is driven by a favorable exothermic enthalpy variation
linked to electrostatic contacts orjand by a favorable
positive entropy variation linked to hydrophobic
interactions.

Table 1. Thermodynamic parameters in the binary system

at 37°C and pH 6.80

DHFRb DHFRc

L1H Ka L1S L1H Ka L1S

MTX -105 1.6 10 7 -200 -67 3.7 1010 -13
TMQ -97 2.2 10 8 -152 -52 8.8 10 9 +14
TMP -58 1.6 10 5 -88 -44 1.7 10 8 +14
L1H in kJ.mol-1, Ka in lf'1 and L1S in J.K-1.mol-1

As is foreseeable by the N1 protonation of the three

compounds, which induces salt bridge formation, the
formation of all complexes is driven by favorable negative
L1H. Also, L1S are strongly unfavorable in the case of
mammalian enzyme, and close to zero or even positive for the
bacterial enzyme. Thus, the less favorable L1H of DHFRc is
compensated by a more positive L1S and, finally, the three
compounds exhibit the highest affinity for DHFRc.
The heat signal measured by microcalorimetry includes
not only the binding part, but also the protein
restructuration part induced by the ligand2. The discrepancy
in ~s between the two enzymes, observed for each ligand, is
probably due to the protein refolding that occurs during
complex formation; this refolding effect depends on the
thermal stability of the enzyme. DHFRc is a more stable
protein than DHFRb and undergoes a smaller conformational
change during the binding process. Indeed one can suppose
that DHFRc is able to establish both electrostatic and non
polar contacts with the ligands, without restructuration of
the active-site cavity. In contrast, in mammalian DHFR the
inhibitor cannot establish these contacts without a large
folding up of the protein.
Relationships between the ligand structure and the
binding part of the thermodynamic parameters :
1) For DHFRc :
The surprisingly high affinity of MTX for DHFRc in
absence of NADPH results from the favorable L1H (-67 kJ.mol-
1). This affinity is due to its hydrophilic character.
Indeed, MTX presents additive electrostatic interactions 3 by
comparison with TMP, for example through its N8.
Furthermore, hydrophobic interactions appear, for instance
between Leu 54 and the p-aminobenzamide moiety of MTX 4
Inversely, the lipophilic character of TMP, induced in
particular by the trimethoxybenzene group, leads to more
hydrophobic interactions than does MTX (L1S=+14 J.K- 1 .mol- 1 ).

522
Nevertheless, these additional hydrophobic contacts do not
compensate the less numerous hydrophilic interactions (~H=-
44 kJ.mol- 1 ), and finally, TMP affinity is 220 times lower
than that of MTX.
TMQ affinity is intermediate between those of MTX and
TMP; like TMP, it presents lipopholic interactions ( ~S=+l4
J.K- 1 .mol- 1 ) but also exhibits additional electrostatic
contacts (~H=-52 kJ.mol- 1 ) which make its thermodynamic
behavior close to that of MTX. This could be explained by
the same location of MTX and TMQ in the protein binding
site5.
2) For DHFRb :
The poor affinity of TMP for bovine liver DHFR might
entail, for example, that the trimethoxybenzyl ring is less
buried, inducing a decrease in hydrophobic interactions 6
(~S=-88 J.K- 1 .mol- 1 ), or might be due to the lack of
hydrogen bond between the 4-amino group and the carbonyl
oxygen of Val 115 which participates in MTX binding3.
TMQ shows a more favorable ~s than MTX due to its
trimethoxybenzene moiety, and a more favorable ~H than TMP.
Hence it exhibits a higher affinity than MTX and TMP.
In conclusion for binary complexes, the folate
analogues present an entropy driven selectivity in favor of
the bacterial enzyme. The selectivity is correlated with a
weak conformational change for this enzyme.

Table 2. Thermodynamic parameters in the ternary system

at 37"c and pH 6.80

DHFRb DHFRc

~H Ka ~s ~H Ka ~s

MTX -72 3 1o10 -35 -52 4 1010 +30

TMQ -67 3 1010 -16 -42 3 1010 +60
TMP -18 3 10 6 +65 -48 1.6 10 11 +53
~H in kJ .mor1, Ka in M"' 1 and ~s in J .rl.morl

II) Ternary complex study - Influence of NADPH.

Table 2 shows that NADPH does not induce a cooperative
effect on the MTX binding to DHFRc, and only a little
cooperative effect on the TMP binding to DHFRb. When NADPH
binds to DHFR, its location and its conformation are similar
whatever the enzyme originl, but a mobile region of DHFR
folds in presence of cofactor. This refolding is more
pronounced in vertebrate DHFRs which present a larger
binding pocket6. The favorable variation of ~s (o~S) between
binary and ternary systems, evidenced for all compounds and
all DHFRs, could be explained by:
1) a smaller conformational change induced by the
folate since the enzyme has already been refolded by NADPH.
2) additional hydrophobic contacts provided by cofactor
between protein and ligands. However it should be noted that
for example, the hydrophobic interaction between Met 20 and
the trimethoxybenzyl group of TMP in DHFRcl does not play a
rna jar role in the favorable MS. Indeed, the lack of this

523
interaction in DHFRb, because of the markedly different
orientation of the trimethoxybenzene ring, does not lead to
a decrease in o~S which actually is higher (153 J.K-1.mol-1
for DHFRb instead of 40 J.K-1.mol- 1 for DHFRc). Thus one can
hypothesize that the difference of o~S between the two
enzymes is due more to the conformational change of the
proteins already induced by NADPH, than to the binding of a
folate analogue. Indeed for DHFRb the o~S are highly similar
for the three compounds whereas their affinities are
drastically different. On the other hand, the most important
factor for the presence or absence of selectivity for MTX
and TMQ is the modulation of the favorable entropic effect
by an unfavorable enthalpic effect. This unfavorable effect
appears essentially for MTX and the bacterial enzyme (o~H=15
kJ.mol- 1 ) and, even more so, for TMP and the mammalian
enzyme (o~H=40 kJ.mol- 1 ). For TMQ, o~S is intermediate
between TMP and MTX values as could be predicted by its
structure, but the cooperative effect of NADPH in DHFRb is
allowed by a less unfavorable MH. Thus, as for MTX, no
selectivity is observed in ternary complexes for TMQ.
In conclusion this study evidences that the
cooperativity between NADPH and antifolate drugs has an
enthalpic origin. Moreover it reveals that the most
important role of NADPH in the species selectivity of
antifolate compounds is not to induce antibacterial agent
selectivity for bacterial DHFR, but only to increase it.
Surprisingly, NADPH cancels the selectivity of anticancer
agents for bacterial enzyme in binary complexes by
drastically increasing drug affinity for vertebrate enzyme.

FOOTNOTES

The authors thank Dr E.Howell (Agouron Institute, La Jolla,

California) for the gift of E.coli strain.
* This work was supported by grants from ARC, FNCLCC and
FEGEFLUC.

REFERENCES

1. J.N. Champness, D.K. Stammers and C.R. Bedell, FEBS Lett. 199:61
(1986).

2. R.M. Gilli, J.C. Sari, C.L. Lopez, O.S. Rimet and C.M. Briand,
Biochem. Biophys. Acta 1040:245 (1990).

3. J.H. Freisheim and D.A. Matthews, in Folate Antagonists as

Therapeutic Agents (F.M. Sirotnak, J.J. Burchall, W.D. Ensminger and
J.A. Montgomery, eds.), 1, p. 69, Academic, New York, (1984).

4. L. Li and S.J. Benkovic, Biochemistry, 30:1470 (1991).

5. A. Hempel, N. Camerman and A. Camerman, Cancer Biochem. Biophys.

10:25 (1988).

6. D.K. Stammers, J.N. Champness, C.R. Beddell, J.G. Dann, E.

Eliopoulos, A.J. Geddes, D. Ogg and A.C.T. North, FEBS Lett. 218:178

524
COMPARISON OF BINDING AND ACTIVITY OF 8-ALKYL-PTERINS
AND 8-ALKYL-NS-DEAZA-PTERINS WITH DIHYDROFOLATE
REDUCTASE

Michael T.G. Ivery and Jill E. Gready

Department of Biochemistry, University of Sydney

NSW 2006 Australia

INTRODUCTION

The 8-alkyl-pterins 1 and 8-alkyl-NS-deaza-pterins 2 constitute two new classes of

mechanism-based substrates and inhibitors, respectively, for the enzyme dihydrofolate
reductase.

R, R2 R3 R4

0 1a -CH 3 -H -H
1b -CH2CH 3 -H -H
N) : N
.... I Ra
1c -CH 2CH2CH3 -H -H
H NJ!.N NJ(R2 1d -CH(CH)a -H -H
2 I 1e -CH2-CH=CH2 -H -H
R1 1f -CH3 -H -CHa

1 2a -CH 3 -H -H -H
2b -CH 2CH3 -H -H -H
2c -CH 2CH2CHa -H -H -H
0 R4 2d -CH(CH)a -H -H -H
NnR3 2e -CH 3 -CH 3
I ....: I 2f -CH 2CH3
-H
-H -CH 3
-H
-H
H NJ!.N N R2 -CH3
2 I 2g -CH2CH2CH3 -H -H
R, 2h -CH(CH)a -H -CH 3 -H

21 -CH3 -CH 3 -H -H
2 2j -CH 2CH2CHa -CH3 -H -H
2k -CH 3 -H -H -CH 3
2m -CH2CH2CH3 -H -H -CHa

These compounds have been designed as analogues of folate, 1•2 but with increased
basicities compared with the parent. This increased basicity allows the compounds to readily
form an N3-protonated cation which mimics the assumed enzymically-active form of the
natural substrate. Previous studies 1•2 on the 8-alkyl-pterins have shown them to be good

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 525
substrates of DHFR with Km values in the micromolar range, while initial studies on the 8-
alkyl-N5-deaza-pterins indicate they are good inhibitors with thermodynamic dissociation
constants in the low micromolar range. 3 We report further studies of the binding of these
two classes of compound with both recombinant human and chicken liver DHFRs.

MATERIALS AND METHODS

Instruments and Reagents Perkin-Elmer LS-50 and Shimadzu spectrometers were used
for fluorimetric binding and UV/vis spectral kinetic studies respectively. 8-alkyl-pterins and
deaza-pterins were reported. 3•4 Ellis and Morrison5 MESffRIZMA/NaCl/ethanolamine pH
buffers with 1=0.15 M were used. Recombinant human DHFR was a gift from Prof. J.H.
Freisheim, chicken liver DHFR was from Sigma, and NADPH and NADP+ were from
Boehringer. Enzyme concentration was determined by methotrexate titration. 6

Enzymology Thermodynamic dissociation constants (Kct) were determined by following

the quenching ofthe enzyme fluorescence (excitation 280 nm, emission 320 nm) on addition
of ligand. Data were analysed by the procedures of Birdsall et al. 7 using the non-linear
regression program GraFit. For determinations in the presence of cofactor, molar ratios of
10:1 for NADPH:DHFR and 30:1 for NADP+:DHFR were used, with enzyme
concentrations in the range 0.12-0.5 J.LM. Km's were determined by initial rate
spectrophotometric assay at 30° C following the decrease in absorbance of the assay mixture
at either 340 nm 1•2 (NADPH decrease) or 400 nm (pterin decrease), with saturating NADPH
(60 J.LM) and data fitting with Enzfitter. The Ki of 2e was determined by initial rate assay at
varying concentrations of inhibitor (3-10 J.LM) using 1f (6-50 JlM) as substrate and with data
fitting using GraFit.

RESULTS AND DISCUSSION

The binding of both 8-alkyl-pterins and 8-alkyl-N5-deaza-pterins to DHFR appears to

show strong pH-dependence, with minimum Km values for 8-alkyl-pterins reported
previously 1•2 at -pH 5.8, and minimum Kct values for 8-alkyl-N5-deaza-pterins at -pH 6.6. 3
These values approximately parallel the pKa's of the two classes suggesting that a ligand
protonation is required for binding to enzyme. Structure-activity relationships for 8-alkyl-
pterins and 8-alkyl-N5-deaza-pterins were thus examined at pHs 5.8 and 6.6 respectively.
Kct values for both binary and ternary complexes with human and chicken DHFRs are
given in Table 1 for deaza-pterins 2a-2m. In terms of structure the compounds are divided
into singly and doubly substituted compounds which will be discussed separately. All the
singly (8-) substituted compounds investigated showed some binding with DHFR from both
sources. As the size of the 8-substituents investigated ranged from the small methyl group to
the bulkier isopropyl group, this suggests that the enzyme can accommodate a large degree of
structural variation in the 8-substituent. For chicken DHFR, binding strength in the binary
complex increased with the size of substituent in the order isopropyl (2d) >propyl (2c) >
ethyl (2b) >methyl (2a) suggesting that the enzyme binding pocket for the 8-substituent is
quite large and probably hydrophobic in nature. For the binary complexes with human
DHFR, the binding strength again increases as the size of the substituent increases except that
the propyl compound has the strongest binding. In the ternary complexes, binding is also
stronger for the larger substituents for both enzymes, but for chicken enzyme the ethyl group
has anomalously weak binding while for human enzyme binding is strongest for the
isopropyl compound reversing the order with the propyl compound compared with the binary
complexes. Comparison of the Kct values for each substituent in the binary and ternary

526
complexes indicates that the presence of cofactor increases the strength of binding in each
case with the ratio ranging from 1.2 to 2.1 for the chicken enzyme and 3 to 17.9 for the
human enzyme. This co-operativity effect has been observed previously in the binding of
several different ligands including methotrexate to DHFRs. 6•8 The degree of cooperativity is
dependent on both the substituent and the enzyme with the effect generally much greater for
human than chicken enzyme, especially for the isopropyl compound. This result may partly
be due to the presence of some cofactor in the Sigma chicken DHFR.

Table 1. Dissociation constants K<I (J.LM)a at pH 6.6 for 8-alkyl-N5-deaza-pterins 2a-2m

for binary and ternary complexes with cofactor NADPH and chicken and human DHFRs.

Chicken DHFR Human DHFR

Compound NoNADPH WithNADPH Ratiob NoNADPH WithNADPH Ratiob
2a 58±5 27±1 2.1 118±12 21±2 5.6
2b 38±3 32±6 1.2 60±2 13±2 4.6
2c 18±1 11±1 1.6 12.0±0.1 4.0±0.7 3.0
2d 16±1 10±1 1.6 34±2 1.9±0.2 17.9
2e 16±1 1.1±0.1 14.5 31±1 0.7±0.1 44.3
2f 60±6 74±8 0.8 130±9 16±2 8.1
2g 138±28 25±3 5.5 108±7 4.6±0.5 23.5
2h 44±2 23±3 1.9 60±4 18±1 3.3
2i 11 ± 1 1.1±0.1 10 21±1 2.9±0.3 7.2
2j 3.4 ±0.1 5.9±0.5 0.6 3.4±0.2 2.0±0.3 1.7
2k 20±1 11±2 1.8 21±1 5.1±0.7 4.1
2m 12+1 5.5±0.4 2.2 7.1±0.2 1.2±0.2 5.9
a Standard errors b Ratio of Kd's for binary to ternary complexes

For the doubly substituted deaza-pterins 2e-2m, again, each compound shows some
binding to both DHFRs. However, this binding varies over a much wider range than
observed for the singly (8-) substituted pterins. For the 6-methyl derivatives 2e-2h the
strongest binding in the binary complex is for the 6,8-dimethyl compound (2f) with binding
for the chicken enzyme decreasing in the order methyl >isopropyl> ethyl> propyl and with
significantly weaker binding for the latter three compounds. For the human enzyme binding
of the ethyl and propyl compounds is similar. For ternary complexes with chicken DHFR,
the 6,8-dimethyl compound binds more than 20 times stronger than any of the other 6-
substituted compounds with its binding increased -15 times by the presence of cofactor, but
with cooperativity for the other compounds.in the range of only 0.8 to 5.5. For ternary
complexes with human DHFR the 6,8-dimethyl compound again binds most strongly with
its binding increased -44 times by the presence of cofactor. The other 6-substituted
compounds show significantly greater cooperativity with human enzyme, particularly for the
propyl compound 2g. In general these results suggest both enzymes have difficulty in
accommodating both a large 8-substituent and a 6-methyl- group. However, the greater
binding cooperativity for ternary complexes of the human enzyme implies greater active site
flexibility in accommodating the cofactor and ligand in an optimum relative orientation than
does the chicken enzyme. The results for the 7,8-dimethyl- (2i) and 7-methyl-8-propyl (2j)
compounds show both compounds bind strongly to both enzymes with 2j having the lowest
Kd value of any compound for the binary complexes. In the ternary complexes 2i shows
strong binding cooperativity with both enzymes with "K<! values similar to those for the 6,8-
dimethyl- compound 2e. For the ternary complexes of the 7-methyl-8-propyl- compound
chicken enzyme shows negative cooperativity (0.6), but with modest cooperativity (1.7) with
human enzyme. Both 5,8-dimethyl- (2k) and 5-methyl-8-propyl- (2m) compounds bind

527
well to both enzymes with significantly stronger binding than for the 6-methyl- analogues 2e
and 2g in binary complexes but with weaker binding cooperativity in the ternary complexes
than for 2e and 2g.
In summary: comparison· of binding strengths of all compounds with each enzyme
indicates that human enzyme can bind larger 8-substituents more strongly in the ternary
complex than the chicken enzyme, suggesting the active site of human enzyme has more
space for optimum binding of even large 8-substituents as well as cofactor.

Table 2. Michaelis-Menten Km (JlM) values at pH 5.8 for 8-alkyl-pterins for both human
and chicken DHFRs.

Enzyme Com12ound
la lb lc ld le 1f
chicken DHFR 25a 62±4 9.4±0.9 7.7±0.4 16±11 4.3±0.3
humanDHFRa 130 140 b 19 b 11
a Reported in ref. 2 b Not determined

To start to assess the strength of binding of the 8-alkyl-pterins to DHFRs, a ~value

for the ternary complex of 6,8-dimethyl-pterin , chicken enzyme and NADP+ was determined
to be 16o±30 JlM at pH 5.8. The~ value at pH 6.6 of 14±1 JlM obtained for the same
complex for 6,8-dimethyl-N5-deaza-pterin indicates both that NADP+ has little effect on the
strength of binding (cf binary and ternary complex with NADPH results of -16 and 1 JlM in
Table 1), and that the pterins bind much more weakly in this ternary complex than do deaza-
pterins. This result is consistent with x-ray9 and theoreticaPO studies indicating the
nicotinamide group of NADP+ is poorly bound. To further evaluate the strength of binding
of the 8-alkyl-pterins, Km values with both human and chicken DHFR were determined at
pH 5.8 (Table 2). Comparison of these values with the analogous ~values for ternary
complexes in Table 1 indicates greater similarity for the two data sets and that binding in the
presence of NADPH is more comparable for the pterins and deazapterins than suggested by
the~ values in the presence of oxidized cofactor. The Km data also suggest that NADPH
greatly strengthens binding of 8-alkyl pterins to DHFR. Kinetic inhibition studies using 6,8-
dimethyl-pterin 1f as substrate and 6,8-dimethyl-N5-deaza-pterin 2e as inhibitor indicated
competitive inhibition with a Ki of 0.80±0.06 JlM suggesting that both classes of compound
bind at the same site on the enzyme.

REFERENCES

1. V. Thibault, M.J. Koen and J.E. Gready, Biochem. 28: 6042 (1989).
2. J.E. Gready, in: "Chemistry and Biology ofPteridines 1989," H.-Ch. Curtius, S. Ghisla and N. Blau,
eds., de Gruyter, Berlin (1990), pp. 23-30.
3. J.E. Gready, M.T.G. Ivery, M.J. Koen and H.J. Yang, in: "Pteridines and Related Biogenic Amines and
Folates.1992," N. Blau, H-Ch. Curtius, R. Levine andY. Yim, eds., Hanrim, Seoul (1992), pp. 265-76.
4. M.T.G. Ivery and J.E. Gready, Bioi. Chem. Hoppe-Seyler 373: 1125 (1992).
5. K.J. Ellis and J.F. Morrison, Meth. Enzym. 87: 405 (1982).
6. J.W. Wiiiiams, J.F. Morrison and R.G. Duggleby, Biochem. 18: 3449 (1979).
7. B. Birdsall, R.W. King, M.R. Wheeler, C.A. Lewis, S.R. Goode, R.B. Dunlap and G.C.K. Roberts,
Anal. Biochem. 132: 353 (1983).
8. B. Birdsall, S.V. Burgen and G.C.K. Roberts, Biochem. 19: 3732 (1980).
9. C. Bystroff, S.J. Oatley and J. Kraut, Biochem. 29: 3263 (1990).
10. P.L. Cummins, K. Ramnarayan, U.C. Singh and J.E. Gready, J. Am. Chem. Soc. 113: 8247 (1991).

528
DEVELOPMENT OF A SPECTROFLUORIMETRIC METHOD
FOR DETERMINING THE pKa OF
PTERIN-ANALOGUE LIGANDS BOUND TO DHFR

Soon-Seog Jeong and Jill E. Gready

Department of Biochemistry
University of Sydney
Sydney, NSW 2006, Australia

INTRODUCTION

As part of our interest in new mechanism-based substrates and inhibitors of the enzyme
dihydrofolate reductase (DHFR), a series of 8-alkyl substituted pterins (8-R-Pt's) and 8-alkyl
substituted-N5-deazapterins (8-R-N5d-Pt's) have been synthesizedl-4 and the pH-
dependence of their electronic spectra studied.5,6 The UV/vis and excitation and emission
spectra of neutral and cationic forms of both classes of compound are distinctly different, and
both classes are highly fluorescent, especially for 8-R-N5d-Pt's with very high quantum
yields (<I> > 0.7).5,6 In agreement with values determined by UV/vis spectral methods,?
pKa's determined by fluorimetric methods showed high basic values of ca. 5.5 for 8-R-Pt's
and ca. 6.6 for 8-R-N5d-Pt's.5,6
As these compounds have been designed to bind to the DHFR active site in substrate-
like orientation but in a protonated form, 8 we were interested in studying the ionization state
and effective pKa's of enzyme-bound compounds. Previous reports in the literature of the
charge state of ligands bound to DHFRs by UV/vis spectroscopy9,10 and nmr11,12 studied
mainly methotrexate which displays tight binding. However, as our initial compounds have
much weaker binding with Kd's in the micromolar range UV/vis methods will not be
successful. The strong fluorescence of our compounds and possibility to discriminate ligand
from enzyme and cofactor emission suggested the use of spectrofluorimetry.6 In this paper
we introduce a spectrofluorimetric method for investigating the charge state and determining
the pKa of our ligands bound to DHFR.

MATERIALS AND METHODS

A Perkin Elmer LS50 spectrofluorimeter and a 5mm x 5mm quartz cuvette were used
with excitation and emission slits set at 5 nm for determining the pKa and at 10 nm for
determining the binding constants (KJ'g). Recombinant human dihydrofolate reductase

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 529
(rhDHFR) was a gift from Prof J. Freisheim and its active enzyme concentration was
determined by methotrexate titration.l3 The pKa of 6,8-dimethyl-N5-deazapterin (6,8-diMe-
N5d-Pt) was determined in the presence of NADPH, NADPH plus casein, NADPH plus
rhDFHR, and in buffer only6 (Tris/AcOH/ethanolamine14) in the pH range 5-8 in a total
volume of 300 JlL. Data were analysed using non-linear regression with the PC program
GraFit and experimental spectra compared with spectra simulated using measured Kd's and
pKa's. Kd's were determined from fluorimetric quenching of enzyme fluorescence with
compensation for dilution and the data were fitted as reported previously. 5

RESULTS AND DISCUSSION

Of available compounds 6,8-diMe-N5d-Pt was chosen for study as it had the best
binding to DHFR in the presence of NADPH. Initial examination of experimental conditions
suggested the main technical problems were overlapping absorption by enzyme and cofactor
and UV absorption by components in the Tris/MES/ethanolarnine multicomponent buffer14
previously used in pH-dependent enzyme studies to ensure constant ionic strength.1,8 The
Tris/AcOH/ethanolamine system was found to be satisfactory over the pH range 5.0- 8.0
and the strong fluorescence of the ligand coupled with high fluorimeter sensitivity made it
possible to follow the emission intensity of ligands by exciting at an edge wavelength (380
nm) of an absorption band at which the enzyme and the cofactor neither absorb nor emit
significantly. Similarly, excitation spectra of the ligands were run following emission at an
edge wavelength (400 nm).
6,8-diMe-N5d-Pt has unique peaks at 258 and 278 nm in the excitation spectra for
neutral and cationic forms, respectively, and also the long wavelength band (365 compared
with 354 nm) and emission spectrum of the neutral form (427 compared with 420 nm) are
red shifted. Thus exciting at the edge wavelength (380 nm) increases the relative intensity of
the neutral emission spectrum, allowing greater sensitivity in following intensity shifts for the
variation in the proportions of neutral and cationic forms with pH. Following the emission at
400nm as the pH was varied showed similar excitation spectra in the presence and absence of
DHFR, suggesting the same cationic form for bound ligand. pKa's of 6,8-diMe-N5d-Pt
under the different conditions were determined both from the intensities at 258nm in
excitation and at 431nm in emission spectra: the values are given in Table 1.

Table 1. Comparison of apparent pKa of 6,8-diMe-NSd-Pt bound to rhDHFR.NADPH

with uncorrected values for bound ligand (+NADPH.DHFR), for free ligand and for ligand
in control experiments.

Free ligand +NADPH +NADPH.casein +NADPH.DHFR Bound ligand

258nm 6.69 ± 0.04 6.71 ± 0.04 6.68 ± 0.11 7.61 ± 0.13
431nm 6.63 + 0.03 6.69 + 0.03 6.67 + 0.04 7.87 + 0.06 9.08 + 0.03

As shown in Table 1, there was an increase in the ligand pKa value of about one unit
compared with that of free form or in the control experiments under the experimental
conditions which correspond to a mixture of free and bound form. Kd measurements as a
function of pH, as shown in Table 2, indicated significant pH-dependence. A plot of Kd
versus pH showed a bell-shape curve, from which apparent acidic constants of the enzyme
and ligand, and the pH-independent dissociation constant were determined to be 5.93 ± 0.14,
7.04 ± 0.16, and 0.36 ± 0.10 )lM, respectively, using the method of Morrison.16,17 From
the Kd's values, fractions of bound and unbound forms as a function of pH were calculated
as shown in Table 2. Then the pKa of 6,8-diMe-N5d-Pt bound to rhDHFR.NADPH was

530
determined by correcting the total intensities at the various pH values for the proportion of
unbound form using data from the control spectra for ligand in the presence of NADPH and
casein, and then scaling the intensities for 100% bound form. The resulting value of 9.1 is
about 2.5 units higher than the value for free ligand of -6.7. Figure 1 shows the pH-
dependence of the uncorrected and corrected intensities, together with those for the control
experiment.

600

500
4000
:c z-1
.! 400 m
z
>
1- (/)
Cii
ffi 300
~
~
200

1000

Figure 1. pH-dependent changes of intensities of 6,8-diMe-NSd-Pt in presence of: (a) NADPH and casein
(<1); (b) NADPH and rhDHFR (0) and (c) 100% bound form after correction(.).

Then using the apparent and measured pKa values of 9.1 and 6.7 for bound and free
ligand, respectively, the fractions of cationic and neutral forms of bound and unbound ligand
were calculated: the values are shown in Table 2. Total fractions of cationic and neutral
forms were then calculated by summing bound and unbound fractions of each form (see
Table 2). Simulated spectra at the various pHs were then calculated by multiplying the
excitation spectra of the control experiment in the presence of NADPH and casein at pH 4.6
for cation and pH 8.8 for neutral form with the relevant fractions and summing the two
components. Comparison of the shape and pH-dependence of the experimental (ligand in the
presence of NADPH and DHFR) and simulated spectra showed they were very similar, and
quite different from those for binary complex (ligand in the presence of DHFR only) at the
same pHs. This result is consistent with earlier results at pHs 6.6 and 7.4 showing that 6,8-
diMe-N5d-Pt binds to rhDHFR about 15 to 20 times less strongly in the binary complex
than in the ternary complex.5,15

531
Table 2. The fractions of bound and unbound forms and the total fractions of cationic and
neutral forms of ligand 6,8-diMe-N5d-Pt calculated from measured Kd and pKa values.

pH Kd(!lM) Bound form Unbound form Total cation Total neutral

6.11 0.67 76% 24% 95% 5%
6.43 0.59 78% 22% 92% 8%
6.53 0.59 78% 22% 91% 9%
6.70 0.61 78% 22% 89% 11%
6.80 0.64 77% 23% 87% 13%
6.88 0.68 76% 24% 85% 15%
7.06 0.79 73% 27% 81% 19%
7.24 0.98 70% 30% 75% 25%
7.43 1.28 64% 36% 68% 32%
7.64 1.83 57% 43% 59% 41%
8.58 12.87 18% 82% 15% 85%
9.73 176.71 2% 98% 0% 100%

CONCLUSIONS

The pKa of 6,8-dimethyl-N5-deazapterin when bound to recombinant human DHFR

has been determined to be 9.1 using a newly developed fluorimetric method. This is about
2.5 units higher than for free ligand indicating a strong preference for the enzyme to bind the
cationic form. As for other DHFR inhibitors such as methotrexate and trimethoprim, this is
probably due to the favourable interaction which can form between the conserved active site
acidic residue, glutamate in rhDHFR, and protonated ligand.

ACKNOWLEDGEMENT

We thank Prof. Freisheim for the enzyme and Mr. Michael Ivery for the sample of 6,8-
dimethy1-N5-deazapterin.

REFERENCES
1. V. Thibault, M.J. Koen and J.E. Gready, Biochemistry 28:6042 (1989).
2. M.T.G. Ivery and J.E. Gready, Bioi. Chern. Hoppe-Seyler 373:1125 (1992).
3. S.-S. Jeong and J.E. Gready, Bioi. Chern. Hoppe-Seyler 373:1139 (1992).
4. M.J. Koen and J.E. Gready, J. Org. Chern. (1993) in press.
5. J.E. Gready, M.T.G. Ivery, M.J. Koen and H.J. Yang, in: "Pteridines and Related Biogenic Amines and
Folates 1992," N. Blau, H.-Ch. Curtius, R. Levine and J. Yim, eds., Hanrim, Seoul (1992) pp.265-76.
6. S.-S. Jeong, P. Wormell and J.E. Gready, Pteridines (1993) in press.
7. M.T.G. Ivery, M.J. Koen and J.E. Gready, unpublished results.
8. J.E. Gready, in: "Chemistry and Biology ofPteridines 1989," H.-Ch. Curtius, S. Ghisla and N. Blau,
eds., de Gruyter, Berlin (1990) pp.23-30.
9. K. Hood and G.C.K. Roberts, Biochem. J. 171:357 (1978).
10. S. Subramanian and B.T. Kaufman, Proc. Natl. Acad. Sci. USA 75:3201 (1978).
II. L. Cocco, J.P. Groff, C. Temple, Jr., J.A. Montgomery, R.E. London, N.A. Matwiyoff and R.L.
Blakley, Biochemistry 20:3972 (1981).
12. B.S. Selinsky, M.E. Perlman, R.E. London, C.J. Unkefer, J. Mitchell and R.L. Blakley, Biochemistry
29:1290 (1990).
13. J.W. Williams, J.F. Morrison and R.G. Duggleby, Biochemistry 18:3449 (1979).
14. K.J. Ellis and J.F. Morrison, Meth. Enzymol. 87:405 (1982).
15. M.T.G. Ivery and J.E. Gready, other paper this volume.
16. S.R. Stone and J.F. Morrison, Biochim. Biophys. Acta 745:237 (1983).
17. S.R. Stone and J.F. Morrison, Biochim. Biophys. Acta 745:247 (1983).

532
SELECTIVE INHIBITION OF DIHYDROFOLATE
REDUCTASE FROM PROBLEM HUMAN PATHOGENS

R.L. Thenl, P.G. Hartmanl, I. Kompisl, D. Santi2

lPharmaceutical Research, F.Hoffmann-La Roche Ltd
4002 Basel/Switzerland
2Depts. of Pharmaceutical Chemistry and of Biochemistry
University of California, San Francisco 94143

INTRODUCTION

Considerable changes in the prevalence and epidemiology of human

pathogenic microorganisms have occurred over the last decade. Two main
factors are responsible:
1. Resistance development, which is a particular concern among gram-
positive cocci, e.g. staphylococci!
2. The increased number of patients with impaired immuneresponse,
highlighted by the spread of HIV, who suffer infections with opportu-
nistic pathogens such as Pneumocystis carinii, Toxoplasma gondii, Myco-
bacterium avium and others.
The combination of trimethoprim (TMP) and sulfamethoxazole (co-
trimoxazole) has been recommended as an alternative regimen for infections
with methicillin-resistant staphylococci2 and this agent is also effective in the
prophylaxis and treatment of P.carinii pneumonia3. The combination of
pyrimethamine with sulfadoxine is used in the treatment of malaria and of
T.gondii infections.
Although the use of these inhibitors against P.carinii and T.gondii
proves that dihydrofolate reductase (DHFR) is a valid target, neither TMP nor
pyrimethamine are particularly good inhibitors of the DHFRs of these
organisms. In addition, TMP-resistance in multiresistant staphylococci is not
infrequent nowadays, and is caused by a transposon-mediated DHFR which
has reduced sensitivity towards TMP1,4.
With the cloning, expression and crystallization of the DHFR from
P.cariniiS and the DHFR from Tn4003 in staphylococci4,6, valuable tools were
obtained to aid the design and selection of more potent inhibitors of these
enzymes.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 533
 The availability of the enzymes for screening allowed us to initiate a
programme directed towards selection, both from compounds already avail-
able and from new synthetic efforts, of DHFR inhibitors that have a higher
affinity than TMP for relevant DHFRs together with satisfactory selectivity.
From over 200 compounds tested, some examples of more selective and
potent inhibitors of the enzymes from P.carinii , the TMP-sensitive and the
TMP-resistant staphylococcal DHFR are presented in this study.

MATERIAL AND METHODS

DHFRs from E.coli, P.carinii and the two staphylococcal DHFRs were
overexpressed in E.coli and purified to homogeneity as described5,6,7.
Expression and purification of the human DHFR has been described8.
DHFR inhibitors were either selected from inhibitors available at
F.Hoffmann-La Roche Ltd, Basel, Switzerland, or synthesized de novo to fill
the gaps in SAR.

RESULTS AND DISCUSSION

All compounds were tested against the DHFRs from P.carinii, S.aureus
and the DHFR on transposon Tn4003 from staphylococci. DHFRs from E.coli
and man served as reference. Data are shown in Table 1. Among more than
200 compounds tested for inhibition of the purified DHFR from P. carinii,
several emerged with a distinctly higher activity than TMP. The best
compound among a series of pyrrole-substituted 2,4-diamino-benzyl-pyrimi-
dines was Ro 11-8958, which is 14 times more active than TMP. Ro 11-8958
also has a ten times better specificity with respect to the human enzyme than
TMP. Replacing the ethoxy-group in positions 3 and 5 by a wide range of
substituents resulted in compounds with reduced affinity for the P. carinii
DHFR.
Efforts were undertaken to find inhibitors of the TMP-resistant staphy-
lococcal DHFR, located on transposon Tn4003. A number of compounds
emerged which showed distinctly higher activity than TMP. Among various
substitued 2,4-diamino-benzyl-pyrimidines Ro 11-8958 was the most active
but one (-20 fold lower ICso). Several quinazolines (Ro 18-4802, 18-5771) were
even more active, but devoid of selectivity. None of these compounds over-
came resistance in vitro.
The chromosomal DHFR from S. aureus is fourfold less sensitive
towards TMP than that from E.coli. The enzymes have 36 % amino acid
homology6. Probing both enzymes with a variety of inhibitors showed that
the majority of the 2,4-diamino-5-benzyl-pyrimidines tested were less active
against the staphylococcal than the E. coli DHFR. Substitution in position 2',
however, resulted in compounds with greater affinity for the staphylococcal
DHFR. Several compounds (Ro 11-7396, 15-4672, 21-0057) strongly discrimi-
nated between the two enzymes, pointing to significant differences in the
active site of the two DHFRs.

534
Table 1. Activity of Selected Compounds Against DHFRs from Several Problem Human
Pathogens.
ICSO (iJ.M) for DHFR from
Compound Structure P.carinii Tn4003 S.aureus E.coli man

Trimethoprim 43 16 0.034 0.0078 900

12-5279
HzNANr·Oy(r) _.:I
180 300 0.85 0.012 >300

·~~0
H,N_..4NI "I

11-7496 _.o - 46 10 0.046 0.0065 >100

13-4670
HzNAN·&t:x"0 ,...I
Cl - 23 >100 0.55 0.6 >300

H1N
·&yc~
).,
~N
I
"
I
NL)

13-5705 .-s - 18 8.0 0.0063 0.005 180

11-8960
HzNANI
·r« '-I

6' 16 19 0.044 0.024 300

NH1 lf
H2N'-;.Yc;(
11-5736
-6-- 10 100 1.3 0.46 30

HzNAN
·~r:1 " 0

11-9016 6' 6.0 100 0.22 0.065 200

.,....NY-o:o,
6. '0
..,
46·4561 4.6 6.0 0.027 0.0095 >300
('

H2N
!7'Qto
N ' 't;)
11-8958 r' - 3 2.9 0.011 0.0018 600

08-4484
H1N~N·5L¢c'' I

_.ol
'
I
0

3.4 4.3 0.009 0.023 >300

.,.1~,
18-2049
" 5.5 2.3 0.0065 0.0015 170

18-4802
·35-JJ
Hlf_.l.!_t( ""
4.0 1.5 0.002 0.001 0.004

59-~
Cl

18-5771 HlN1N 0.006 0.33 0.0019 0.007 0.038

16-8820
·r«
Hlf),_N

Hlf_£:
' 8r

so 32 0.16 0.006 >300

.))._
H1N~
•:]:"

•:X>- o+r
11-7396 NHl
nt >100 24 0.071 nt

NH1

r
Hlf...L:::,N I s s
15-4672 120 56 0.18 7.2 >300

21·0057
·W,
H1N_.4N I I " 0
I >100 280 0.7 80 >300

nt, not tested

535
ACKNOWLEDGEMENT

We thank Dr. S. Jolidon for his advice and help during this work.

REFERENCES

1. R.L. Then, I. Kohl and A. Burdeska, /. Chemother. 4: 67 (1992).

2. L.P. Elwell, H.R. Wilson, V.B. Knick, and B.R. Keith, Antimicrob. Agents Chemother.
29:1092 (1986).
3. W.T. Hughes, Ped. Infect. Dis. J. 10:391 (1991).
4. A. Burdeska, M. Ott, W. Bannwarth, and R.L. Then, FEBS Letts 266: 159 (1990).
5. W. Sirawarapom, ].C. Edman, and D.V. Santi, Prot. Expr. Purific. 2: 313 (1991).
6. G.E.Dale, R.L. Then, and D. Stiiber, Antimicrob. Agents Chemother. (submitted)
7. P.G. Hartman, M. Stahli, H.P. Kocher, and R.L. Then, FEBS Letts 242,157 (1988).
8. ]. Stuber, eta!. In: Chemistry and Biology of Pteridines (B.A.Cooper and V.M.Whitehead,
eds.) Walter de Gruyter, 839 (1986).
9. D.P. Baccanari, and S.S. Joyner. Biochemistry 20:1710 (1981).

536
TRANSLATIONAL REGULATION OF THE SYNTHESIS OF DIHYDROFOLATE REDUCTASE

Emine Ercikan, Debabrata Banerjee, Mark Waltham, Barbara Schnieders,

Kathleen W. Scotto, and Joseph R. Bertino
Program of Molecular Pharmacology and Therapeutics
Sloan-Kettering Institute for Cancer Research
New York, NY 10021

INTRODUCTION
Earlier studies showed that administration of MTX to patients leads
to an increase in the level of DHFR protein in both normal and leukemic
leukocytes as well as in erythrocytes within hours to days. 1•2•3 In vitro
studies using a human lymphoblastoid cell line showed that increase in DHFR
protein is not transcriptionally mediated but was abolished by
cycloheximide treatment, suggesting that new protein synthesis is required.r.
The rapid increase observed was attributed to either protection of DHFR
from degradation by bound MTX and/or dihydrofolate, 4 or to an increase in
translation of this enzyme. 5 An increase in thymidylate synthase activity
has been reported after 5-fluorouracil treatment, 6- 8 and it has been
suggested that this increase may also be regulated at the translational
1eve 1. 7•8 In this communication, we show that DHFR protein regulates its
synthesis by exerting an inhibitory influence on its translation and that
addition of MTX or dihydrofolate to the reticulocyte translation system
relieves this inhibition, thus providing a possible explanation for the
rapid increase in this enzyme activity noted in certain cells after MTX
administration. In addition, preliminary experiments using CHO cells
lacking the DHFR gene and transfected with DHFR eDNA lacking the 5' leader
sequence and treated with MTX showed an increase in DHFR, indicating that
this region may not be required for the DHFR protein/RNA interaction.

METHODS
Preparation of eDNA Template and In Vitro Transcription
A 141 bp fragment of 5' untranslated leader sequence was amplified
by PCR from genomic DNA isolated from a T47D human breast cancer cell line
using the following set of primers:
PI GGATCCTAATACGACTCACTATAGGGAGAGGGGGGGCGGAAGCTTGCCTGCACAAATAGG
PII AGTTTTAGCGAACCAACCATGGCAGCAGCGGGAGG
PCR conditions were 94·C x 1 min., 50·C x 1 min., and 72·C x 1 min.
for 40 cycles. Another eDNA template was amplified by PCR using the DHFR
eDNA present in the plasmid, pKT7HDR (containing the 639 bp wild type human
DHFR eDNA Ncoi and Hindiii fragment) with the following primers:

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 537
Pili ATCATCCCATGGTTGGTTCGCTAAACTGCATGG
PIV CGATCGAGGATCCCAAGCTTACCTTTT
PCR conditions were 94·C x 1 min., 55·C x 1 min., and 72·C x 1 min.
x 40 cycles. The products of these two PCR amplifications were then Gene
cleaned, combined in a 1:1 ratio, and used as template for a third PCR
amplification using the primers I and IV to amplify the T7 promoter+ 141
bp leader + DHFR eDNA 639 bp fragment.
In Vitro Transcription and In Vitro Translation
The product of the third PCR amplification was used as the template
to make the DHFR mRNA, using T7 RNA polymerase. The RNA was purified by
extraction with phenol/chloroform and precipitated with potassium acetate
and ethanol. The RNA was then used for in vitro translation using a
nuclease treated rabbit reticulocyte lysate system obtained from Promega.
35-S methionine (translation grade) was obtained from NEN. Radiolabeled
products obtained after translation were analyzed by 15% SDS-PAGE and
visualized by autoradiography.
Effect of MTX on DHFR Activity in Wild Type CHO Cells and in CHO DG44 Cells
Transfected with DHFR eDNA
A human DHFR eDNA (639 bp) cloned in the mammalian expression vector,
pSV5 (construct pSV5HDR), was used for transfecting CHO DG44 cells which
lack DHFR. 9 CHO wild type and DG44 cells transfected with pSV5HDR were
treated with MTX for various times, and the DHFR activities in the cell
lysates were measured spectrophotometrically. 10

RESULTS
The in vitro reticulocyte lysate translation system was optimized
both for the concentration of DHFR mRNA and for the time of translation.
7.5 pmols of mRNA and 20 min. of translation time at 37·C were found to
give the best yield of DHFR protein.
These conditions were used to study the effect of exogenously added
recombinant normal human DHFR protein 10 on the translation of DHFR mRNA.
Figure 1 shows that 238 pmols of human DHFR inhibits translation of DHFR
mRNA. This inhibition was relieved by the addition of 43 ~M MTX-Glu 4 or 43
~M FH.f!' suggesting that inhibition was a direct effect of added DHFR
prote1n. MTX and FH 2 alone appeared to increase the translation of DHFR.
DG44 cells transfected with the mammalian expression vector, pSV5HDR (see
Materials and Methods), and treated with either 500 or 1000 nM drug MTX
exhibited a 3-4-fold increase in DHFR activity at 24 and 48h. Wild type CHO
cells had no or only a slight increase in DHFR activity after MTX treatment
(Figure 2).

DISCUSSION
In this reticulocyte lysate assay, DHFR protein is shown to inhibit
translation of DHFR mRNA. The reversal of this inhibition by MTX or FH 2 may
explain in part the observed "induction" of DHFR activity in normal and
leukemic cells after MTX treatment. Other proteins that have been shown to
regulate their translation in eukaryotes are ferritin and thymidylate
synthase. A 90 kDa ferritin repressor protein has been shown to bind to the
iron responsive element within the 5' UTR of ferritin mRNA. 12 The TS protein
has recently been shown to bind to its own mRNA and inhibit translation,
although the exact location and sequences in TS mRNA that the protein binds

538
 1 2 3 4 5 6 7 8 9

Figure 1. Inhibition of DHFR synthesis by exogenously added recombinant hDHFR protein can be reversed
by addition of MTX·Glu4 and FH 2 • No mRNA (lane 1); 7.5 pmol DHFR mRNA alone (lane 2) and 1n the presence
of human recombinant DHFR protein (240 pmol 1n lane 3) and with MTX-Glu4 (43 /.IM and 86 /.IM in lanes 4 and
5, respectively) and w1th FH 2 (43 /.IM and 86 /.IM 1n lanes 7 and 8, respectively). Lanes 6 and 9 show that
MTX-Glu4 (43 f.IMJ and FH 2 (43 /.IM) alone d1d not 1nh1bit translat i on.

2.00
- 24 h (ilii'ill 48 h

LOO

0.80
- 24 h mEtlll 48 h

1.60 w
..
a: ?.
~ ..
a:
l: 0 .60

·~
l:
0
1.20 f. 0
!! 2
"'
E
:~

:~ ~it ~. "'e
!i
~(
5
e ;~
~
IT 5
e
0.40 ~
!~ ,.
~·
0. 80 1%
H {:
~~ j
~ ~ "
'~
t¥1..
ijl i~
Iii ~~~
~·'
I
0.40 :g ~::
0 .20

I
;~
:1!
"'~
~:.:-:
,~
"
w
'1}
1ft
~.
~ *
.
~t.
0.00
if it 7:
0.00
0 0 '*
.5

".. .. ,x
0 .5 1 0 .5 .5

"M MTX

Figure 2. DHFR activity in ce ll free lysate of MTX treated CHO wild type cell s (l e ft panel) and
transfected DG44 cells (right panel).

539
to have not been reported. 8 Transfection studies in CHO DG44 cells with the
pSV5DHFR construct containing the human DHFR eDNA suggest that the 5'
leader sequence is not involved in DHFR binding. Studies by Cowan et al.
also using CHO cells lacking the DHFR gene and transfected with a human
DHFR minigene concluded that sequences other than the coding region affect
regulation by MTX. 11 This raises the possibility that the 3' untranslated
region (approximately 80 bp downstream of the TAA stop signal) may be
involved in the autoregulation process. Our current studies are directed
to understanding the sequences in DHFR mRNA involved in binding the DHFR
protein and the sequences in the protein that bind to the mRNA.

REFERENCES
1. J.R. Bertino, D.M. Donohue, B.W. Gabrio, R. Silber, A. Alenty, M.
Meyers, and F.M. Huennekens, Increased level of dihydrofolate
reductase in leukocytes of patients treated with aminopterin, Nature
193:140-141 (1962).
2. J.R. Bertino, D.M. Donohue, B. Simmons, B.W. Gabrio, R. Silber, and
F.M. Huennekens, The "induction" of dihydrofolate reductase in
leukocytes and erythrocytes of patients treated with methotrexate,
J. Clin. Invest. 43:466-475 (1963).
3. J.R. Bertino and B.l. Hillcoat, Regulation of dihydrofolate reductase
and other folate requiring enzymes, in: "Advances in Enzyme
Regulation," Pergamon Press, New York (1968).
4. B.l. Hillcoat, V. Swett, and J.R. Bertino, Increase of dihydrofolate
reductase activity in cultured mamma 1ian ce 11 s after eJCposure to
methotrexate, Proc. Natl. Acad. Sci., U.S.A. 58:1632-1637 (1967).
5. K. Bastow, R. Prabhu, and Y.-C. Cheng, The intracellular content of
dihydrofolate reductase: possibilities for control and implications
for chemotherapy, Adv. Enz. Reg. 22:15-26 (1984).
6. K.D. Danenberg and P.V. Danenberg, Activity of thymidylate synthase and
its inhibition by 5-fluorouracil in highly enzyme-overproducing cells
resistant to 10-propargyl-5,8,-dideazafolate, Mol. Pharmacal. 36:219-
223 (1989).
7. E. Chu, J.C. Drake, D.M. Koeller, S. Zinn, C.A. Jamis-Dow, G.C. Yeh,
and C.A. Allegra, Induction of thymidylate synthase associated with
multidrug resistance in human breast and colon cancer cell lines,
Mol. Pharmacal. 36:136-143 (1990).
8. E. Chu, D.M. Koeller, J.l. Casey, J.C. Drake, B.A. Chabner, P.C.
Elwood, S. Zinn, and C.J. Allegra, Autoregulation of human
thymidylate synthase messenger RNA translation by thymidylate, Proc.
Natl. Acad. Sci., U.S.A. 88:8977-8981 (1991).
9. G. Urlaub, E. Kas, A.M. Carothers, and l. Chasin, Deletion of the
diploid dihydrofolate reductase locus from cultured mammalian cells,
Cell 33:405-412 (1983).
10. B.I. Schweitzer, S. Srimatkandada, H. Gritsman, R. Sheridan, R.
Venkataraghavan, and J.R. Bertino, Probing the role of two
hydrophobic active site residues in the human dihydrofolate reductase
by site directed mutagenesis. J. Biol. Chern. 264:20786-20795 (1989).
11. K.H. Cowan, M.E. Goldsmith, M.D. Ricciardone, R. levine, E. Rubalcaba,
and J. Jolivet, Regulation of dihydrofolate reductase in human breast
cancer cells and in mutant hamster cells transfected with a human
dihydrofolate reductase minigene, Mol. Pharmacal. 30:69-76 (1990).
12. E.C. Theil, Regulation of ferritin and transferrin receptor mRNAs, J.
Biol. Chern. 9:4771-4774 (1990).

540
EXPRESSION OF THE TRIMETHOPRIM RESISTANT DIHYDROFOLATE
REDUCTASE ENCODED BY TRANSPOSON TN4003 IN A SOLUBLE
FORM AND ITS SUBSEQUENT PURIFICATION TO HOMOGENEITY

Glenn E. Dale, Dietrich Sti.iber, Clemens Broger, and Hanno Langen

F. Hoffmann-La Roche Ltd.

Pharmaceutical Research -New Technologies
CH-4002 Basel, Switzerland.

INTRODUCTION

In recent years resistance to trimethoprim (Tmp) has increased and several Tmp-
resistant (Tmpf) dihydrofolate reductases have been described in gram-negative bacteria. In
staphylococci, however, only one Tmpr dihydrofolate reductase (DHFR) has been found so
far and this is located on transposon Tn40031. To understand the mechanism of resistance
we are interested in determining the three dimensional structure of this enzyme (type Sl
DHFR). However, the expression level of the recombinant enzyme in E. coli was rather low
and the expressed protein was poorly soluble. Furthermore, as a result of an internal start of
translation a truncated derivative was produced which copurified with the full length
enzyme. In contrast to the type S 1 DHFR, the chromosomal Tmp-sensitive (TmpS) enzyme,
termed SaDHFR, of Staphylococcus aureus ATCC 25923 could be highly over-produced in
E. coli in a soluble and active form.
Based on the differences in the DNA and the amino acid sequences of these DHFRs we
increased the expression level of the type S 1 DHFR and eliminated the internal start of
translation. Furthermore, we changed amino acids on the surface of the type S 1 DHFR
resulting in a soluble and active mutein of the enzyme. This recombinant Tmpr enzyme was
purified nearly to homogeneity.

METHODS

Site Directed Mutagenesis

Mutageneses were performed using either PCR or the oligonucleotide-directed in vitro

mutagenesis system of Amersham (UK), following the protocol which is supplied by the
manufacturer. In the latter case, EcoRI-BamHI fragments carrying the coding region for
DHFRs were subcloned into the double stranded DNA of bacteriophage Ml3mp18. The
single stranded DNA of the resulting M13mp18(DHFR) derivatives was used as template for

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 541
mutagenesis reactions with mutagenic primers. Mutant double stranded M13mp18(DHFR)
DNA was isolated and the EcoRI-BamHI fragment was transferred into the expression
vector pDS56/RBSII,Ncoi2. Internal Pvui sites were introduced into the coding regions for
type S 1 DHFR and SaDHFR by PCR with mutagenic primers following standard PCR
amplification protocols3.

Expression of S. aureus DHFRs in E. coli

E. coli M15(pREP4) cells2 harboring expression plasmids were grown to A600 = 0.9
in super-medium2 containing 100 j.lg/ml ampicillin and 25 j.lg/ml kanamycin, before gene
expression was induced by addition oflPTG. After 4 h of induction the cells were harvested
by centrifugation. Samples for SDS-PAGE were prepared by either directly resuspending
the cells in sample buffer or by resuspending the supernatants of cells disrupted by
sonication in sample buffer. The DHFR assay has been described4. A unit ofDHFR activity
was taken as the conversion of 1 j.lM of dihydrofolate per minute.

RESULTS

Expression of Type Sl DHFR and SaDHFR

The expression of SaDHFR and type S1 DHFR in E. coli was accomplished by

integration of the respective coding regions into the pDS expression system2. In the resulting
expression plasmids pSaDHFR and pS 1 the DHFR genes are under control of a regulatable
promoter, which is repressed in presence of the lac repressor but can be induced by addition
of IPTG, and a synthetic ribosomal binding site. E. coli M15(pREP4) cells containing
plasmids pSaDHFR and pS1 over-expressed SaDHFR and type S1 DHFR, respectively
(Table 1).
Although the proteins encoded by pS1 and pSaDHFR are 80% identical (unpublished
results) and the promoter and Shine-Dalgamo sequences are completely identical, their level
of expression in E. coli is quite different. Namely, SaDHFR was expressed -10 times better
than the type S 1 DHFR. Furthermore, expression in E. coli of type S 1 DHFR results in the
over-expression of a additional polypeptide of -14 Kd. N-terrninal amino acid sequencing of
this protein revealed that it is a truncated derivative of type S1 DHFR starting with amino
acid 43. Since position 42 is occupied by a methionine and on the RNA level the codon for
this methionine is preceded in a proper distance by a putative Shine-Dalgamo sequence, we
assumed that the 14 Kd polypeptide could represent the product of an internal start of
translation. To prove this assumption we destroyed the putative Shine-Dalgamo sequence by
site directed mutagenesis. In the resulting plasmid pS 1(Is) the sequence GGGAA is replaced
by the sequence GGCAA, which is believed not to contribute to the binding of ribosomes.
In fact, E. coli cells containing plasmid pS1(Is) do no longer produce the truncated
derivative of type S1 DHFR. Interestingly, most of the recombinant SaDHFR was produced
in a soluble form whereas by far most of the recombinant type S 1 DHFR was obtained in an
insoluble form.

The RNA Sequence Downstream of the Initiation Codon Determines the

Expression Levels of Type Sl DHFR and SaDHFR

To analyse the basis why SaDHFR is expressed to a far higher level than type S 1
DHFR we constructed plasmids containing chimeric genes composed of defined regions of
the two DHFR genes. Both genes contain a convenient Styi site at position 66, which
corresponds on the amino acid level to position 22. This site and a Pvui site introduced by
site directed mutagenesis into the genes at position 150 (amino acid position 50) were used

542
for construction of four plasmids containing chimeric genes. Both of the plasmids containing
the 5' end of the gene for SaDHFR direct a high level expression of the encoded proteins,
whereas the remaining two plasmids containing the 5' end of the gene for the type S 1 DHFR
direct only a low level expression. Obviously, the nucleotides encoding the first 22 amino
acids of SaDHFR exhibit a positive effect on the expression level of the chimeric proteins.
Within this region SaDHFR and the type S 1 DHFR differ by 4 amino acids and the
corresponding genes by 18 nucleotides. Consequently, to eventually increase the expression
level of the type S1 DHFR we mutated the coding region present in plasmid pS1(Is) by
replacing as many of the 18 nucleotides as possible by nucleotides occurring in the gene for
SaDHFR. In fact, E. coli cells containing the resulting plasmid pS1(RNA) produce type Sl
DHFR to the same high level, which is achieved for SaDHFR.

Table 1. Solubility muteins of the type S 1 DHFR.

DHFR Expression Solubility 2 Activity 3 ICso

mutein Ievell (U/ml) forTmp
(mg/1) (J.!M)

Type Sl DHFR 6,60 1 0.02 16.0

Sl DHFR[Q17E] 6 15 n. d. n. d.
S1 DHFR[Q33K] 6 1 n. d. n.d.
S1 DHFR[N48E] 6 25 n. d. n.d.
S1 DHFR[Q104K] 6 1 n. d. n. d.
Sl DHFR[N130D] 6 10 n. d. n. d.
Sl DHFR[N48E,Q104K] 6 10 n. d. n. d.
Sl DHFR[N48E,N130D] 6, 60 100 5.8 8.8
Sl DHFR[Q104K,N130D] 6 1 n. d. n. d.
Sl DHFR[N48E,Q104K,N130D] 6 15 n. d. n. d.
SaDHFR 80 100 4.2 0.012

1 Production conditions: 10 ml shaking culture, LB medium, 37'C, 210 rpm, induced at 0060()=0.9 for 4h.
The second value gives the expression level obtained with a coding region for the enzyme optimized for
expression.
2 Relative solubilities (SaDHFR = 100; Type Sl DHFR = 1) after sonication of E. coli cells in SO mM
phosphate buffer, pH7.
3 Enzyme activity in the supernatant of sonicated producing E. coli cells.

Towards Soluble Muteins of Type Sl DHFR

According to in-house hypothetical models built by homology to the E. coli DHFR, 24

of the amino acids which differ between the insoluble type S 1 DHFR and the soluble
SaDHFR are located on the surface of the proteins. SaDHFR contains one lysine, one
aspartate and two glutamate residues more than the resistant enzyme, thus carries two more
negative charges at neutral pH. Consequently, the isoelectric points of the soluble SaDHFR
and the insoluble type S1 DHFR were calculated to be 6.3 and 6.7, respectively.
To eventually obtain the type S 1 DHFR in a soluble form, 9 muteins of the enzyme
have been constructed, in which surface amino acids (at 5 different positions) have been
replaced by the corresponding amino acids of the soluble SaDHFR (Table 1). Muteins S 1
DHFR[Q33K] and S1 DHFR[Q104K], which contain additional positively charged amino
acids, did not show a improved solubility. However, all the muteins with more negatively
charged amino acids than the type S 1 DHFR showed improved solubility compared to the
type S 1 DHFR. The highest solubility was obtained with the mutein S 1
DHFR[N48E,N130D]. Even after high level expression in E. coli using a plasmid with a

543
gene optimized for expression (internal start of translation removed, RNA optimized for
expression) this mutein showed a solubility comparable to SaDHFR (Table 1). The increase
in solubility of S1 DHFR[N48E,N130D] versus the type S1 DHFR is best demonstrated by
comparing the enzymatic activities present in the supernatants of cultures after disruption of
the cells (Table 1). Interestingly, the mutein S1 DHFR[N48E,N130D] exhibited a similar
IC50 for Tmp as type S1 DHFR indicating that the activity of the mutein towards the
substrate is not affected by the mutations.

Purification of Sl DHFR[N48E,N130D]

The soluble Tmpr mutein S1 DHFR [N48E,N130D] was produced in a 100 1

fermenter. E. coli cells present in 7 1 of the culture were harvested by centrifugation and
disrupted with the French press before the resulting suspension was centrifuged. Most of the
recombinant protein was in the supernatant, which was passed over DEAE-Sepharose Fast
Flow to remove DNA. The recombinant protein did not bind to the ion exchange column,
therefore, the flowthrough was passed over a Blue-Sepharose column, to which the mutein
bound. From this column the recombinant protein was eluted with 1 M KCl I 2 mM folate.
Finally, the recombinant protein was subjected to a chromatography on Superdex 200.
In total 25 mg of S 1 DHFR [N48E,N130D] were purified with a purity of greater than
97 % as judged by SDS-PAGE and a specific activity of 87 units per mg of protein. The
molecular weight of the purified protein was checked by electrospray mass spectrometry.
We measured a molecular weight of 18477 ± 2, which is in good agreement with the
calculated molecular weight of 18478. About 30% of the protein molecules carried aN-
terminal methionine residue. Only a single peak was observed in reversed phase HPLC and
by capillary zone electrophoresis.

SUMMARY

A high level expression in E. coli of the Tmpr type S1 DHFR was achieved by: (1)
elimination of an internal start of translation within the RNA, and (2) optimization of gene
expression by replacing nucleotides at the 5' end of the gene by nucleotides present in the
highly expressible gene for SaDHFR.
In addition, by replacing amino acids supposed to be on the surface of the protein, the
mutein S1 DHFR[N48E,N130D] was constructed, which can be expressed in E. coli to
high levels in a soluble and active form.
The mutein S1 DHFR[N48E,N130D] was purified nearly to homogeneity. The enzyme
is highly active and remains soluble even at a protein concentration of 10 mg/ml.

REFERENCES

1. D.A. Rouch, L.J. Messerotti, L.S.L. Loo, C.A. Jackson, and R.A. Skurray, Trimethoprim resistance
transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and
thymidylate synthetase flanked by three copies of IS257, Molec. Microbiology 3:161 (1989).
2. D. Stiiber, H. Matile, and G. Garotta, System for high level production in E. coli and rapid purification
of recombinant proteins: Application to epitope mapping, preparation of antibodies and structure-
function analysis, in: "Immunological Methods", I. Lefkovits and B. Pemis, eds., Academic Press,
Orlando (1990).
3. M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White. "PCR Protocols: A Guide to Methods and
Applications", Academic Press, San Diego (1990).
4. D.P. Baccanari and S.S. Joyner, Dihydrofolate reductase hysteresis and its effect on inhibitor binding
analyses, Biochemistry 20:1710 (1981).

544
EFFECT OF GENOMIC POSITION ON AMPLIFICATION OF THE DFR1 GENE IN
SACCHAROMYCES CEREVISIAE

B.J. Barclay,1* N.K. Ondrusek,1 Y.D. Wildenhain,1 T. Huang,1

R.L. Carlone, 1 J-M. Clement,2 ** G.M. Wahl2

1Department of Biological Sciences

Brock University, St. Catharines, Ontario
Canada L2S 3A 1

2Gene Expression Laboratory

The Salk Institute for Biological Studies
La Jolla, California 92037 USA

INTRODUCTION

Among the best studied examples of DNA amplification, as a response to drug

selection, is the increased DHFR gene copy number observed in tumor cells resistant to
methotrexate (MTX) 1,2.. In spite of numerous studies into the mechanism of DHFR gene
amplification in a variety of cell types, we do not yet have a detailed understanding of the
molecular events which initiate the amplification process. Moreover, in spite of a
relative wealth of information about the distribution and arrangement of amplicons
observed in MTXR cells, we know very little about endogenous factors which affect the
frequency of DHFR amplification-nor do we understand, at the molecular level, the
process of resolution of early events into the final amplicon structures observed in
drug-resistant cells.
The cumulative results of studies in mammalian systems have shown that DHFR
amplification events can be broadly catagorized into two types: those in which the
amplified genes are located as tandem or inverted repeats (or a combination of both)
arrayed on chromosom•3S and those which have additional DHFR gene copies on extra-
chromosomal elements 1.2. Rather than mutually-exclusive events, these two genotypic
alterations may be causally related2. Analysis of very early events in mammalian cells
has revealed new mechanistic insights into the DNA amplification process3. These
studies have suggested that extra-chromosomal elements may be generated early in the
amplification process by chromosomal fragmentation. After replication and
disproportionate distribution into daughter cells during mitosis, these acentric
structures may provide additional substrates for integration into chromosomes by
homologous recombination3.

Present Address: *Dept. of Genetics, University of Alberta, Edmonton, Alberta,Canada

T6G 2E9 **lnstitut Pasteur, 28 rue de Dr. Roux 75724 Paris, Cedex 15, France.

Chemistry and Biology of Pteridines and Folates, E<hted by

J.E. Ayling et al., Plenum Press, New York, 1993 545
 The smaller genome size and genetic tractability of S.cerevisiae makes this
simple eukaryote an ideal organism for the study of DNA amplification. We supposed that
the development of a DHFR amplification system in yeast would allow for the molecular
dissection of early DNA amplification events and the resolution of intermediates into
final amplicon structures. The availability of a large catalog of yeast mutants which
affect cell cycle progression, DNA replication, repair and recombination would also
allow us to study the effects of these mutations on the amplification process. In this first
study, we examined genomic position effects on DFR1 gene amplification. We took
advantage of the facility with which exogenous DNA could be targeted in yeast by
homologous recombination to defined chromosomal loci. We report here a 2000 fold
variation in the frequency of DFR1 gene amplification events with genomic position.

MATERIALS AND METHODS

Drug Selection
MTXR strains were selected by plating 1-2 x1 o7 stationary phase cells on
complex medium (YEPD) agar plates containing 100 Jlg/ml MTX and 5mg/ml
sulfanilamide (Sigma). Cells were grown on selection plates for 5 days at 3ooc which
produced a confluent lawn and then replica-plated onto fresh drug plates. MTXR colonies
were then scored as discrete colonies after 5-6 days further incubation. ~-gal activity
in drug-resistant isolates was assayed qualitatively on buffered SDZ agar plates
containing 1oo Jlg/ml X-gal4 and quantitatively according to the culture assay method of
Rose and BotsteinS. Reaction mixtures were incubated at 37oc for 25-40 min and
stopped by addition of 1M Na2 C 0 3. Units of activity were defined as
OD42o/ODsoo/min/ml measured in a Beckman model DU7 spectrophotometer.

Transformations
Spheroplasts of strain M1-2B (genotype: Mata., trp1-289, ura3-52) were
transformed by the spheroplast method. The plasmid DNA used for transformations was
linearized by restriction enzyme digestion at a site yielding ends homologous with the
target genomic DNA. Ura+ transformants having the predicted Southern hybridization
pattern and ~-gal activities consistent with a single cassette insertion event were used
for further study (data not shown).

RESULTS
Haploid yeast cells were transformed with a DNA cassette containing the DFR1
geneS and a fusion of the yeast LElJ2 upstream DNA sequence with the lacz coding region
of E. coli. Since the DFR1 gene and the /eu2::/acz fusion were regulated independently we
considered an increase in the activity of both gene products by a single mutational event
to be unlikely. Thus, by using MTX resistance as a selected marker and increased ~-gal
activity as a secondary reporter activity we supposed that DNA amplification events
could be easily distinguished from other genetic alterations, which might have given rise
to a drug-resistant cell.
When strains containing the DNA cassette in one of a variety of different genomic
positions were plated on MTX medium a large variation in the frequency of MTXR
outgrowths was observed (Table1 ). The highest frequency of MTXR clones was seen in
the strain in which the cassette had been integrated at the RDN1 locus. Other genomic
locations which increased the frequency of drug resistance were: proximity to a telomere
or integration into an actively transcribed region (Table1 ). When drug-resistant
outgrowths were grown in culture and assayed for ~-gal activity 80-90% had eiiOlvated
enzyme levels compared to the host (Fig.1 ). This suggests that DNA amplification is a
relatively common mechanism of MTX-resistance.

546
 Table 1 Effect of genomic position on MrxR frequency

Locus Genomic Position? MTXRtcell Plated

DFR1 CHM XV R host 1.0 x 1o-s
DFR1 CHM XV R (1.8) 2.1 x 1o-7
SUC2. CHM IX L tel (7.5) 2.0 x 1o-5
HIS1 CHM V R (5.4) 2.2 x 1o-7
RDN1 CHM XII R rDNA (6.4) 5.2 X 10-4
URA3 CHM V L can (1 .1) 2.5 x 1o-5
LEI.J2 CHM Ill L cen (2.3) 2.7 X 10-7
HIS3 CHM XV R (6.1) 2.1 x 1o-7
HIS3 CHM XV R dis (0) 1.1 x 1o-5
TRP1 CHM IV R cen (7.2) 1.2 x 1o-7
HISS CHM IX L (5.5) 3.3 x 1o-7
HISS CHM IX L dis (0) 1.1 x ws
PGK1 CHM Ill R cen (2.9) 2.2 x 1o-7
The number in parentheses represents the size of flanking repeat DNA in Kbp after integration
of the cassette. Conditions for selection of MTXR clones are described in "Methods".
Chromosomal context elements close to the site of integration are: can= centromere.
tel=telomere. dis= gene disruption.

15

;;...
....
>
.....
....
10
u
<
..J
<
0
....<
5
w
=
0
SUC2 HIS! RDN1 URA3
GENOMIC POSITION

Figure1_ Effect of genomic position on ~-gal activity of MTXR clones. The target locus
for cassette integration for each strain is indicated. ~ -gal activities were determined as
described in "Methods". Each bar represents an independent isolate.

547
 20

15
;><
u
7-
W;l
;:::> 10
c;
W;l
Qii
~
5

0
2 3 4 5 6 7
BETA-GAL ACTIVITY

Figure2. Frequency distribution of ~-gal activity of MrxR clones at the URA3 locus. MTXR
colonies were selected a random and assayed for ~ -gal activity as described in "Methods".

40

><
....
..... 30
>
.....
....
u
<
...l 20
<
"....<
W;l
;:!:~
10

100 200 300 400 500 600

MTX CONCENTRATION (ug/ml)

Figure3. Effect of increased MTX concentration on ~-gal activity at the RDN1 locus. MTXR
colonies were selected on agar plates as described in "Methods". Colonies were
replica-plated onto medium containing 5mg/ml sulfanilamide and step-wise increases in MTX
concentration. 20 colonies were selected at random from each series of drug plates and
assayed for ~-gal activity as described in "Methods". The data represents the mean ~ - gal
activity for each series.

548
 Ml/28 Ml3 5 4

DFRJ_,
E 4.5 E 3.2 E
genomic

E 10.9 E 1.9 E 2.4 E cassette

s E B
probe
F1gure 4. Southern hybnd1zat1on analys1s of MTXR clones at the RDN1 locus
M1-2b=host strain, M13= cassette mtegrated at RDN1 , samples 4 and 5= MTXR clones havmg
mcreased ~-gal actiVIties of 13 8 and 12 6 fold, respectively Genomic DNA was extracted
from cultured cells and digested w1th EcoR1 2 5 ug DNA was loaded 1n each lane and probed
w1th a 1 8 kbp Sa/1-BamH1 genomic fragment conta1mng the DFR1 cod1ng reg1onB Fragment
s1zes are g1ven for cassette DNA sequence

The frequency distribution of ~-gal activities of MTXR clones at the URA3 locus is
shown in F1g.2. The mean increase in enzyme activity was about 3 fold. Similar results
were found for several other genomic locations (data not shown). When cells from
several of these MTXR clones were growth in stepwise increases in drug concentration
the mean ~ -gal activity of the population increased about 6-fold (data not shown). In
contrast, cells contaming the cassette at RDN1, which were subjected to the same drug
regimen, showed an mcrease in enzymatic activity of about 30 fold (Fig.3}. Southern
hybridization analysis showed that the cassette DNA was amplified in these strains
(F1gA). However the gene copy number as determined by Southerns was consistently
lower (1.5-6 fold} than that calculated on the basis of ~-gal activity. This may indicate
that the enzyme assay method is a very sensitive indicator of DNA amplification events
or that newly amplified reg1ons have increased levels of gene expression.

549
DISCUSSION

In this study we have amplified the DFR1 gene at at several genomic locations in
S. cerevisiae. The frequency of MTXR outgrowths varied over a 2000 fold range at the
different chromosomal loci. Similar chromosomal position effects have been observed in
animal cells8. The RDN1 locus gave the highest frequency of drug resistance. This
genomic location is a special case as it contains a large number of repeated DNA
sequences (ca. 140}. Our results are in agreement with reports published previously
for other genes integrated at the RDN1 locus9.
Although there is considerable variation in MTXR frequencies at other genomic
locations (Table 1), at the present time we cannot ascribe these differences with any
certainty to any particular chromosomal structure. Further more detailed study is
required to determine whether these or other DNA context elements, such as retroposon
(Ty) sequences or DNA replication origins influence the amplification process. There is
a hint from this data that differences in amplification frequency may be due, at least in
part, to accessibility of the cassette DNA by the transcription apparatus, regions known
to have higher recombination rates in yeast, or to proximity to a telomere (Table1 ).
The finding that 80-90% of primary drug resistant clones had elevated ~-gal
levels suggests that DNA amplification is a relatively common mechanism for MTX
resistance in yeast. In addition, all primary drug resistant clones examined were highly
unstable, as measured by the loss of ~-gal activity in the absence of selection. This may
suggest that nascent DNA amplification structures are inherently unstable ( as would be
expected if chromosome fragmentation was an early step in the amplification process3)
or that increased DHFR activity in amplified strains destabilizes the genome. A recent
report suggests that the latter is the case in MTXR Chinese hamster V79 cells 1o.
If this genetic instability observed in MTXR yeast and Chinese hamster cells is
also true of human tumor cells, these findings may have important clinical
considerations. It will imply that MTXR cells not only represent a clinical problem
because of the outgrowth of drug-resistant cells but also because the genomic instability
of drug-treated cells may generate new variants at higher rates. Thus, the inherent
instability of MTX-resistant cells may contribute to more rapid tumor progression and
the generation of cells with increased metastatic potential. The series of strains
described here will provide a useful model system, in a simple eukaryote, for the study
of the molecular mechanism of DFR1 gene amplification and the effect of these events on
the stability of the genome.

REFERENCES

1. G.R. Stark, M. Debatisse, E. Giulotto and G.M. Wahl, Cell 57:901 (1989}.
2. G.M. Wahl, Cancer Res. 49:1333 (1989}.
3. B.E.Windle and G.M. Wahl, Mutat. Res.
4. M.J. Casadaban, A. Martinez-Arias, S.K. Shapira, and J. Chou, Methods in Enzymol.
100:293 (1983).
5. M. Rose, and D.Botstein, Methods in Enzymol. 101:167 (1983).
6. B.J. Barclay, T.Huang, M.G. Nagel, V.L. Misener, J.C. Game, and G.M. Wahl, Gene
63:171 (1988).
7. R.K. Mortimer, C.R. Contopoulou and J.S. King, Yeast 8:817 (1992}.
8. G.M. Wahl, R. de Saint-Vincent and M. DeRose, Nature 307:516 (1984).
9. J. W. Szostak and R. Wu, Nature 284:426 (1980).
10. M. Roy, S. Sengupta, R. Ghosh, N.P. Bhattacharyya, S.K. Dey and S.B.Bhattacharjee
Mutat.Res. 291:43 (1992).

550
DIHYDROFOLATE REDUCTASE IS NOT THE TARGET OF TRIMETHOPRIM IN
SACCHAROMYCES CEREVISIAE

B.J. Barclay1,2, M.G. Nagel, T. Huang1

Dept. of Biological Sciences

Brock University
St. Catharines, Ontario
Canada l2S 3A 1

INTRODUCTION

Unlike wild-type strains of S. cerevJSJae which are resistant to growth

inhibition by trimethoprim (TRM), two mutants defective in error-prone DNA repair
are sensitive to the drug 1,2,3. Since first reported almost twenty years ago, the
sensitivity of rad6 and rad18 mutants to TRM has been puzzling, in the absence of any
apparent connection between folate metabolism and gene products involved in DNA
repair. A role for a reduced folate in light-dependent DNA repair was first suggested
when 5,1 0-methenyltetrahydrofolate (MTHF) was found to be associated with purified
photolyases4,5 from E.coli and S.cerevisiae. In vitro studies, designed to define the
function of the folate chromophore in photoreactivation, have led to the suggestion that
MTHF acts as a light-gathering "antenna" of the enzyme6. Subsequently, it was found
that yeast cells depleted of reduced folates are defective in both light and dark DNA repair
in vivo? ,8. This has suggested a more general involvement of tetrahydrofolates in the
repair response of yeast cells to the DNA damage induced by UV light.
As TRM is a well-characterized inhibitor of dihydrofolate reductase (DHFR), we
supposed that the drug sensitivity of the two rad mutants was causally related to
inhibition of this enzyme activity. In order to test this, we compared the effects of the
drug and those of another well-characterized DHFR inhibitor; methotrexate (MTX), in
RAD+, rad6 and rad18 cells, containing additional copies of the DFR1 gene. We examined
the effects of the two drugs on growth inhibition, loss of viability and the induction of
cytoplasmic petites9, 1o, 11. We report here that yeast cells treated with TRM respond
differently than those exposed to MTX, with respect to all of these parameters.
Moreover, transformants containing additional copies of DFR1 are resistant to MTX but
remain TRM-sensitive.
We conclude from these results that the catalytic activity of DHFR is not the
target of TRM in S. cerevisiae.

Present Address: 1Department of Genetics, University of Alberta, Edmonton, Alberta,

Canada T6G 2E9 and 2 Molecular Oncology Program, Cross Cancer Institute, 11560
University Ave., Edmonton, Alberta, Canada T6G 1Z2

Chemistry and Bwlogy of Pteridines and Folates, EdJ.ted by

J.E. Ayling et al., Plenum Press, New York, 1993 551
RESULTS AND DISCUSSION

rad6 and rad18 strains, cultured in complex medium containing MTX, show dose-
dependent inhibition of cell growth, with the same kinetics and at similar drug
concentrations as wild-type cells (Fig.1). Thus, the TRM sensitivity of the DNA repair
mutants does not appear to be due to an impairment in folate biosynthesis or to a
decrease in the level of DFR1 gene expression. Transformants harboring additional
copies (8-1 0) of the DFR1 gene, are significantly more MTX-resistant than cells
containing a single copy of the gene (Fig.1). Similar results obtained for MTX-treated
cells in defined medium have been published previously?.
The results of parallel experiments using TRM as the inhibitor are in contrast to
those obtained with MTX. Firstly, the rad mutants have different growth inhibition
kinetics than wild-type, and are more sensitive to low concentrations of the drug
(Fig.2). Secondly, there is no detectable effect of increased DFR1 gene copy number on
the TAM-sensitivity of any of these strains (Fig.2). lastly, unlike MTX, TRM caused
significant lethality in medium lacking all one-carbon metabolites12 ( Table1).

Table 1 loss of Viability and Induction of Petites by TRM and MTX

Survival (%) Petites C%\

Strain Control MTX TAM Control MTX TAM

AAO+ 97.2 1.3 70.4 1.4 86.8 1. 1

+DFR1 98.6 56.1 83.4 1.2 90.1 0.8

rad6 87.0 0.9 0.2 NO NO NO

+DFR1 87.9 11.0 0.3 NO NO NO

rad18 87.6 3.3 7.9 1.2 72.4 1.0

+DFR1 87.1 12.8 7.5 1 01 68.8 1.3

Cells were incubated for 20h in medium containing 500 J.Lglml MTX or TRM as described in the
legend to Fig.1. Viability was determined by colony forming ability on YEPD agar plates. After 3-4 days
incubation at 30°C, colonies were overlaid with 2,3,5-triphenyltetrazolium chloride9 and scored for
respiratory-activity. ND = none detected. +DFR1 = transformant containing multi-copy plasmid.

The two drugs also affect the respiratory competence of treated yeast cells quite
differently. Anti-folate drugs induce respiratory-deficient mutants (petites) in yeast
by two mechanisms: by depletion of intracellular pools of 10-formyltetrahydrofolate,
required for the initiation of mitochondrial protein synthesis 10 , and by thymidylate
stress-induced mutagenesis of the mitochondrial genome 11. In both RAO+ and rad1 8
strains, MTX induced petites at high frequency, as expected (Table1). In contrast, TAM
had no detectable effect on the frequency of respiratory-deficient cells, in the
population.

552
 100
Rad + rad 6 rad 18
,. !'i
6

X 75
1- fj

;;: 6
0 6
a:
(!)
w 6
> 50
i=
<(
.....
w
a:

25

--~

0
0 2 3 4 0 2 3 4 5 0
[Methotrexate] (f.Lg/ml x 1o-l)

Figure 1. Transformants containing additional copies of DFR1 are MTX-resistant. Exponential

cells were grown for 20h in complex (YEPD) medium containing various concentrations of MTX.
Cell numbers were determined in a model Z81 Coulter counter. The genotypes of haploid yeast
strains used in this study are: AH2: Mata /eu2-3, 112, his4 (kindly obtained from Dr.James
Freisen), LP2730-1 a: MA Ta, /eu2-3, 112, his3-1, trp1-289, ura3-52, rad6-1 and LP2729-
4b: MATa, /eu2-3,112, his3-1, trp1-289, ura3-52, rad18-2 (kindly obtained from Dr. Louise
Prakash). The plasmid used for transformations was constructed by cloning of an 8.8kbp Bam
H1 genomic fragment containing the DFR1 coding region? into the autonomously replicating 2JJ.m
vector, pYF91. Open symbols denote transformants containing the DFR1 plasmid.

100?+----0 --- .......

Rad +
\
\
\
\
X 75
1-
;;:
0
a: \
\
(!) \
\
w
> 50 \
i= \
<(
.....
w \
a: \
\
\
\
25 \
\
\
\
\
\
_o n

0
0 1 2 3 4 5
[Tnmethopnm] (f.Lg/ml x 10-2)

Figure 2. Transformants containing additional copies of DFR1 are TAM-sensitive. Exponential

cells were grown for 20h in synthetic (SO) medium contaming various concentrations of TRM.
Wild-type cells were pre-incubated overnight in saline according to the procedure of Mayer and
Goin2. The genotypes of plasm ids and strains are given in the legend to Fig.1. Open symbols
denote transformants contammg the DFR1 plasmid.

553
 TRM has been used clinically as an effective anti-microbial agent for over three
decades and is well-characterized as a DHFR inhibitor13. However, the results presented
here suggest another target for the drug in S. cerevisiae. Although the precise nature of
any alternate target for the drug is unknown at the present time, the drug-sensitivity of
DNA repair mutants suggests that DNA damage might be implicated. TRM may cause DNA
damage directly, repaired by the error-prone pathway, in wild-type cells. Conversely,
the RAD6 and RAD18 gene products might be complexed with proteins involved in semi-
conservative DNA replication. The TAM-sensitivity of the mutants could be a
consequence of increased affinity for the drug by a folate binding protein, (perhaps a
structural function of DHFR), of the DNA replication complex. A similar explanation has
been proposed for the increased sensitivity of DNA synthesis mutants of T 4
bacteriophage to the DHFR inhibitor pyrimethamine14.
Although the detailed roles of the RAD6 and RAD18 genes are not known, they are
thought to have closely related functions in error-prone DNA replication 15. Mutants
defective in these genes, fail to respond to the mutagenic action of UV-Iight16 and DNA-
damaging chemicals 17. The RAD6 gene product has been identified as a ubiquitin
conjugating enzyme 18 and has been reported to play a role in cell-cycle progression 19.
Why a defect in either of these activities would sensitize yeast cells to TRM is unclear.
A possible connection between folate metabolism and the product of the RAD6 gene
may be via thymidylate synthase as it has been suggested that rad6 mutants fail to
recognize a thymine nucleotide signal in the induction of error-prone repair20.
The identification of an alternate target for TRM, may help to clarify this
potential relationship between folate metabolism and the mutagenic DNA repair pathway.

REFERENCES
1. J.C. Game, J.G. Little and R.H. Haynes, Mutat. Res. 28:175 (1975).
2. C.W. Lawrence and R. Christensen, J. Bacterial. 139:866 (1979).
3. V.W. Mayer and C.J. Gain, Genetics 106:577 (1984).
4. J.L. Johnson, S. Hamm-Aiverez, G. Payne, G.Sancar,and G.B. Rajagopalan, Proc. Nat/. Acad.
Sci. USA 85:2046 (1988).
5. G.B. Sancar Mutat. Res. 236:147 (1990).
6. M.S. Jorns in: "Chemistry and Biology of Pteridines" H-Ch.Curtius, S. Ghisla and N. Blau
eds. Walter de Gruyter Berlin, New York (198g).
7. T. Huang, B.J. Barclay, T.l. Kalman, R.C. von Borstel, and P.J. Hastings, Gene
121:167 (1992).
8. B.J. Barclay, K.J. Silva, T. Huang, S.M. Rosenberg and P.J. Hastings, (submitted).
9. B.J.Barclay, and J.G. Little, Mol. Gen. Genet. 160: 33 (1978).
10. U. Wintersberger and J. Hirsch, Molec. Gen. Genet. 126:61 (1973).
11. B.J. Barclay, B.A. Kunz, J.G. Little and R.H. Haynes, Can. J. Biochem. 60:172 (1982).
12. B.J. Barclay and J.G. Little, J. Bacterial. 132:1036 (1977).
13. G.H. Hitchings, ed., "Inhibition of Folate Metabolism in Chemotherapy" Springer-Verlag, New
York. (1983).
14. P.M. MacDonald and D.H. Hall, Genetics 107:343 (1984).
15. C. Cassier-Chauvat and F. Fabre, Mutat. Res. 254:247 (1991 ).
16. C.W. Lawrence, J.W. Stewart, F. Sherman and F.L.X. Thomas, Genetics 64:S36 (1970).
17. L. Prakash Genetics 78:1101 (1974).
18. S. Jentsch, J.P. McGrath and A. Varshafsky, Nature 329:131 (1987).
19. K.S. Ellison, T. Gwozd, J.A. Pendergast, M.C.Paterson and M.J. Ellison, J. Bioi. Chern.
266:24116 (1991 ).
20. B.J. Barclay and J.G. Little, Mol. Gen. Genet. 181:279 (1981).

554
POINT MUTATIONS IN THE DIHYDROPTEROATE SYNTHASE GENE
CAUSING SULFONAMIDE RESISTANCE

G6te Swedberg, Christian Fermer, and Ola Sk6ld

Department of Biological Sciences, Division of Microbiology,

Biomedical centre, Uppsala University, Box 581, S-751 23
Uppsala, Sweden

INTRODUCTION

Sulfonamides act by competing with p-aminobenzoic acid (PABA) in

the formation of dihydropteroate by the enzyme dihydropteroate synthasel.
In many bacteria, as well as protozooans and fungi, this inhibition leads to a
block in the synthesis of dihydrofolate, and eventually to a stop in DNA
synthesis due to lack of dTTP.
Plasmid-borne sulfonamide resistance in gram-negative bacteria is
caused by the production of drug-resistant variations of the enzyme
dihydropteroate synthase.6 These plasmid-mediated enzymes have a
remarkable capacity to distinguish between substrate and inhibitor. When
compared to nucleotide sequences of chromosomal genes coding for
dihydropteroate synthase from different bacteria, the plasmid-mediated
enzymes show a large number of amino acid differences and it is difficult
from a direct comparison to deduce what amino acid differences are
important for the resistance phenotype.
In order to study the importance of amino acid changes and plan for
site-directed mutagenesis, the nucleotide sequences of the dihydropteroate
synthase gene from a laboratory selected sulfonamide resistant E. coli strain,
as well as from clinical isolates of Streptococcus pyogenes and Neisseria
meningitidis were determined and compared. The comparisons point to a
limted number of amino acid changes that might be important for
resistance.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 555
RESULTS AND DISCUSSION
Ooning and sequencing of the dihydropteroate synthase gene from a
sulfonamide resistant and thermosensitive strain of Escherichia coli

The sulfonamide resistant mutant C-167ts2Q4 was used as a source of

DNA. The chromosomal DNA was partially digested with Sau3A and
ligated to BamHI-digested pBR322. Several colonies were selected on plates
containing ampicillin and sulfathiazole, and 20 of these were cultivated to
obtain pure clones. However, only one clone gave substantial levels of a
drug-resistant enzyme activity. This clone, designated C1, was subcloned,
using its Pstl cleavage sites and the dhps gene was localized by comparison
with the previously sequenced dhps genes. One Psti site was found to be
congruent with a Psti site in the plasmid-encoded gene sul!.S A partial
amino acid sequence from the amino terminal end has been published
recently7 and was used to verify the established nucleotide sequence. The
sequence obtained from C-167ts20 was used to design oligonucleotide
primers for PCR amplification of the dhps gene from the wild type strain C-
167, and from an E.coli K-12 strain. A comparison of all sequences is shown
in Fig 1. Only a single nucleotide was changed in comparison with the wild
type strain C-167. This change alters Phe-28 in the wild type to anIle residue
in the mutant. The determjnation of thP- E.coli K-12 sequence showed only
one nucleotide difference when compared with the E.coli C sequence. This
alteration did not alter the amino acid sequence.

M K L F A Q G T S L D L S H P H
(a) ATG AAA CTT TTT GCC CAG GGT ACT TCA CTG GAC CTT AGC CAT CCT CAC
(b)
(c) --- --- --c --- --- --- --- --- --- --- --- --- --- --- --- ---
V M G I L N V T P D S I S D G G
(a) GTA ATG GGG ATC CTC AAC GTC ACG CCT GAT TCC ATT TCG GAT GGT GGC
(b) --- --- --- --- --- --- --- --- --- --- --- T-- --- --- --- ---
(c) --- --- --- --- --- --- --- --- --- --- --- T-- --- --- --- ---

Figure 1. The first 96 nucleotides of the tlhps coding sequence from the E. coli strains C-167ts20
(a), C-167 (b), and K12 (c). Identical nucleotides are marked with hyphens(-).

Ooning and sequencing of the dihydropteroate synthase gene from a

sulfonamide resistant strain of Streptococcus pyogenes

The resistance determinant from a resistant strain of S. pyogenes

(G56) was cloned by partial digestion of streptococcal DNA with Sau3A and
cloning the resulting fragments in the plasmid vector pUC9 digested with
BamHI. The resulting mixture of recombinant plasmids was used to
transform the E. coli strain JM83 to ampicillin and sulfathiazole resistance.
After subcloning, a 2.5 kb Hindiii-BamHI fragment was obtained. The
nucleotide sequence of the dhps gene was determined and was used to
design oligonucleotide primers for PCR amplification of the corresponding
sequence from other strains of S. pyogenes, both resistant and sensitive. The
complete nucleotide sequence from the sensitive strain G1 and from the

556
resistant strain G72 were determined and a comparison of the deduced
amino acid sequences are shown in Fig 2. The number of differences
between the three strains is remarkable but not evenly distributed. In the 5'
part Gl and G72 are almost identical, while G56 differs in many positions.
From position 38, G56 and G72 are almost identical and differ from Gl.
Towards the 3' end the three sequences again becomes more similar with
still some but markedly less differences.

G56 MKIGKFVIDGNAAIMGILNVTPDSFSDGGSYTTVQKVLQQVDQLIAGGAKIIDVGGESTR
Gl .... R.. VE ........................... A.DH. E .M .. D............ .
G72 .... R.. VE.K ......................... A...................... .

G56 PGYQFVSAADEIERVVPMIKAIKAKYDVLISIDTYKTETARAALEAGADILNDVRAGLYD
Gl .. CR .... T ••• D •••• V ••••• EN •• I •••••••••••••••.•••••••••• W•••••
G72 ••••••••••••••••••• I •••••••••••••••••••••••••••••••••• W•••••

G56 GEMLALAAEYDVPIILMHNQKEEVYQDVTQDVCDFLSARAQAAIDAGVPKDNIWIDPGFG
Gl .Q.F ....... A •••••••• D ••••• E ••••••••• GN ..... L •••••• K •••••••••
G72 ........................................................ .....
G56 FPKSVQHNMELLKGLDHVCQLGYPVLFGISRKGVVDALLGGNTKAKERDGATAALSAYAL
Gl .A •••. Q••••••••• R •••••••••.••••• R •.••••••••••.••••.•••••••••
G72 .A •.•••••••••... R ••••.•••••••••. HI ......................... .

G56 GKGCQLVRVHDVKANQEIVAVLSQLM*
Gl ••••• I •••..•.••• D .•.•••.••
G72

Figure 2. Comparison of amino acid sequences for dihydropteroate synthase of the

Streptococcus pyogenes strains G56 (Sui), Gl (SuS) and G72 (Sur). Dots(.) indicate identical
amino acids.

Cloning and sequencing of dhps genes from sulfonamide resistant and

sulfonamide sensitive strains of Neisseria meningitidis

The nucleotide sequences of the dhps genes from the sulfonamide

resistant strains M0035 and 418, as well as from the sensitive strain 1014
have been published3. The strains BT227 and 3976 are two resistant strains
which differ from the previously characterized resistant strains by not
showing hybridization to a probe specific for strain M0035. The nucleotide
sequence of the dhps gene from these two strains showed a much more
limited number of amino acid differences compared to the sensitive strains
than was recorded for M0035 (Fig 3). These two strains give more
information about the minimal requirements for changing from
sulfonamide sensitivity to sulfonamide resistance.

The E. coli ts mutant had a F to I change at pos 28. Also another E. coli
mutant with a changed amino acid at pos 25 has been reported2. Also the
Neisseria meningitidis clinical isolates BT227 and 3976 has amino acid
changes at the corresponding position. However, the resistant isolates
M0035 and 418 as well as the S.pyogenes resistant strains has the wild type F
at this position. In the isolate 418 the first 130 amino acids are identical to
those found in sensitive isolates, while the following amino acids differ

557
substantially from the sensitive strains and are instead identical to those
found in other resistant strains. Crucial differences should thus be found in
the part of the sequence that is unique for the resistant strains. One
remarkable difference between the sensitive and resistant strains of N.
meningitidis is an SG insertion next to a conserved part of the enzyme.
However, the sulfonamide resistant strain BT227lacks the SG insertion and
there is no obvious difference between the sulfonamide resistant and
sensitive strains of S. pyogenes in this part of the sequence. Around pos 230
another conserved sequence SRK is found in all strains, both sensitive and
resistant. The amino acid following this conserved sequence is in most
sensitive strains an R, whereas many resistant strains has an S instead. In
the S. pyogenes strains, G1 has an R, while the resistant strains G56 has G
and G72 has H, respectively. Of all possibilities, these differences seems to be
most likely to be important for resistance, although this has to be shown by
directed mutagenesis.

M003 5: sur MARHVWQAGRFEIGLDKPKIMGIVN1TPDSFSDGGVYSQNAQTA1AHAEQ11KEGADI1DIGGESTRSGADYVSP 75.

418 :sur -----------------------------------A-------- -----R- ----------------1-------
1014 :sus -----------------------------------A---------- ---R-------- -------- -P--- ---S
BT227: sur ~VGC------------ ------------- -LP-- -A------------ -R----------------- P---- ---

3976 :sur -VGC-------- ------------------1--- -A---- -R------ -R-------------- --- P----- --

M0035: sur EEEWARVEPV1AEVAGWGVPIS1DTRRTVIMEKA1A1GGIDIINDVAA1NDEGAVE11ARQADTGIC1MHMQG1P 150.

418 :sur -----------E--------v------------------v-----------------------------------
1014 :sus ---------- -v--- ---- -v----- ---v----- ------------ --T----1- ---c-------- -- -R---
BT227: sur ------- -s- --------- -v---- -H--V--- ------------- ---T- ---1----c------- --------
3976 :sur ------- -s-- -------- -v---- -H--V---- ------------- --T----1-- --c------- --------

M003 5: sur KTMQINPKYQDWGEVARY1KARSAECIAAGIAPQRII1DPGFGSGFGKP1QHNIA1MRH1PE1MAETGFP11IG 22 5.

418 :sur
1014 :SuS EN----------- ----------A-------------T------ -- -T--------------- ----------
BT227:Sur -N---------------------A-------------T-----C ---T-----T-------------Y-----
397 6 :Sur -N------- --------------A-------------T---- -C ---T- ----T-- -------- ---Y- ----

M003 5: sur VSRKSTIGE1TGEANAAERVHGSVAAA1ASVARGAQIVRVHDVKATADA1KVWEA1GIN1 285.

418 :sur ------------------------------------------------------------
1014 :SuS ----RMV------TD--A-----------A------------------------------
BT227:Sur -----M-------TD--A-G---------A-----K---------------A--------
3976 :sur -----M-------TD--A-G---------A-----K---------------A--------

Figure 3. Comparison of amino acid sequences for dihydropteroate synthases from strains of
Neisseria meningitidis showing resistance or sensitivity to sulfonamides. Hyphens (-)
denotes identical amino acids. Asterisks (*) denotes conserved amino acids.

REFERENCES

1. Brown G. M. 1962. J. Biol. Chern. 237:536-540.

2. Dallas, W.S., J.E. Gowen, P.H. Ray, M.J. Cox, and I.K. Dev. 1992. J. Bacteriol.
174:5961-5970.
3. Radstrom, P., C. Fermer, B-E. Kristiansen, A. Jenkins, 0. Skold and G.
Swedberg. 1992. J. Bacteriol. 174: 6386-6393.
4. Skold, 0. 1976. Antimicrob. Agents Chemother. 9: 49-54.
5. Sundstrom, L., P. R<1dstr6m, G. Swedberg, and 0. Skold. 1988. Mol. Gen.
Genet. 213: 191-201.
6. Swedberg, G., and 0. Skold. 1980. J. Bacteriol. 142: 1-7.
7. Talarico, T. L., I. K. Dev, W. S. Dallas, R. Perone, and P. H. Ray. 1991.
J. Bacteriol. 173: 7029-7032.

558
FREQUENT AMPLIFICATION OF A SHORT CHAIN DEHYDROGENASE
GENE IN METHOTREXATE RESISTANT LEISHMANIA

Barbara Papadopoulou, and Marc Ouellette

Service d'Infectiologie du CHUL and

Departement de Microbiologic
Universite Laval
2705 boul. Laurier
Quebec, Canada G 1V 402

METHOTREXATE RESISTANCE IN LEISHMANIA

Acquired resistance to methotrexate (MTX) is readily induced in the kinetoplastid

protozoan flagellate Leishmania under laboratory conditions. The mechanisms of resistance
have been extensively studied and at least three different events have been associated with the
development of resistance to MTX, in Leishmania!. Coderre et al.2 first showed that
resistance to MTX can be associated with the amplification of a genomic region, the R region,
encoding for the structural bifunctionnal gene for dihydrofolate reductase-thymidylate
synthetase (d~fr-ts)3,4. The second event found to be associated with MTX resistance was
the decreased uptake of the drugS that was attributed to mutations in the high affinity folate
cmTier that also represents the main route for MTX uptake in Leishmania 6,7_ The third event
associated with MTX selection was the amplification of the H region as part of
extrachromosomal circlesL6. Using gene transfection experiments, the H locus associated
MTX-resistance gene has been isolated and characterized. Sequence analysis of the gene
present on the H locus revealed that its predicted amino acid sequence has significant
similarities to a family of short chain dehydrogenases, enzymes that are involved in several
oxido-reduction reactions in a wide range of organisms8,9. We present results to show here
that the H-locus encoded short chain dehydrogenase (LTDH) might be involved in an
alternative pathway for the synthesis of reduced folates and that, although ltdh amplification
represents a novel mechanism for resistance to antifolates, it is a frequent event in Leishmania
cell lines selected for MTX resistance in vitro.

PUTATIVE ROLE OF LTDH IN ANTIFOLATE RESISTANCE

Amplification of the H locus does not correlate with elevated levels of DHFR-TslO,
with decreased uptake of the drug 0 or with increased MTX hydrolysisll. These studies were
done in mutants selected for MTX resistance in a step by step fashion. In L. tarentolae, the
transfected ltdh gene, due to its better expression, gave much higher resistance than when
amplified as part oflarge amplicons8. We have therefore reexamine whether a decrease in the

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 559
uptake of the drug could be observed in ltdh transfectants. Figure 1 confirms that LTDH,
even when properly expressed, is not involved in a reduction of the steady-state accumulation
of radiolabeled MTX.
The similarity ofLTDH with enzymes involved in the modification or degradation of
various molecules, raises the possibility that LTDH might modify MTX to render it inactive.
A MTX hydrolyase activity has been described in Leishmania1 but this activity was not
increased in cell lines where the ltdh gene was amplifiedll. Our analysis ofTLC plates on
which MTX and its putative derivatives were resolved, after contact with partially purified
extracts derived from ltdh transfectants, confirmed that MTX was not grossly, if at all,
modified (unpublished). Decreased polyglutamylation of MTX is a mechanism of resistance
in mammalian cellsl2 but although folates are polyglutamylated in Leishmania 13, MTX is
notll.

~ neo
pNeo40.8

minutes
Figure 1. Steady-state accumulation of radioactive MTX (expressed as pmol MTX/mg protein) versus time
in control cells (neo) and ltdh tran~fectant (pNeo40.8). Experiments were carried essentially as described6,7.
Each time point is the mean of three independent measures.

It is therefore clear that ltdh confers resistance to MTX by a novel mechanism. Our
working hypothesis is that ltdh. once expressed at high levels, is capable of providing
reduced folates to the cells even when the DHFR is blocked8,14. Leishmania were thought
to be auxotrophic for folates but there is now clear evidence that Leishmania is capable of de
novo folate synthesis from biopterinl5. The overproduced LTDH could either replace
DHFR-TS similarly to the plasmid encoded DHFR in ttimethroprim resistant bacteria, or
could be involved in the pathway responsible for the conversion of biopterin to reduced
folates. Interestingly, some enzymes that are pa11 of the bioptetin metabolism belong to the
same short chain dehydrogenase family as LTDH16,17.
If ltdh overexpression is capable to replace DHFR, it should, in principle, confer
resistance to all molecules acting as DHFR inhibitors in Leishmania. Ltdh transfectants were
indeed found to be resistant to ttimetrexate and aminopterin 8. The sensitivity measurements
are usually done in tich medium in which thymidine is present, hence reducing the need for
reduced folates. The ltdh transfectants were also tested in various medium, including
completely defined medium IS, lacking folates and thymidine (Table 1). The less folate or

560
thymidine the medium contains, the lower is the inhibitory concentration for MTX (Table 1).
However, the ltdh transfectant (pNeo40.8) was always highly resistant to MTX even in poor
medium where reduced folates are absolutely needed for the synthesis of thymidylate
precursors (Table 1). Wild type cells are capable to grow for 10-15 generations in fdDMEL,
living on their stored folate pools hut will eventually die. The ltdh transfectants are capable to
grow for more than 60 generations, albeit at much reduced rates than in DMEL (unpublished)
indicating either that LTDH might synthesize, inefficiently, reduced folates from a medium
lacking folates or biopterin, or that the pool of reduced folates is much larger in pNeo40.8.

Table 1. Ltdh confers high level resistance to MTX in rich and poor medium

Medium I Relative MTX resistance2

Tarn pNeo3 Tarn pNeo40.83

SDM79 1 (50 !lM)4 >20

M199 1 (lO!lM) >20
DMEL 1 (5!lM) >20
fdDMEL 1 (0.5!lM) >20

lsDM-79 is a rich medium containing M199 (Gibco), DMEM (Gibco), and fetal calf serumlO.
M199 is Ml99 medium pin:- fetal calf serum. DMEL medium is a defined medium for
Leishnurnia18 and fdDMEL is a DMEL medium deficient in folates.
2The relative MTX resistance values were obtained by dividing the 50% growth inhibition value
of the strain tested by the :-mne value for the wild type cells. A value of 1 indicates that the
strain tested and the wild type have the smne sensitivity for MTX.
3rm·II pNeo. a wild type Leishmania strain transfected with the vector alone; Tm-II pNeo40.8, a
strain o·ansfected with the ti"agment containing the ltdh gene.
4Ec 50 values for MTX in the different medium m·e in pm-enthesis.

In collaboration with Jolivet (Montreal) and Priest (South Carolina) we have measured
the level of oxidized and reduced folates in Leishmania control and transfectant cells.
Preliminary results indicate that the level of dihydrofolate is much lower in ltdh transfectants
treated with MTX than in control cells treated with MTX (unpublished). This observation
would argue for a direct role of LTDH in the fmmation of reduced folates through the DHFR
pathway. Experiments to show that dihydrofolate is the substrate for LTDH have failed so
far, however. In order to better study the biochemistry of LTDH in resistance, we have
statted its overexpression and purification in E. coli cells.

AMPLIFICATION OF LTDH

In order to see whether ltdh amplification was a common mechanism of MTX

resistance in Leishmania, we have selected more than twelve independent mutants for MTX,
or both MTX and arsenite resistance, the latter being also a potent inducer of gene
amplification in Leislunania19. More than 60% of all mutants resistant to MTX amplified

561
their ltdh gene as part of circular or linear amplicons of various size with different
rearrangement points. The amplicons were usually stable when cells were selected with the
drug, but were lost rapidly in revertants. In some cell lines the amplicon appeared early
during the selection process whereas in others the amplified ltdh gene appeared only late.
The amplification of ltdh represents therefore a common mechanism for MTX resistance in
Leishmania. None of our MTX resistant mutants contained an amplified dhfr-ts gene.
We used steady-state accumulation transport studies with radiolabeled MTX to look at
its transport in Leishmania MTX resistant mutants with and without ltdh amplification. The
highly MTX resistant mutants (resistant to 1 mM MTX in SDM-79) all showed a decreased
steady-state accumulation of the drug, but could be separated in two classes. The first one
showed a five-fold decreased accumulation of MTX when compared to wild type. No
accumulation of radio labeled MTX could be measured in the mutants of the second class.
Interestingly, the mutants of the first class had ltdh amplified whereas no gene amplification
could be observed in the mutants that are part of the second transport category. It seems that
ltdh amplification is observed only in mutants where the transport mutations do not abolish
MTX accumulation.
The level of MTX accumulation is commensurate with the level of resistance in the
mutants that showed a five-fold decrease in accumulation. More MTX accumulates in the
mutants selected at the earlier passage at low dmg concentration than in later passages at
higher drug concentration. The mutation responsible for the lack of accumulation in the
mutants without ltdh amplification arises early during the selection process since no detectable
accumulation could be measured even in the mutants that were adapted to low drug
concentration. This explains well why these mutants were adapting much more rapidly to
higher MTX concentration than mutants that had ltdh amplification. We will now try to
isolate the gene(s) responsible for MTX entry and characterize the mutations responsible for
the differential accumulation of MTX in MTX resistant mutants.

ACKNOWLEDGEMENTS

Work in the lab is supported in part by NSERC, MRC (MT-11331) and an

Etablissement de nouveau chercheur from FCAR to MO. This investigation received
financial support from the UNDP/World Bank/WHO Special Programme for Research and
Training in Tropical Diseases. MO is a chercheur boursier junior of the FRSQ.

REFERENCES
1. Ouellette, M. and Papadopoulou, B. (1993). Pru·asitol. Today. in press.
2. Codene, .T.A. eta!.. (19R3). Proc. Nat!. Acad. Sci. USA 80:2132-2136.
3. Grumont, R. eta!., (19R6). Proc. Nat! Acad. Sci. USA 83:5387-5391.
4. Beverley, S., Ellenberger. T.. ru1d Cordingley. .T. (1986). Proc. Nat!. Acad. Sci. USA 83:2584-2588.
5. Dewes, H., O;tergamd, I-LL., ru1d Simp~oon, L. (1986). Mol. Biochem. Pru·asitol. 19:149-161.
6. Ellenberger, T.E .. and Beverley. S.M. (1987) . .T. Bioi. Chern. 262:13501-13506.
7. Kaur K. eta!. (1988) . .T. Bioi. Chern. 263:7020-7028.
8. Papadopoulou, B., Roy, G., and Ouellette M., (1992). EMBO J., 11:3601-3608.
9. Callahan, H.L. and Beverley. S.M. (1992) . .T. Bioi. Chern. 267:24165-24168.
10. White, T.C'.et al. (1988) . .T. Bioi. Chern. 263:16977-16983.
11. Ellenberger, T.E. et al. (1989) . .T. Bioi. Chern. 264:15960-15966.
12. Cowan, K.H. and .Tolivet, .T..T. (1984) . .T. Bioi. Chern. 259:10793-10800.
13. Santi, D.V., Nolan, P., and Shane, B. (1987). Biochem. Biophys. Res. Comm. 146:1089-1092.
14. Ouellette M., & BoN P. (1991). Rei>. Microbiol. 142:737-746.
15. Beck, .T.T., and Ullman, B. (1991). Mol. Biochem. Pmasitol. 49:21-28
16. Ichino~oe, H.et a! (1991). Biochem. Biophys. Res. Comrn. 179:183-189.
17. Pru·k, Y.S.et a!. (1991). Biochem Biophy,. Res. Comm. 175:738-744.
18. Iovanni~oci. D.M., and Ullman, B. (1983) . .T. Pmasitol. 69:633-636.
19. Grondin, K., Papadopoulou, B., and Ouellette, M. (1993). Nucleic Acid Res. in press

562
ENZYME INTERACTIONS INVOLVING T4 PHAGE-CODED THYMIDYLATE
SYNTHASE AND DEOXYCYTIDYLATE HYDROXYMETHYLASE

Christopher K. Mathews, Linda J. Wheeler, Christian Ungermann,

J. Patrick Young, and Nancy B. Ray
Department of Biochemistry and Biophysics
Oregon State University
Corvallis, OR 97331-7305

INTRODUCTION

The enzymatic machinery for DNA replication functions at a high rate and
with extremely high accuracy. Typically, an Escherichia coli cell replicates its 4000-
kilobase pair genome in 40 minutes, using two replication forks, each of which
extends two chains. Thus, the rate of chain growth is about 800 nucleotides per
second at 37o. The four deoxyribonucleoside triphosphates (dNTPs) must be
provided efficiently at these few sites, in order to meet the demand. At the same
time the four dNTPs must be maintained at balanced concentrations. An excess or
deficiency of any one of the four leads to inaccurate DNA replication, thereby
stimulating spontaneous mutagenesis. Evidence suggests that steady-state dNTP
concentrations at replication sites are severalfold higher than intracellular
concentrations, as estimated from dNTP pool measurementsl. Thus, dNTP
concentration gradients must be maintained in the face of enormous rates of dNTP
pool turnover.
Because of these kinetic factors and because most of the dNTPs produced in a
cell are used for DNA replication (as opposed to DNA repair, recombination, or
transposition), it seemed reasonable to ask whether the enzymes of dNTP
biosynthesis interact with each other, and with DNA replication enzymes, to
facilitate synthesis of dNTPs and their movement from sites of production to sites
of utilization. Evidence supporting this concept emerged from experiments on T4
phage-infected E. coli, carried out in G. R. Greenberg's laboratory2,3. T4 encodes
nearly all of its own enzymes for both dNTP synthesis and DNA replication.
Figure 1 summarizes present knowledge of pathways of dNTP synthesis in T4
phage infection. Note that T4 encodes its own thymidylate synthase. A structurally
related enzyme, dCMP hydroxymethylase, catalyzes the synthesis of the modified

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 563
 dCTP
UDP COP
ll
deamona11
rNDP jllrNDP
reducto" ~rtductou

dUDP dUTP dCDP NOPkonalt dCTP

I
NOPiunalt

~CTPaa'}/t.'
/""'"•t)~:~ dC:monalt /dCTPalt·dUTPaao

FH
4 synthetase TdR
reduc~FH2 ~khymodone
'[i.:. 'l;._. . . :;,·lr r·
'/
/k•n-~se

'gl)."" ...~... dA::. + + +

'""' ("" ~ /~~.

r . . .T
:~~~ hm·!~;,

dATP dTTP
""f.... . .... 'T
hm-dCTP dGTP
I I
lJ I
DNA polymerase
I

DNA
l DNA Qlucotyltrontferau&, methyloat

modified DNA

Figure 1. Reactions of dNTP biosynthesis in T4 phage-infected E. coli. Reactions catalyzed by virus-

coded enzymes are identified with heavy arrows, and those catalyzed by preexisting host cell
enzymes are denoted with light arrows.

nucleotide, 5-hm-dCMP, from dCMP, and this accounts for the substitution of 5-
hydroxymethylcytosine for cytosine in phage DNA.
Greenberg and his colleagues devised the tritium release assay, widely used
since then in many laboratories to estimate intracellular flux through thymidylate
synthase. Administration to infected cells of 5[3H]deoxyuridine creates a pool of
labeled dUMP, from which radioactivity is displaced by the activity of dTMP
synthase. The rate of transfer of label to water is related to intracellular dTMP
synthase flux, and simple modifications to the procedure allowed simultaneous
measurement of dCMP hydroxymethylase flux rates. The ratio of synthase to
hydroxymethylase flux rates was found to be 2:1, consistent with the fact that the
AT-rich T4 genome requires thymine nucleotides to be synthesized at twice the
rate of hydroxymethylcytosine nucleotides2. That 2:1 ratio was maintained over a
wide range of physiological conditions3, suggesting either physical or functional
associations between these two enzymes, associations that could help regulate
their activities relative to each other.

THE T4 dNTP SYNTHETASE COMPLEX

Several other lines of evidence, both in the Greenberg laboratory and in ours,
supported the concept of enzyme interactions, and in 1977 we4 reported the
isolation of a multienzyme aggregate containing several phage-coded enzymes of
dNTP synthesis. We have been able to purify this aggregate, which we call the
dNTP synthetase complex, by several hundredfold5. All of the phage-coded
enzymes of dNTP synthesis shown in Figure 1 are present in this complex, plus at

564
least two enzymes of host origin-(deoxy)adenylate kinase and nucleoside
diphosphokinase. The complex has a molecular mass of about 1.5 million, as
shown by gel filtration. The purified complex carries out multi-step reaction
pathways with virtually no lag, and with negligible accumulation of intermediates,
as expected if it is designed as a substrate shuttle. One example of such a coupled
reaction pathway is the five-step sequence shown below.

dCTP ~ dCMP ~dUMP~ dTMP ~ dTDP ~ dTTP

In addition, experiments with mutants support the specificity of the enzyme

interactions involved. For example, cells infected with gene 42 amber mutants,
which synthesize truncated forms of dCMP hydroxymethylase, showed no kinetic
coupling when we analyzed the sequence from dCTP to dTMP6 (note that the
hydroxymethylase reaction per se is not involved in this sequence).
However, the enzyme purification approach has limitations for study of the
structure, biological functions, and regulation of this complex. Recoveries of
individual enzymes are variable, and overall recovery of the complex is low. In
addition, the complex as purified contains no detectable DNA replication proteins.
Yet there is reason to think of this complex as a delivery system shuttling dNTPs to
replication sites, as suggested below.

host cell DNR

synthesis

regulatory
dNTPs

Figure 2. A speculative view of the relationship between dNTP synthesis and DNA replication in
T4 phage-infected E. coli. The dNTP synthetase complex is represented as a "funnel", taking raw
materials from ribonucleotide reduction (the predominant pathway) or from host cell DNA
breakdown. Phage proteins functioning at the replication fork include the products of genes 43
(DNA polymerase); 44, 45, and 62 (processivity-enhancing proteins); 32 (single-strand DNA-
binding protein); and 41 and 61 (helicase-primase complex).

Accordingly, we have turned to different approaches for identifying

individual protein-protein interactions within the dNTP synthetase complex and
for asking whether the complex is associated with proteins of the DNA replication
apparatus. We describe three such approaches here, all of which require the
availability of the relevant T4 genes cloned into expression vectors, and the gene
products purified to homogeneity from E. coli strains carrying these recombinant

565
plasmids. Most of the enzymes that we have detected in the T4 dNTP synthetase
complex are now available as the products of cloned genes.

AFFINITY CHROMATOGRAPHY WITH IMMOBILIZED PROTEIN LIGANDS

The first approach involves immobilization of a purified enzyme on a column

of Affi-Gel (BioRad) and analysis of phage or host proteins that bind specifically to
the column. Typically a column is prepared with 5 mg of protein affinity ligand. A
[35S]methionine-labeled protein extract is passed through the column, which is
then eluted with stepwise increases in salt concentration. Control columns with
immobilized bovine serum albumin allow identification of nonspecifically bound
proteins. Although this approach was first carried out in our laboratory with
dCMP hydroxymethylase as the affinity ligand7, the experiment of Figure 3
depicts results with immobilized dCTPase-dUTPase, th8 product of gene 56.

0
One dimensional analysis of radioactive proteins
(35S] methioKne labeled T 4 proteins 1E£2!!. proteins

E.coll
FT Q2 06 2.0
loaded on the aHinity column
circulated overnight al 0.1 mVmin

0. 2 Msa~sallcut
43

b} tJ
I I
211

18

Figure 3. SDS-P AGE analysts of T4 or E col! protems eluted from tmmobihzed T4 dCTPase-
dUTPase. Protems, [35S]methwnme-labelcd from 3 to 8 mmutes after mfectwn or mock mfectwn,
wpre eluted at the mdicated NaCI concentratiOns, with each fraction analyzed by electrophoresis
and radwautography FT, flow-through fractions

Note that of the tightly bound proteins (0.6 M and 2.0 M salt eluates) far more
phage proteins than E. coli proteins are bound, and that the bound proteins are
distinct subsets of the total populations of labeled proteins. Specifically bound
proteins can be identified by two-dimensional gel electrophoresis. Figure 4 shows
a radioautogram of a 20 gel display of total labeled T4 proteins. Most of the spot
identifications derive from analysis either of cells infected with T4 mutants missing
known gene products or of purified proteins.
To date we have analyzed proteins bound to five different enzymes of the T4
dNTP synthetase complex- dCMP hydroxymethylase (gp42, the product of gene
42), dTMP synthase (gptd), dCTPase-dUTPase (gp56), dCMP deaminase (gpcd),
and E. coli nucleoside diphosphokinase. Analysis of T4 deoxyribonudeoside
monophosphokinase (gpl) is under way. Table 1 summarizes our Identifications of
proteins bound specifically to each ligand. Note that each column retains between

566
Table 1. Association of T4 proteins with immobilized enzymes (0.6 M salt eluates)
Gene Gene product Retained by Immobilized

dCMP dTMP dCTPase- dCMP E. coli

HMase synthase dUTPase dcaminase NDPkinase
42 dCMP + +
HMase

td dTMP + + + +
synthase

frd DHF + + + + +
reductase

56 dCTPase- +
dUTPase

nrdA rNDP reduc- + +

tase Rl

nrdB rNDPreduc- +
tase R2

32 ssDNA-bind- + + + + +
ing protein

44 polymerase
+
accessory

45 polymerase + + +
accessory

61 DNA + + +
primase

62 polymerase +
accessory

uvsX repair, re- + + + +

combination

uvsY repair, re- + + +

combination
+

f3gt DNAP-glu- + + +
cosyltrans-
ferase
pseT DNA kinase- + +
phosphatase

regA translation- + + +
a! control

6 and 12 T4 proteins. The bound proteins include both dNTP synthetase complex
enzymes and DNA replication and repair proteins.
Do these data mean that each immobilized enzyme binds each of the dozen or
so proteins bound to the column? Not at all; some of the interactions are

567
 97
,.,,
68
•
43 1
S.1
~t
,.
kDa
t4
. T
29
62.

18

14

Figure 4. Two-dimen~wnal electrophoresis and radioautography of total T4 protems 35S-labeled

from 3 to 8 mmutes after mfechon

undoubtedly secondary, with a retained protein binding to a protein that in turn

binds to an immobilized enzyme By analyzing extracts of cells infected with
particular T4 mutants, we can Identify such indrrect mteractlons For example, in
an extract of cells infected with a dTMP synthase-negative mutant, gp32 (single-
stranded DNA-binding protein) no longer binds to a dCMP hydroxymethylase
column, suggesting that gp32 binds to dTMP synthase, which in turn binds to the
immobilized dCMP hydroxymethylase7 Many such indirect associations are now
being identified.

ANALYSIS OF ANTI-IDIOTYPIC ANTIBODIES

The second of our three approaches mvolves molecular mimicry of protem-

protein interaction domains, usmg anti-idiotypic antibodies. A polyclonal
antiserum against a purified enzyme should contain antibodies against such
interaction domains. Antibodies generated against these antibodies should contain
antigen-combining sites whose surface structure m1m1cs the interaction domain of
the original protein, and hence, which can be used as probes to identify interacting
proteins. In our first application of this approach we developed an antiserum to
purified dCMP hydroxymethylase antlbodies8. This serum precipitated both
dCMP hydroxymethylase itself and T4 dTMP synthase Separate antibodies in this
serum reacted with each protein, rulmg out antigenic cross-reactivity. Thus, the
approach confirms what our protem affmity expenments suggest-that dTMP
synthase and dCMP hydroxymethylase mteract with one another in the cell, just as
was predicted by them vzvo flux rate measurements of Flanegan and Greenberg3.
More recently, this approach has yielded evidence for an interaction between
gp56 (dCTPase-dUTPase) and gpl (dNMP kmase). As shown in Figure 5, a

568
polyclonal antiserum against purified gp56 immunoprecipitated two T4 proteins,
with molecular weights suggesting identities as gp56 (18 kDa) and gpl (27 kDa).
These identifications were confirmed by a dilution experiment; addition to an
immunoprecipitation mixture of excess nonradioactive gp56 caused the 18-kDa
band to disappear, while addition of purified gpl caused the 27-kDa band to
disappear. These results suggest that the rabbit immunized against gp56
developed an anti-idiotypic immune reaction against gpl, supporting the idea that
these two proteins interact in vivo.

2 3 4 5

43

29

18

14

'i) ~
+pure +pure
gp56 gp1

Figure 5. Identification of anti-idiotypic antibodies to T4 gpl in a polyclonal rabbit serum generated

against gp56. An extract of radiolabeled T4 proteins was incubated with serum in the absence
(lanes 2 and 5) or presence (lanes 3 and 4) of excess purified nonradioactive gp56 or gpl, as
indicated. The immune complexes were analyzed by SDS-P AGE and radioautography.

RECONSTITUTION OF SUBCOMPLEXES FROM PURIFIED ENZYMES

A long-range goal of our work is to reconstitute the T4 dNTP synthase

complex from purified enzymes, for detailed understanding of its ability to
facilitate multi-step reaction sequences. As a start toward that goal, we are seeking
evidence for partial complexes, by electrophoresis on nondenaturing agarose gels9.
Figure 6 presents preliminary evidence for such a subassembly, involving enzymes
th;Jt catalyze the sequence dCTP ~ dCMP ~ dUMP (gp56, gpcd, and gp42).
t
hm-dCMP
A mixture of the three enzymes migrated into the gel and was detected with an
antiserum prepared in our laboratory against purified T4 dCMP deaminase
(kindly supplied by Dr. Frank Maley). No such interaction was detected with a
pairwise combination of gpcd and gp56; however, in a separate experiment (not
shown) gpcd + gp42 yielded evidence of interaction. In comparable experiments
with dTMP synthase, we saw evidence for a subcomplex with synthase, dCMP

569
 1 2 3

...

cd + + +
9P56 - + +
gp42 - +
Figure 6. Identification of a gp56-gpcd-gp42 subassembly, by Western blot analysis of purified
protein mixtures, using polyclonal antiserum against purified T4 dCMP deaminase.

deaminase and dCTPase-dUTPase; however, this latter entity migrated as a smear,

so evidence for this interaction is not yet convincing.
From these three approaches we are developing a picture of the direct protein-
protein interactions stabilizing the dNTP synthase complex, as well as some
indirect associations and a route to identify subassemblies of the entire complex.
The interactions noted by affinity chromatography between dNTP synthetic
enzymes and several DNA metabolic enzymes suggest that the speculative model
in Figure 2 may have some validity. We are extending our approaches to other
biological systems. Recently, for example, we identified an association between the
ribonucleotide reductase encoded by vaccinia virus and a single-strand-specific
DNA-binding protein encoded by the virus10. Therefore, these experiments show
promise for identifying protein-protein interactions in deoxyribonucleotide
synthesis in a variety of organisms.

Acknowledgments

We thank colleagues for clones or purified proteins-Drs. Gisela Mosig (gene 56),
Maurice Bessman (gene 1), Masayori Inouye (NDP kinase gene), William
Konigsberg (genes 32 and 43), and Frank Maley (purified dCMP deaminase).
Financial support came from NSF Research Grant No. 9218618.

References

1. C. K. Mathews and N. K. Sinha, Proc. Nat/. Acad. Sci. USA 79:302 (1982).
2. P. K. Tomich, C-S. Chiu, M.G. Wovcha, and G. R. Greenberg,]. Bioi. Chern. 249:7613 (1974).
3. J. B. Flanegan and G. R. Greenberg, f. Bioi. Chern. 252:3019 (1977).
4. G. P. V. Reddy, M. E. Stafford , A. Singh, and C. K. Mathews, Proc. Nat/. Acad. Sci. USA 74:3152
(1977).
5.L. K. Moen, M. L. Howell, G. W. La~ser, and C. K. Mathews, J. Mol. Recogn. 1:48 (1988).
6. C. Thylcn and C. K. Mathews,]. Bwl. Chern. 264:15169 (1 989).
7. L. J. Wheeler, Y. Wang, and C. K. Mathews, f. Bioi. Chern. 267:7664 (1992).
8. J.P. Young and C. K. Mathews, J. Bioi. Chern. 267:10786 (1992).
9. H. J. Hoffmann, S. K. Lyman, C. Lu, M-A. Petit, and H. Echols, Proc. Nat/. Acad. Sci. USA 89:12108
(1992).
10. R. E. Davis and C. K. Mathews, Proc. Nat/. Acad. Sci. USA 90:745.

570
ISOlATION OF CDNAS ENCODING THYMIDYLATE SYNTHASE FROM
SOYBEAN SEEDLINGS AND EXPRESSION OF TilE PROTEIN IN _6. COLI

Minghan Wang\ Shobha Ratnam2, and James H. Freisheim1

Department of Biochemistry and Molecular Biology

1

Medical College of Ohio

Toledo, OH 43699-0008

Department of Biological Chemistry

2

University of Michigan
Ann Arbor, MI 48109

INTRODUCTION

Thymidylate synthase (5,10-methyltetrahydrofolate: dUMP C-methyl-transferase,

EC 2.1.1.45) (TS) catalyzes the conversion of deoxyuridylate to thymidylate. Because
of its important role in the synthesis of DNA precursors, this enzyme has been
extensively studied. The primary structure of thymidylate synthase is highly conserved
as revealed by comparison of amino acid sequences of the avian\ mammalian 2, and
bacteriaP enzymes. Most of these enzymes are homodimers made up of about 35
kDa subunits. However, the enzyme from protozoal parasite, Leishmania major is
contained in a bifunctional polypeptide with both thymidylate synthase and
dihydrofolate reductase (DHFR) activity. Little is known about TS from plants,
although there has been a report of the isolation of a protein complex containing
both TS and DHFR activities 4• Whether plant TS is a bifunctional enzyme with
DHFR or the two enzymes are non-covalently associated can be addressed by eDNA
cloning and sequencing. Isolation and expression of the plant TS gene will facilitate
characterization of the enzyme at the molecular level, providing biochemical evidence
for the study of macromolecule evolution.

RESULTS AND DISCUSSION

A .Agtll eDNA library constructed using polyA+ RNA from soybean seedlings
was screened with antiserum directed against soybean dihydrofolate reductase. One
clone (D2) was isolated and subsequent sequence analysis suggested that it encoded
thymidylate synthase. Further screening of the eDNA library with D 2 as a probe
resulted in the isolation of a full length eDNA. This eDNA was subcloned into
pBluescript II sk (-). A restriction map of the insert was determined by various

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 571
restriction digestions. The fragments produced by digestion with Nsil, Sacl and Kpnl
were subcloned and sequenced. Several primers were synthesized based on the
sequence obtained and further sequencing resulted in the alignment of the DNA
fragments. The total length of the eDNA is 1,099 nucleotides with an open reading
frame encoding a 33 kDa protein. We concluded that the clone encodes soybean
thymidylate synthase because the deduced amino acid sequence was remarkably
similar to TS from a number of organisms. Soybean TS shares 66% and 54%
sequence identity with the human and E. co 7i enzymes respectively. It is also
extremely conserved in the sequence in the folylpolyglutamate and 5'-fluoro-2'-
deoxyuridylate binding sites, when compared with the human, Lactobaci 77 us casei
and E. co 7i enzymes (Figure 1).

Nucleotide Binding Site Folate Binding Site

Soybean MA L P P C HMF A Q P L L T T K K V F WR
Human MA L P P C H A L C Q P L L T T K R V F WK
L. casei MA L P P C H T L Y Q P L L T T KKV P F G
E. co 7i MA L A P C H A F F Q P L VT T KR C H L R
F.gwe 1. Comparison of substrate and co-factor binding sites of thymidylate synthases from different sources.
J:. casei:
Lactobacillus casei.

To obtain reasonable quantities of enzyme for structural and functional studies,

the protein coding region of the eDNA was cloned into pGEX-KG expression vector6
which directs synthesis of protein interested in E. co 7i as a fusion protein with
glutathione s-transferase (GST). High level expression of the GST-TS fusion protein
was observed based on SDS-polyacrylamide gel electrophoresis but the fusion protein
was insoluble. The insoluble protein was dissolved in 1.5% N-lauroyl sarcosine in
PBS buffer and applied to a glutathione-sepharose affinity column7• The high affinity
of the GST portion of the fusion protein for glutathione cross-linked to the column
enables the fusion protein to bind on the column specifically. After washing with PBS
buffer, the fusion protein was eluted with 50 mM Tris-HCl (pH 8.0) containing 5 mM
reduced glutathione. The purified fusion protein was concentrated using microcon-
centrators and then dialyzed against 50 mM Tris-HCl (pH 8.0), 150 mM NaCI. The
use of 1.5% N-lauroyl sarcosine to solubilize the insoluble protein was essential and
did not affect the chromatography on the glutathione-sepharose column. Taking this
advantage, the fusion protein can be purified by a single step chromatography. Since
there is a specific protease cleavage site in the linker region between GST and TS,
digestion of the purified fusion protein with the protease removes GST and generates
TS.

The results presented in this paper show that plant TS is very conservative as
those from other origins. Since little information is available about plant TS, cloning,
sequencing and expression of plant TS would provide a basis for structural and
functional studies of the plant TS. We have purified TS after removing GST by
protease cleavage and presently examining conditions for refolding the enzyme in an
active form.

572
REFERENCES

1. M.Y. Lorenson, G.F. Maley, and F. Maley, The purification and properties of
thymidylate synthase from chick embryo extracts, J. Bi o 7. Chem.
242:3332-3344 (1967).
2. H. Hornishi and D.M. Greenberg, Purification and properties of thymidylate
synthase from calf thymus, Biochem. Biophys. Acta 258:741-752 (1972).
3. M. Friedkin, E.J. Crawford, E. Donovan, and E.J. Pastore, The enzymatic
synthesis of thymidylate. III. The further purification of thymidylate
synthase and its separation from natural fluorescent inhibitors, J. Bio7.
Chem. 237:3811-3814 (1962).
4. B. Bachmann and H. Follmann, Deoxyribonucleotide biosynthesis in green
algae: characterization of thymidylate synthase-dihydrofolate reductase in
Scenedesmus ob7iquus, Arch. Biochem. Biophys. 256:244-252 (1987).
5. A.H. Rosenberg, B.N. Lade, D-S. Chui, S-W. Lin, J.J. Dunn, and F.W. Studier,
Vectors for selective expression of cloned cDNAs by T 7 RNA polymerase,
Gene 56:125-135 (1987).
6. D.B. Smith and K.S. Johnson, Single-step purification of polypeptides
expressed in Escherichia coli as fusions with glutathione S-transferase,
Gene 67:31-40 (1988).
7. K. Liang and J.E. Dixon, Eukaryotic protein expressed in Escherichia co 7i:
An improved thrombin cleavage and purification procedure of fusion
proteins with glutathione S-transferase, Ana 7. Bi ochem. 192:262-267
(1991).

573
USE OF 10-PROPARGYL-5,8-DiDEAZAFOLATE AND DIRECTED
MUTAGENESIS TO PROBE THE CATALYTIC MECHANISM OF
THYMIDYLATE SYNTHASE

H. Trent Spencer,t J. Ernest Villafranca,2 James R. Appleman I

!Department of Molecular Pharmacology

St. Jude Children's Research Hospital
Memphis, TN 38101, U.S.A.
2Agouron Pharmaceuticals, Inc.
3565 Atomics Court
San Diego, Ca 92121, U.S.A.

INTRODUCTION

Thymidylate synthase (TS) catalyzes the conversion of dUMP and CHzH4folate to

dTMP and H 2folate by methyl group and hydride transfer. The enzyme has been well
characterized from many sources including a 2.1 Aresolution X-ray structure of E. coli TS
(TS).l With the exception of TS from protozoans the enzyme consists of two identical
subunits with a dimeric molecular weight of approximately 60,000. Although an active site is
present on both subunits, there is debate as to whether the active sites function
independently, if catalytic activity alternates between subunits, or to what degree of
coordination exists between subunits. It is believed that substrate binding and product
release are ordered with dUMP binding first, initially to a single subunit, followed by
CH2HJolate binding to the same subunit occupied by dUMP.2 Recently, however, it was
suggested that the dUMP binding site is accessible in the TS-CHzHtolate-polyglutamate
binary complex. 3 These results suggest that TS complexed with the polyglutamated form of
CH2l:4folate may represent an intermediate along the TS reaction pathway.
The binding of ligands triggers the transition between free TS (open form) and bound
TS (closed form) resulting in conformational changes that sequesters both ligands from the
solvent.! Most notable changes are observed at the C-terminus. In the absence of ligand the
C-terminus, approximately 4 residues, shows very little constraint. Upon binding of
CH2E4folate or the analogue 10-propargyl-5,8-dideazafolate (PDDF), the C-terminus folds
over the folate binding site and forms a hydrogen bonding network with several atoms of the
bound cofactor.
Although much is known about the structure and chemical reactions catalyzed by TS,
little is known about the details of the kinetic scheme, specifically transient state kinetcs. To
further understand and defme the kinetic scheme we have generated mutant enzymes, C146A
and P261Amber, by oligonucleotide-directed mutagenesis. C146A has the active site
cysteine replaced with alanine which prevents covalent attack by TS on dUMP, resulting in a
catalytically inactive enzyme. However, processes preceding attack by Cysl46, eg.ligand
binding and C-terminus closure over the substrate binding pocket, are not affected.
P261Amber lacks the four C-terminal amino acid residues. Comparisons of transient state
kinetics of this mutant with the wild-type enzyme has allowed us to identify spectral changes
associated with C-terminus movement. The use of PDDF, an analogue of CH 2H4folate,
has also benefitted our understanding of the kinetics of individual steps in catalysis. This

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling eta/., Plenum Press, New York, 1993 575
 PDDF 82 ~ PDDF

TS +dUMP = TS.dUMP
\_.
-
ayv-cllf<§>-~-glu
0 ~a2
C~B
o
TERNARY COMPLEX
B WITH SUBSTRATES
dUMP -~.146
-o{o :_)
H
H2~ ~f· TS
~~l-glu o-
~~B 0 nOB
Bf
o

TERNARY COMPLEX
0 s TS (TS.dUMP.PDDF) AFTERCWSURE
! B OF PROTEIN C-TERMINUS
TERNARY COMPLEX
AFTER ATTACK OF
NUCLEOTIDE BY CYs146

Figure 1: Schematic showing the processes believed to occur at one of the two binding sites
during the interaction of dUMP and the CH2H4folate analogue PDDF with TS.

analogue undergoes reactions similar to the first few catalytic steps. (See figure 1 for a
schematic depicting the binding of PDDF toTS.) Interpretation of the kinetics with this
ligand and the crippled mutants is much simpler than with the wild-type enzyme and the
natural substrates because the reactions do not proceed past ternary complex formation.

MATERIALS AND METHODS

Production of wild-type and mutant enzymes: The E. coli TS gene was mutated by
the method of Kunkel using the Bio Rad MUT-A-GENE M13 in vitro Mutagenesis Kit.4
Wild-type thymidylate synthase (wt TS), C146A, or P261Amber were isolated from E. coli
transformed with a high amplifying expression plasmid containing the thymidylate synthase
or mutated gene. Each enzyme was purified by FPLC on Q-Sepharose (eluted with a 150
mM to 400 mM KCl gradient) and Phenyl Sepharose (eluted with an initial gradient of 1M
(NH4)zS04 to 0.7 M (NH 4)zS04 and a second gradient of 0.7 M (NH4)zS04 to 0 M
(NH4)zS04. Residual dihydrofolate reductase activity was removed by methotrexate affinity
chromatography. Enzyme concentrations were determined using an extinction coefficient for
the dimeric enzyme of 113 mM-1 cm-1 at 280 nm for both wild-type and mutant thymidylate
synthases.
Stopped-Flow Spectroscopy: The progress of PDDF interaction with wt TS,
C146A, or P261Amber was followed by stopped-flow fluorescence spectroscopy as
previously described.5

RESULTS AND DISCUSSION

Stopped flow spectroscopy was employed to determine the kinetics of PDDF binding
to the enzyme.dUMP binary complex. Figure 2A and B show representative time courses
over a short (A) and extended (B) time range for the interaction ofPDDF with wt TS.dUMP,
C146A.dUMP, and P261Amber.dUMP. The data was fitted as previously described5 and
the recorded data and best fit curves are shown.

576
 Figure 2: Time course and best fit curves for the binding of PDDF to wt TS.dUMP,
Cl46A.dUMP, and P261Amber.dUMP. The wt TS time course was fitted to a three
exponential equation, the C146A and P261Amber time courses were fitted to a two
exponential equation. The final concentration of enzyme, dUMP, and PDDF were 1 J.LM,
500 J.!M and 6 J.!M, respectively.

The time course for wt TS is best described by 3 phases. Two rapid processes result
in decreases in fluorescence and a slower process results in an increase in fluorescence. The
time course for the interaction with P261Amber was similar to that of wt TS. The
fluorescence decrease, however, is described by only a single phase and the process
resulting in the fluorescence increase is more rapid compared to wt TS. For C146A the two
phases resulting in fluorescence decreases are observed and have similar rate constants
compared to wt TS but the fluorescence increase phase is not observed. These results are
summarized in Figure 3.
For wt TS and the two mutants the first phase is linearly dependent on the
concentration of PDDF, indicating that this process represents ligand binding. The second
phase, observed only for wt TS and C146A, shows a hyperbolic dependence on PDDF
concentration, indicating a post binding process. Because this second phase is not observed
with P261Amber this phase most likely represents conformational changes dependent on the

Identification of First Three Phases in PDDF Binding

to TS.dUMP from Study of Mutant Enzymes

--
Phase 1
-
Phase 2

--
Phase3 Enzyme
wild type

- -
P261Amber
truncated C·terminus
C146A
TS.dUMP~TS.dUMP.PDDF~ TS.dUMP.PDDF ~TS-dUMP.PDDF
PDDF C-terminus Conformational
. . c 146
binding closure ch anges requmng ys
attack on dUMP

Figure 3: Summary of the phases observed by stopped flow fluorescence monitoring of the
interaction of wt TS, C146A, or P261Amber with PDDF. An arrow indicates that the phase
was observed.

577
C-tenninus. Phase 3 is observed with wt TS and P261Amber, is hyperbolically dependent
on the concentration of PDDF and reaches a maximum of approximately 4 s-1 for wt TS and
approximately 16 s-1 for P261Amber. This phase is not seen for C146A, which indicates it
is due to processes, eg. confonnational changes, dependent on attack of the nucleotide by the
active site thiol.
These results clearly show that the binding processes of an analogue of the folate
cofactor to the binary complex of TS.dUMP is easily distinguished from conformational
changes involving movement of the C-tenninus. These results also show that a process
requiring the active site cysteine can be observed spectrophotometrically. This third process,
which requires attack of the bound nucleotide by the active site thiol, is likely confonnational
changes involved in the collapsing of the active site around the bound ligands.

REFERENCES
1. Perry, K.M., Fauman, E.B., Finer-Moore, J.S., Moatfort, W.R., Maley, G.F., Maley,
F., and Stroud, R.M., 1990, Plastic adaptation toward mutations in proteins:
structural comparison of thymidylate synthase,Proteins: Struct., Funct., Genet. 8,
315-333.
2. Galivan, J.H., Maley, G.F., and Maley, F., 1976, Factors affecting substrate binding in
lactobacillus casei thymidylate synthetase as studied by equilibrium dialysis,
Biochemistry 15, 356-362.
3. Kamb, A., Finer-Moore, J.S., and Stroud, R.M., 1992, Cofactor triggers the
confonnational change in thymidylate synthase: implications for an ordered binding
mechanism, Biochemistry 31, 12876-12884.
4. Kunkel, T.A., Roberts, J.D., Zakour, R.A., 1987, Rapid and efficient site-specific
mutagenesis without phenotypic selection, Methods Enzymol. 154,367-382.
5. Appleman, J.R., and Villafranca, J.E., 1992, in: "Techniques in protein chemistry llf'
(Angelletti, R.H., ed.), Academic Press, Inc. Orlando Florida, 407-416.

578
y-LINKED DIPEPTIDE ANALOGUES OF 2-DESAMIN0-2-METHYL-
N10-PROPARGYL-5,8-DIDEAZAFOLATE AS ANTITUMOUR AGENTS

A.L. Jackman, G.M.F. Bisset, D.I. Jodrell, W. Gibson, R. Kimbell, V.

Bavetsias, A.H. Calvert, K.R. Harrap, T.C. Stephens,* M.N. Smith* and
F.T. Boyle*

Drug Development Section, The Institute of Cancer Research, 15

Cotswold Road, Sutton, Surrey, U.K. - *ZENECA Pharmaceuticals
(ICI), Mereside, Alderley Park, Macclesfield, Cheshire, U.K.

INTRODUCTION

Quinazoline analogues of folic acid have been developed successfully as

thymidylate synthase (TS) inhibitors which possess clinical activity. CB3717 (N 10-
propargyl-5,8-dideazafolic acid), the first of these agents to receive clinical study, was
withdrawn because of nephrotoxicity1• The more water-soluble (and consequently non-
nephrotoxic) analogue (ICI D1694) is currently in Phase II clinical study worldwide.
ICI D1694 is more potent as an in vitro and in vivo antitumour agent than CB3717
because of its cellular pharmacology2•3• ICI D1694 is actively transported into cells via
the reduced-folate/MTX cell membrane carrier (RFC) where it is immediately
polyglutamated by the enzyme, folylpolyglutamate synthetase (FPGS). CB3717 either
uses this carrier poorly or not at all and therefore enters cells slowly. This, together
with poorer substrate activity for FPGS, results in relatively slow formation of
polyglutamates. Polyglutamation of quinazoline TS inhibitors has three important
consequences: it allows accumulation of drug inside the cell, substantially reduces drug
efflux and, because polyglutamates are more potent TS inhibitors than the parent drug,
results in improved TS inhibition.
An alternative class of agents has been identified. Compounds of this type are
not substrates for FPGS but still rely on the RFC for cellular uptake and closely
resemble in structure classical folate-based TS inhibitors. Such compounds should be
active in tumours expressing low levels of FPGS or with an acquired change in FPGS
expression. The lack of prolonged drug-retention through polyglutamation may allow
for greater control over the duration of TS inhibition if given by continuous infusion.
The work from the laboratories of Sirotnak has provided evidence that compounds with
favourable kinetic parameters for the RFC may offer a tumour-selective advantage, at
least in murine experimental tumour models4•
Compounds not subject to "metabolic activation" through polyglutamation need
to be intrinsically potent TS inhibitors, and, for this reason 2-desamino-2-methyl-N10-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 579
propargyl-5,8-dideazafolic acid (ICI 198583; TS Ki = 10nM) was chosen as the model
compound5• We have previously published data demonstrating that methylation of the
C7 of ICI 198583 prevents FPGS substrate activity so this compound itself fulfills the
above criteria for this new class of agene. However we were also interested in pursuing
other structural analogues but with modifications to the glutamate moiety. One
approach is to develop y-linked dipeptide analogues of ICI 198583 diglutamate. This
concept originated from the observation that polyglutamates of ICI 198583 (and other
TS inhibitors) have up to 100-fold improved TS inhibition over the parent compound
(Table 1)5• The single, largest incremental increase in activity (30-fold) results from the
addition of one extra glutamate. This did not translate into increased growth inhibition,
a possible result of the increased anionic charge of the diglutamate, impeding good cell
membrane transport (Table 1). However ICI 198583 diglutamate was transported via
the RFC as demonstrated by the cross-resistance of the L1210:1565 cell line (defective
RFC) to this agent (Table 1).
Described below is the development of y-dipeptide analogues with Ki values for
TS as low as 0.2nM, that enter the cell via the RFC and are active in ICI D1694
resistant L1210 cells (L1210:MB3) that cannot polyglutamate ICI D1694, ICI 198583
and other antifolates.

Table 1. Inhibition of cell growth by ICI 198583 polyglutamates in sensitive and

resistant L1210 cells

L1210 TS L1210IC50 , L1210:1565IC50 , L1210:MB31C50 ,

Ki (nM) (JLM) (JLM) (JLM)

ICI198583 10 0.1 8.2 2.7

diglu 0.35 0.1 4.6 2.8

triglu -0.1 0.3 - -

tetraglu -0.1 >10 - -
ICI 01694 60 0.009 1.1 70
L1210:1565 cell line- defective reduced-folatejMTX transport carrier. L1210:MB3 cell line -defective
polyglutamation of antifolates

METHODS

The synthesis of these compounds is described in an accompanying manuscript

(Bavetsias et al). The measurement of the inhibition of isolated TS and the inhibition
of Ll210 cell growth has been described previousll. The L1210:1565 cell line has a
severely impaired RFC and the L1210:MB3 cellline7, with acquired resistance to ICI
D1694, cannot polyglutamate other quinazoline and pteridine-based antifolates.
The stability of y-dipeptides in male DBA2 mice was measured by injecting
100mg/kg of the TFA salts i.p., removing plasma, liver and kidneys after 1hr and
measuring the levels of the dipeptide and any hydrolysis product (ICI 198583 or D-

580
 Table 2. Inhibition of TS and L1210 cell growth by y-dipeptide analogues of ICI
198583

-L-L-aa TSIC50 L1210 IC50

(nM) (JJM)

-L-glu-L-glutamate NHCH(COOH')CH 2CH 2COOH 2 0.12

-L-glu-L-aspartate NHCH(COOH')CH2 COOH 10 2.4
-L-glu-L-adipate NHCH(COOH')CH2 CH 2 CH 2 COOH 2 0.33
-L-glu-glycine NHCH2COOH' 11 0.11
-L-glu-L-alanine NHCH(COOH')CH3 18 0.56
-L-glu-L-norvaline NHCH(COOH')CH2 CH 2CH 3 16 1.2

-L-glu-L-valine NHCH(COOH')CH(CH 3) 2 20 2.5

-L-glu-L-serine NHCH(COOH')CH20H 22 1.2
-L-glu-L-glutamine NHCH(COOH')CH2 CH 2 CONH 2 16 0.6
-L-glu-L-phenylalanine NHCH(COOH')CH2Ph 18 10

-L-glu-phenylglycine NHCH(COOH')Ph 14 0.6

-L-glu refers to the g lutamate of 1(.;1 198583

glutamate equivalent) by HPLC (manuscript submitted). A 1hr time point was chosen
because of the rapid plasma clearance of ICI 198583 and dipeptide analogues of ICI
198583 diglutamate (t 112f3 -15mins).

RESULTS AND DISCUSSION

-L-L-Dipeptide Analogues of ICI 198583

The terminal glutamate of ICI 198583 diglutamate (ICI 198583-y-L-glu)

was replaced by a number of natural and unnatural amino acids. Only the -L-glu-
glycine analogue had equivalent activity to the -L-glu-L-glu dipeptide in L1210 cells
(despite its 5-fold poorer isolated TS inhibition) but the dipeptides generally had good
activity in both systems. Their Ll210 growth inhibition was prevented by thymidine,
confirming their TS locus.

The In Vivo Stability of Dipeptide Analogues

Representative examples of this class of agent were tested for activity against the
Ll210:MB3 cell line. This resistant line did not demonstrate significant cross-resistance
to these analogues, suggesting that they are not active through polyglutamation (data
not shown). This suggests that a) they are not substrates for FPGS, and, b) they are
stable in vitro and not significantly broken down to ICI 198583, a substrate for FPGS.
However it is well known that an ubiquitous class of hydrolytic enzymes exists in
mammalian tissues, the y-glutamyl hydrolases, which hydrolyse amide bonds of
polyglutamates8• In this manner the terminal amino acid of the y-linked dipeptides
could be removed, releasing the FPGS substrate, ICI 198583. This would defeat the
object of this new class of agent. It was therefore necessary to determine whether these

581
 Table 3. Plasma and liver concentrations of the dipeptides and their hydrolysis
product, ICI 198583, 1hr post-injection of 100mgjkg (5 mice per group)

Plasma concentration, J,£M Liver content, nmolesjg tissue

-L-L-aa
Dipeptide l1c1 198583 Dipeptide 11CI198583

-glu-glutamate 27 .±. 5 14.±.2 56.±. 28 1421 .±. 461

-glu-alanine 19.±.12 -2 .±. 1 135 .±. 108 115.±.28
-glu-phenylalanine 5.±.1 4.±.1 43 .±. 20 48 .±. 10
-glu-glycine 26 .±. 12 4.2 .±. 0.4 28 .±. 12 342 .±. 103

dipeptides were hydrolysed in vivo. HPLC analysis of plasma, liver and kidneys of mice
treated with a number of dipeptides revealed that significant hydrolysis to ICI 198583
occurred. Examples are given in Table 3.
The unsuitability of these y-linked dipeptides as in vivo antitumour agents led
to new chemical synthesis concentrating on the development of dipeptides stable to in
vivo hydrolysis. Two approaches were explored 1) removal of the carboxyl on the a-
carbon of the second amino acid (defined here as a' -COOH) and 2) replacement of the
first and/or second amino acid with an unnatural D-amino acid.

Table 4; Removal of the carboxyl from the a-carbon of the terminal amino acid

0 ~
N-{}-----li~,~~)).,coou
H
HN:Xr-
~ ~ HL
I
II H .•,,/"cooH
~ ~ o L
e.g. -L-glu-L-glu and -L-glu-GABA

Stable L1210TS Inhibition of L1210

in mice IC50 , nM cell growth
(IC50 , J.£M)

-L-glu-L-glu NHCH(COOH')CH2CH 2COOH NO 2 0.1

-L-glu-GABA NHCH2 CH 2CH 2COOH YES 14 0.4

-L-glu-L-ala NHCH(COOH')CH 3 NO 13 0.62

-L-glu-ethylamide NHCH2 CH 3 YES 70 5.2

Data from two pairs of compounds are shown in Table 4 where the o:' -COOH
has been removed to give the corresponding L-glu-y-amide. In both cases removal of
the a'-COOH resulted in a compound stable to in vivo hydrolysis. For example the
L-glu-GABA analogue, when injected into mice gave a plasma and liver concentration
at lhr of 20J,£M and 125nmolesjg respectively with no ICI 198583 being detected. Only
a 5-fold loss in activity (over the -L-glu-L-glu analogue) was observed against isolated
TS. Removal of the a-carbon on the first glutamate e.g. GABA-glu was far more

582
 Table 5. First or second glutamate replacement of ICI 198583-y-diglutamate
with its D-enantiomer

Stable to L1210TS L1210 IC50 , L1210:MB31C 50

hydrolysis in mice IC50 , nM JLM (at 48hrs) L1210 IC50

-L-glu-L-glu NO 2 0.1 23
-0-glu-L-glu NO 36 2.9
-0-glu-0-glu YES 26 1.3
-L-glu-0-glu YES 5 0.22 3
7-CH 3 , 2'F -L-glu-0-glu YES 0.9 0.2 2

deleterious, with a 40-fold loss in TS inhibitory activity. Whereas the L-glu-GABA

retained comparable activity to the parent dipeptide, this was not the case for the L-glu-
ethylamide (Table 4). It would seem that the absence of a COOH group on the
terminal amino acid compromises this activity, possibly by reducing uptake via the RFC.
Replacement of the terminal glutamate by its unnatural D-enantiomer e.g. L-glu-
D-glu also bestowed stability upon these dipeptides (Table 5). The plasma and liver
levels of the L-glu-D-glu at 1hr were 34J.£M and 881nmoles/g respectively, with no ICI
198583 being detected. The reverse configuration i.e. D-glu-L-glu was not resistant to
hydrolysis. The minimal loss of either TS inhibition or L1210 growth inhibition of -L-
glu-D-glu compared with -L-glu-L-glu suggested that this approach was viable.

In vitro activity of -L-glu-y-D-glu dipeptides

In order to enhance TS inhibition, further structural modifications were made

to the -L-glu-D-glu analogue. The 6-fold increased inhibition found with the addition
of a 2'F to the benzene ring and a CH3 to the 7-position of the quinazoline ring of ICI
198583 (data not shown) was also observed when these modifications were made to the
-L-glu-D-glu dipeptide (Table 5). Unfortunately this increased TS inhibition did not
translate into greater L1210 growth inhibition, possibly because the 7-CH3 slightly
interferes with uptake via the RFC (Jansen et al; Personal communication).
Nevertheless the 7-CH3, 2'F analogues of -L-glu-D-glu had an IC50 value of 6.8J.£M (34-
fold cross-resistance) for the Ll210:1565 cell line indicating that the RFC is a major
mechanism for cell entry for this dipeptide.
The activity against the L1210:MB3 cells also suggests that changing the
enantiomeric configuration of the second amino acid prevents polyglutamation as the
Ll210:MB3/Ll210 ratio falls from 23 to 3 with this structural change (Table 5). A
ratio of 2-5 is always observed for compounds that use the RFC due to a small defect
in the RFC in this resistant cell line.
The presence of a 7-CH3 in the dipeptide analogues may have an advantage
other than increasing TS inhibition, that is the prevention of polyglutamation of any
product of hydrolysis that may occur which is below the detection limit of the HPLC
method ( -0.2-0.5J.£M in plasma and 2-5nmoles/g in liver).

In vivo antitumour activity of the 7-CH3, 2'F-L-glu-y-D-glu dipeptide analogue ofiCI

198583

Activity was demonstrated against the L5178Y TKI- (thymidine salvage

583
 Table 6. The in vivo antitumour activity of the 7-CH3, 2'F-L-glu-y-D-glu
analogue

L5178Y TK" 1· (1 day sub- L5178Y TK+ !- (7 day sub- HX62 xenograft (14 day
cutaneous infusion) cutaneous infusion) sub-cutaneous infusion)

4.2 day growth delay at 3.7 day growth delay at 1OOmgjkg 30 day growth delay at
30mgjkg 80mgjkg
5/5 cures at 50mgjkg 11 day growth delay at 150mgjkg
(20% body weight loss)

incompetent) mouse lymphoma, the L5178Y TK+I- (thymidine salvage competent)

tumour and the HX62 human ovarian tumour xenograft (Table 6).

SUMMARY

Our search for water-soluble quinazoline TS inhibitors that are transported into
cells via the RFC, but are not substrates for FPGS, led us to the synthesis of dipeptide
analogues of ICI 198583 diglutamate. Although a number of dipeptide analogues were
active against isolated TS and L1210 cells in vitro, lack of in vivo stability was a
problem. This was circumvented by the synthesis of modified dipeptides where either
the a-carboxyl of the second amino acid was removed (a'-COOH) e.g. -L-glu-GABA
or where the second amino acid was the unnatural D-enantiomer e.g. -L-glu-D-glu.
Further studies were performed with the -L-glu-D-glu and its 7-CH3, 2'F modified
analogue, demonstrating that they use the RFC for cell entry but are not active through
polyglutamate formation. The latter compound was tested against experimental
tumour models and found to have good activity.

REFERENCES

1. S.J. Clarke, A.L. Jackman and I.R. Judson. In: "Novel approaches to selective treatment of human solid
tumours:laboratory and clinical correlations". (Y. Rustum. Ed.) Plenum Press, in press.
2. A.L. Jackman, G.A. Taylor, W. Gibson, R. Kimbell, M. Brown, A.H. Calvert, I.R. Judson and L.R.
Hughes. Cancer Res. 51: 5529-5586, 1991.
3. A.L. Jackman, W. Gibson, M. Brown, R. Kimbell and F.T. Boyle. In: "Novel approaches to selective
treatment of human tumours: laboratory and clinical correlations". (Y. Rustum. Ed.) Plenum Press,
in press.
4. P.M. Sirotnak, and J.I. DeGraw. In: Folate Antagonists as Therapeutic Agents. Vol.2. (P.M. Sirotnak
et a!. eds.) Academic Press Inc. pp 43-95, 1984.
5. A.L. Jackman, D.R. Newell, W. Gibson, D.I. Jodrell, G.A. Taylor, J.A. Bishop, L.R. Hughes and A.H.
Calvert. Biochem. Pharmacol. 42:1885-1895, 1991.
6. A.J. Barker, F.T. Boyle, A.L. Jackman, A.H. Calvert and S.J. Pegg. Proc. Amer. Assoc. Cancer Res. 32:
327, 1991.
7. A.L. Jackman, L.R. Kelland, M. Brown, W. Gibson, R. Kimbell, W. Aherne and I.R. Judson. Proc.
Amer. Assoc. Cancer Res. 33: 406, 1992.
8. J.J. McGuire, and J.K. Coward. In: Folates and Pterins, Vol.l (R. Blakley and S.J. Benkovic, eds.) J.
Wiley and Sons Inc. pp 135-190, 1984

584
SUBSTITUTED-2-DESAMIN0-2-METHYL-QUINAZOLINONES. A SERIES
OF NOVEL ANTITUMOUR AGENTS

F.T. Boylel, Z.S. Matusiak!, L.R. Hughesl, A.M. Slaterl,.

T.C.Stephensl, M.N.Smithl, M.Brown2,R Kimbell2 and A.L. Jackman2

lzENECA Pharmaceuticals, Mereside, Alderley Park, Macclesfield,

Cheshire, UK.
2The Institute of Cancer, Research,Sutton, Surrey, U.K.

INTRODUCTION
ICI D1694, a quinazolinone based inhibitor of thymidylate synthase (TS) was
developed to act in part through efficient metabolism to polyglutamates by the
enzyme folypolyglutamyl synthetase (FPGS)l. The addition ofy-glutamates to agents
such as ICI D1694 has two major consequences- the polyglutamates are up to 100-
fold more active against isolated TS and the increased polyionic character results in
intracellular retention leading to extended TS inhibition even after drug removal. In a
cell line with altered expression levels of FPGS these classical agents have been
shown to be less effective and cytotoxicity then generally correlates with TS
inhibitory activity.(See accompanying paper A.L.Jackman et al .). In our search for
agents to compliment ICI D1694, we therefore wanted compounds which were potent
inhibitors of TS, used the reduced folate I methotrexate carrier (RFC) for cell entry
but did not depend on intracellular polyglutamation for activity. Because of its
excellent TS inhibitory activity we chose 2-desamino-2-methyl-NlO-propargyl-5,8-
dideazafolic acid (ICI 198583)2 as the basis of this investigation.

CONFORMATIONAL ANALYSIS OF SUBSTITUTED QUINAZOLINONES

The starting point for this study was the finding3,4 that in the crystal structures
of E. coli TS ternary complex with FdUMP and CB3717 indicate that CB3717 binds
in a partially folded conformation with the para-aminobenzoic acid ring inclined at
65° to the quinazolinone.Molecular mechanics analysis of CB3717 using SCANOPT5

~(2)

"~~/ 1~'"'~'-·•
following stepwise rotations around the torsion angles defining bonds (1) and (2)
indicate 3 pairs of populations. Conformational pairs are a consequence of 180°

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 585
quinazolinone ring rotations. Minimum energies of the conformations identified by
SCANOPT were calculated using the semi-empirical quantum mechanical energy
programme AMPAC6. Minima for each pair of conformations were very close to
those predicted by molecular mechanics analysis and one of these conformations
closely resembled that observed in the ternary complex . The impact of nuclear and
C9-substitution on this conformation was then assessed.

Scheme 1. Synthesis of Substituted 2-Desamino-2-Methyl-Quinazolinones

Method A R1 R1
(1) I I

======~> Br~CH 2 ====~> NC~H2

NH2~R (2,3) AcNH~R

0 R1
H N~N-oCONH-Giutamate
Me..kN~R ~

NC~GN
Method C

AcNH~R
Conditions

(1) Br2 : AcOH: 10-15°C. (2) Ae;.O: Pyridine: EtoAc: 25°C. (2A) Ac20: 100°C. (3) CuCN : NMP: 120°C.
(3A) CuCN : NMP : 150°C. (3 8 ) CuCN : NMP: 175°C.(4) Hp2 : NaOH :EtCH : Hp.(5) POMCI : KOBu 1 : DMSO.
(6) NBS: CCI 4.(7) CaC03 : DMA: 110°C.(8)TFA: 25°C.(9) Pentafluorophenol: DCCI : DCM: 25°C.
(10) NH-Ligand: Et3 N: DMF: HOST :25°C.(11) 1N NaOH: 25°C.(12) Raney Nickel: AcOH: 50°C.
(13) Propargyl Bromide: CaC03 : DMA: 110°C.

586
 C7-methyl substitution reinforced the binding conformation whereas C5-methyl
favoured a non-binding conformation as its preferred minimum. Analysis of C7,9-
methylated analogues gave mixed results. In the C7 ,9R enantiomer two almost
equivalent low energy conformations were predictive of the optimum TS binding
conformation, however for the C7 ,9S enantiomer neither pair of conformations
matched that found in the ternary complex.

SYNTHESIS
A convergent synthesis (Scheme 1) was developed which allowed the
production of a series of C5, C7 and racemic C9 methyl substituents as well as a
range of sterically differing, electron withdrawing and donating substitutions at C7 of
the quinazolinone nucleus. The general synthesis (Method A)was applicable to most
compounds and modifications in the sequencing (Method B for 7-CN, Method C for
7-Et and CF3,) were applied when required. Method D (for C7,9 substitution) was
necessary due to the steric bulk of the incoming nucleophile reducing the reaction rate
to unacceptable levels

BIOLOGICAL RESULTS
TS inhibition was assessed using partially purified Ll210 TS. In vitro growth
inhibitions were assessed using LU210 cell lines. The impact of structural changes on
uptake into L1210 by the reduced folate/methotrexate carrier (RFC), and ability to
form intracellular polyglutamates were determined using the L1210 mutants 1565
(impaired RFC) and MB3 (impaired antifolate polyglutamation) respectively.
198583 is a moderately potent growth inhibitor, a consequence of good uptake
by the RFC (1565 cross resistance ratio of 62) and its converson to polyglutamates as

Table 1 In vitro activity of Quinazoline T S Inhibitors

Inhibition of Cell Growth(ICsoJ1M)

(Resistance Factors in Parentheses)
Ll210 TS
R Rl (ICsonM) L1210 1565 MB3
H H 50 0.13 8(62) 2.7(21)
7-Me H 28 0.19 8(42) 0.59(3)
5-Me H 3040 0.14 17(121) >100(>588)
H Me 180 0.92 29(32)
7-Me Me 340 1.8 >50(>28) 20(11)
7-Cl H 40 1.0 5(5)
7-Br H 17 2.4 36(15) 7.8(3)
7-Et H 30 2.1 40(19) 6.4(3)
7-CF3 H 116 >20
7-CN H 680 >20
7Me0 H 250 22
ICI D1694 670 0.0088 0.92 >100

587
 evidenced by MB3 ratio of21 (confirmed by HPLC analysis ofH3 ICI 198583). C7-
methyl substitution improved TS inhibition (28 vs SOnM) and equivalent growth
inhibition to ICI 198583 results from good uptake by the RFC despite no conversion
to intracellular polyglutamates. (Detailed kinetics of this compound by Professor R
Moran confirm that it is has no FPGS substrate activity). CS-methyl substitution as
predicted by modelling produced a poor TS inhibitor, however very high MB3 cross
resistance ratios suggest that growth inhibition results entirely from intracellular
polyglutamated species. Racemic C7,9 dimethyl substitution gives a 5 and 10 fold
worse inhibitor of TS and cell growth. Sub-optimal conformation and poor
intracellular conversion to polyglutamates, is the likely reason for the reduced
activity.

Extending the range of C7 substituents in ICI 198583 gave the following results:
C7-Br (TS ICso=17nM), Et (30nM), and C1 (40nM) showed improved TS enzyme
inhibition and CF3 (116nM) OMe (250nM) and CN (680nM) were worse. For this
class of agents relatively poor growth inhibition was observed. As evidenced by
significant cross resistan:ce against 1565 most of the compounds enter cells on the
RFC but the low MB3 cross resistance ratios suggest that the major reason for the
poor cytotoxicities is a failure to form intracellular polyglutamates.

Activity in vivo was assessed in a range of acute and chronic models using
Alzet mini-pump infusion protocols (See accompanying paper Stephens et al.).
Profiling 7-methyl ICI 198583 by bolus protocols gave no activity, however using
Alzet mini pumps to maintain enzyme cover, good activity was seen in mice bearing
the L5178Y TK-/- and TK+/- murine lymphoma at 80 and 300 mg/kg/day
respectively. Statistically significant growth delays were also seen against the HX62
human xenograft with a 14 day infusion at 80 mg/kg/day. In all these studies no
toxicity was observed.

CONCLUSIONS

The consequence of structural changes to ICI 198583, originally targeted to

improved TS inhibition has been also to identify novel compounds unable to form
intracellular polyglutamates but retaining affinity for the RFC. This class of
compounds clearly goes some way to satisfying our biochemical profile for a
complimentary agent to ICI D1694.

REFERENCES

(1) A.L. Jackman, G.A.Taylor, W. Gibson, R. Kimbell, A.H. Calvert, I.R. Judson and
L.R. Hughes. Cancer Research 51: 5529-5536 (1991).

(2) A.L..Jackman, D.R. Newell, W.Gibson, D.I. Jodrell, G.A. Taylor, J.A. Bishop,
L.R. Hughes and A. H. Calvert Biochem. Pharmacol. 42: 1885-1895 ( 1991 ).

(3) D.A. Matthews, J.E. Villafranca, C.A. Jonson, W.W. Smith, K.Welsh and S.Freer.
Mol. JBiol. 214: 937-948 (1990).
(4) W.R. Montfort, K.M. Perry, E.B. Fauman, J.S.Finer-Moore, G.F. Maley, L.
Hardy, F. Maley, and R.M. Stroud. Biochemistry 29: 6964-6977 (1990).

(5) Zeneca Pharmaceuticals Internal Program written by Dr N. Stutchbury.

(6) Quantum Chemistry Program Exchange, Indiana University, Bloomington, In.

588
USE OF MURINE L5178Y LYMPHOMA THYMIDINE KINASE MUTANTS
FOR IN VITRO AND IN VIVO ANTITUMOUR EFFICACY EVALUATION
OF NOVEL THYMIDYLATE SYNTHASE INHIBITORS

TC Stephensl, MN Smithl, SE Waterman I, ML McCloskeyl, AL Jackman2

and Ff Boylel

1ZENECA Pharmaceuticals, Macclesfield, Cheshire, UK

2Jnstitute of Cancer Research, Sutton, Surrey, UK

INTRODUCTION

Antitumour efficacy studies with thymidylate synthase (TS) inhibitors are

complicated in mice by a high level of thymidine in plasma (-lj.I.M) which may be salvaged
by tumour cells and prevent cytotoxicity when the de novo pathway of nucleotide synthesis
is blockedl. Thymidine salvage involves the enzyme thymidine kinase (TK) which
phosphorylates the nucleoside to produce thymidine monophosphate (dTMP) which is then
further phosphorylated to the triphosphate (dTTP) for incorporation into DNA.
To overcome the salvage problem we have used a TK deficient mutant of the murine
L5178Y lymphoma tumour cell line (TK-/-) for both in vitro cytotoxicity and in vivo
anti tumour screening of more than 1500 novel folate-based TS inhibitors. By using a more
intensive dosing regime in mice we have also been able to utilise a salvage competent variant
(TK+/-) of the L5178Y tumour to provide a more stringent efficacy test.
The compounds that have been studied are mostly quinazoline analogues of the TS
cofactor 5,10-methylene tetrahydrofolate. Some, such as ICI D16942 (currently in Phase Il
clinical evaluation), are termed "classical" compounds. They rapidly enter cells using the
reduced folate carrier (RFC) and are retained by polyglutamation leading to prolonged
inhibition of TS and good cytotoxicity after a brief drug exposure. Other compounds,
including dipeptides and 7-methyl substituted glutamates, are termed "non-classical". They
still use the RFC for cellular uptake but do not appear to be retained in cells and prolonged
compound exposure is required to achieve good cytotoxicity.

MATERIALS AND METHODS

Cell lines:

The L5178Y TK+/- cell line used in these studies (3.7.2C) was derived from a
"wild-type" TK+/+ parent by ethyl methanesulphonate mutagenesis and bromo-
deoxyuridine selection by Clive3. The TK-/- line was a spontaneous mutant selected with
trifluoromethylthymidine by Dr M Cross (ZENECA Central Toxicology Laboratory,
Macclesfield.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 589
In vitro cell survival assay

L5178Y TK-/- cells in logarithmic growth were harvested and replated at a density
of 5xl05 cells per Sml culture medium (RPMI 1640 supplemented with 10% foetal calf
serum and antibiotics). After incubation for 24 hours, compounds were added in a small
volume of 0.15M sodium bicarbonate or DMSO. Four or 18 hours later the drug was
removed by gently centrifuging the cells, and after washing once with fresh culture medium
they were counted and diluted for cell viability measurement by colony formation in soft-
agar4. Macroscopic colonies were obtained following incubation for about 12 days and were
counted by image analyser. Surviving Fraction (SF) was calculated as the ratio of Plating
Efficiencies (PE's) of treated and control groups (PE =number of colonies per dish/number
of cells plated per dish). In the Table compound activity is expressed as SF following
treatment with a fixed compound concentration of lOJ.!M.
L5178Y in vivo antitumour assays

Although the L5178Y tumour is a lymphoma it was found to grow very well as a
solid tumour if implanted intramuscularly.
DBA2 mice were injected with 5x106 cultured L5178Y cells (TK-/- or TK+/-) into
the gastrocnemius muscle on day 0. Treatment began on day 3 when the tumour was visible
as a slight swelling of the leg. Compounds were formulated in 0.15M sodium bicarbonate
for intraperitoneal (ip) bolus administration or subcutaneous (sc) infusion using ALZET
osmotic minipumps (models 2001D and 2001). Tumour growth was monitored by daily leg
diameter measurements using a perspex disc with calibrated holes that was passed up the
tumour bearing leg to find the size of hole that would just traverse the tumour.
In the initial studies it became apparent that L5178Y cells were slightly immunogenic
in the host mouse strain. Tumours would not grow when less than 1x104 cells were
implanted into previously untreated mice and in animals where a tumour had been "cured"
by drug treatment the take-rate was very poor if they were re-challenged with 5x106 cells.
Thus, for the TK-/- tumour a cure endpoint (no tumour growth within 3 weeks) was
adopted. Cure is thought to reflect 2-3 decades of cell kill, plus the immune effect. Cures
were less common with the TK+/- tumour so an endpoint of 5 days growth delay was
adopted (approximately 4 tumour doubling times). In the Table compound potencies are
expressed as dose per day (mg/kg/day) to produce ?:.3/5 cures in the TK-/- model, or 5 days
growth delay in the TK+/- model.
RESULTS
The in vitro cytotoxicity and in vivo antitumour activity of a range of classical and
non-classical TS inhibitors, against L5178Y TK-/- and TK+/- cells, are tabulated.
For compounds designed to be in the classical class an exposure time of 4 hours was
routinely used in the in vitro L5178Y TK-/- cell survival assay since compounds that are
rapidly transported and retained by cells should show good cytotoxicity with a brief
treatment. As expected, however, several compounds designed to be non-classical showed
little activity with a treatment time of 4 hours, but were highly cytotoxic when a prolonged
exposure of 18 hours was used. The SF at 4 hours for a typical non-classical compound is
shown in the Table. The high value of 0.25 is consistent with no polyglutamation or other
mechanism of retention occurring within 4 hours. Other compounds classified as "non-
classical" were shown by various specific methods not to be retained in cells.
In antitumour studies with TK-/- cells classical compounds were routinely
administered ip twice on one day with 8 hours between doses. Non-classical compounds,
however, were administered by continuous sc infusion over 24 hours using minipumps.

590
Table 1. ACTIVITY OF TS INHIBITORS AGAINST L5178Y TK MUTANTS

TK-1- TK-1- TK+/-

R1 R2 "X" ''Y'' SF@ cure 5dGD
10uM dose* dose*
CLASSICAL COMPOUNDS 4h 0,8h 0,8 hr
exposure X 1 day x 5 days

--o-
H Me <0.0035 -200 -300

H Et -GLU 0.017 <300** -200***

H Pg <0.005 -300** -540***
H Me F <0.001 -30 30

4
-GLU
H Et <0.001 <50 120

--o- s
H Me (ICI 01694) <0.0001 -5 6.6
-GLU
H Et <0.0005 -25 90
H Me <0.0006 -30 70
s
~r
H Et -GLU <0.0008 25 110

H Pg 0.0005 250 380

NON-CLASSICAL COMPOUNDS 18h 1day ?day

--o-
exposure infusion infusion
Me Pg 0.0096 80 -300
-GLU
4h 0.25
Me Me F NT -80 >200

4
-GLU
Me Pg 0.00075 30 275

Me Me F - NHCH(COOH) 0.0014 -30 >150

4
I

Me Pg (CH2)3COOH 0.0003 -10 -150

Me Pg F - NHCH(COOH) 0.0001 50 -150

4
I
(CH2)4COOH

Me Me - NHCH(COOH) NT -30 >150

F I

4
(CH2)3CONHS02CH3

Me Pg - NHCH(COOH) NT <30 -150

I
(CH2)4CONHS02CH3

Me
Pg

Pg
--o- F
-IGLU-Y-dGLU
0.0046

0.00075
50

30
150

-120

4
* = mg/kg/day, ** = 8h x 5 doses, *** = 8h x 15 doses, NT= not tested, Pg = propargyl

591
In both cases cures could be achieved with low doses in the range 10-50mg!kg/day.
To obtain activity with L5178Y TK+/- tumours compounds needed to be
administered over about a week. By analogy with previous studiesl it is believed that during
this period circulating thymidine levels gradually fall until they are below a protective level
and antitumour activity is then achieved in a salvage competent tumour.
Classical compounds were therefore administered ip twice daily for 5 days and non-
classical compounds were infused sc for 7 days. Growth delays of 5 days were achieved
with some compounds from both classes at doses of 10-50mg/kg/day, but some other
compounds required much higher doses to achieve the chosen endpoint.

DISCUSSION

Although it does not indicate the mechanism of retention, the data demonstrate that
the 4 hour exposure L5178Y TK-/- clonogenic assay described here can readily identify TS
inhibitors that are rapidly taken up and retained in tumour cells. By increasing the cell
exposure time it can also be useful in ranking non-classical compounds in terms of
potency. Using the 4 hour exposure assay we have identified more than 50 classical,
glutamate containing, compounds that killed >2 decades of L5178Y TK-/- cells when
dosed at a concentration of 1011M for 4 hours, and by extending the exposure period to 18
hours about 100 non-retained compounds with similar activity have been identified for in
vivo study. In vitro studies using mutant cell lines have been used to establish compound
uptake on the RFC and whether compounds are capable of polyglutamation.
The L5178Y TK-/- antitumour test provides a rapid and simple primary in vivo
screen forTS inhibitors. Classical compounds such as ICI D1694 are highly active when
bolus dosed ip on a single day, and non-classical compounds show similar activity when
infused sc over 24 hours. More than 25 classical compounds have produced cures with the
bolus dosing protocol and -70 compounds that are not efficiently retained in cells have
shown activity by sc infusion.
Some compounds are also active against the thymidine salvage competent variant,
L5178Y TK+/-, although a much more intensive treatment dosing regime is required and
growth delays are more usually observed. With the daily bolus dosing protocol 8
promising classical compounds were identified, from which ICI D1694 was selected for
development. By prolonged infusion for 7 days we have so far identified 8 promising non-
classical compounds. These are now being evaluated for efficacy against a panel of human
tumour xenografts, and toxicity to normal murine tissues is also being assessed.
We conclude that the TK mutants of the L5178Y tumour are very useful pre-clinical
models for efficacy evaluation of TS inhibitors.
REFERENCES

1. AL Jackman, GA Taylor: AH Calvert & KR Harrap. Modulation of anti-metabolite

effects: Effect of thymidine on efficacy of the quinazoline based thymidylate synthase
inhibitor, CB3717. Biochemical Pharmacology 33,3269-3275 (1984).
2. AL Jackman et al. ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that
is a potent inhibitor ofL1210 tumour growth in vitro and in vivo: A new agent for clinical
study. Cancer Res. 51, 5579-5586 (1991).
3. D Clive & P Voytek, Evidence for chemically-induced structural gene mutations at the
thymidylate kinase locus in cultured L5178Y mouse lymphoma cells. Mutation Res. 44,
269-278 (1977).
4. TC Stephens, J Peacock & PW Sheldon, Influence of in vitro assay conditions on the
assessment of radiobiological parameters of the MT tumour. British J. Radiology 53,
1182-1187 (1980).

592
QUINAZOLINE ANTIFOLATES INHIBITING THYMIDYLATE
SYNTHASE: SYNTHESIS OF y-LINKED PEPTIDE AND AMIDE

ANALOGUES OF 2-DESAMIN0-2-METHYL-Nl 0-PROPARGYL-5,8-

DIDEAZAFOLIC ACID (ICI 198583)

Vassilios Bavetsias, 1 Ann L.J ackman, 1 Tim J. Thornton, 1 Krzysztof

Pawelczak, 1 F. Thomas Boyle,2 Graham M.F. Bisset I

1The Institute of Cancer Research, 15, Cotswold Rd, Sutton, Surrey, U.K.
2Zeneca Pharmaceuticals (ICI), Alderley Park, Macclesfield, Cheshire, U.K.

INTRODUCTION

Thymidylate Synthase (TS) inhibitors such as ICI D16941 and to lesser extent ICI
1985832 rely on their poly-y-glutamate metabolites for their antitumour activity. The
polyglutamate metabolites are more potent inhibitors of TS than the parent monoglutamate
forms and in addition, their polyionic nature leads to prolonged retention within the cells.
However, drugs dependent on polyglutamation may have some disadvantages such as a) lack
of activity in tumours expressing low levels of, or an altered expression of,
folylpolyglutamate synthetase (FPGS) or b) prolonged normal tissue toxicities caused by
polyglutamate retention. For these reasons, we were interested in designing and
synthesising tight-binding TS inhibitors which would not be dependent on polyglutamation
for antitumour activity. Addition of one glutamate residue on the y-carboxyl of 2-desamino-
2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583), i.e. dipeptide (1), resulted in
stronger binding toTS by approximately 30-fold2. This finding was the starting point in our
search for a tight TS inhibitor that could not be a substrate for FPGS. We report here the
synthesis of22 dipeptide and 6 y-arnide analogues ofiCI 198583-y-L-Glu.

RESULTS-DISCUSSION
ICI 198583 dipeptide analogues (or quinazoline antifolate dipeptides) were synthesised

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 593
by a route similar to the one employed for the synthesis of ICI 198583 poly-y-glutamates. 3
The first key intermediate, the pteroate analogue (5), was prepared by coupling p-amino
benzoate derivative (3) to either 6-bromomethyl-2-methylquinazolinone (2a) or 6-
bromomethyl-2,7-dimethylquinazolinone (2b), followed by acid hydrolysis (Scheme 1).

Scheme 1

1}2 ~X

Xc
0 HN~C02Bu1 O X

Xr l}~
!
HN BrR2 =Pg,EtorMe,X=H,F HN ?' N COB t
,4 ,4I A ~ ~ 2 u
H:J(: N R1 CaC03 /DMF or H:J(: N R1
2 2,6-Lutidine/DMF 4
aRl =H TFA
b.R1 =Me
o 1}2 Ax
HN N~COOH

H:J(:,4N R1
5
R1 =H or Me R2 = Pg, Me or Et
X=HorF

The preparation of the second key intermediate, the dipeptide (10), is shown in Scheme
2. Amino acids (6) were first protected as their ten-butyl esters (7) either by treatment with
isobutylene I cone. H 2S04 or by transesterification with ten-butyl acetate. Subsequent
coupling to a-ten-butyl-N-benzyloxycarbonyl-L-glutamate (8) using the mixed carbonic
anhydride coupling method4 afforded the Z-protected dipeptide (9), from which (10) was
obtained by catalytic hydrogenation.

Scheme2

Finally, the dipeptide free base (10) was condensed with the pteroate analogue (5) using
diethyl phosphorocyanidate (DEPC) as carboxyl activating reagentS to give the quinazoline
antifolate dipeptide ester (11), which was converted to the final product (12) upon treatment
with trifluoroacetic acid (Scheme 3).

594
 The quinazoline antifolate L-L dipeptides (1, 12a-12n) synthesised by this procedure are
shown in Table 1.

Table 1. Quinazoline Antifolate L-L Dipeptides

Cmpd Rl R2 X -HNCH(COOH)-R3
2nd Amino Acid

1 H Pg H -CH 2CH2COOH (Glu)

12a H Pg H -H (Gly)
12b H Pg H -CH3 (Ala)
12c H Me H -CH 3 (Ala)
12d H Et H -CH3 (Ala)
12e H Pg F -CH3 (Ala)
12f H Pg H -CH2CH 3 (Abu)
12g H Pg H -CH20H (Ser)
12h H Pg H -CH2CH2CONH2 (Gin)
12i H Pg H -CH2CH2CH3 (Nva)
12j H Pg H -CH(CH3)2 (Val)
12k H Pg H -CH(CH3)CH2CH 3 (lie)
121 H Pg H -Ph (Phg)
12m H Pg H -CH2Ph (Phe)
12n H Pg H -CH2COOH (Asp)

Nearly all the quinazoline antifolate L-L dipeptides were potent inhibitors of TS and of
L1210 cell growth (see accompanying paper, A.L. Jackman et al.). However, when these
dipeptides were injected into mice they were partially degraded to the monoglutamate forms
by y-hydrolase enzymes, which act by cleaving the glutamyl y-amide bond. As a result,
further effort was concentrated on making this amide bond resistant to enzymatic hydrolysis.
To achieve this objective we have chosen two main strategies:

595
 i) Replacement of the second amino acid by a primary amine lacking a.-carboxyl groups
ii) Replacement of the ftrst and I or second amino acids by their D-enantiomers.
ICI 198583 y-linked amide analogues (13a-13f) were prepared by a similar route to the
one described above but primary amines were used in place of amino acids.

In general, ICI 198583 y-linked amides were poorer inhibitors ofTS and ofL1210 cell
growth compared with their corresponding parent L-L dipeptides. The advantage of these
compounds, however, lies in their resistance to enzymatic hydrolysis (see accompanying
paper, A.L. Jackman eta/.).
Quinazoline antifolate L-D dipeptides (14a-14f) and the D-D and D-L stereoisomers of
(1) were prepared by an analogous route to that described above for the synthesis of the L-L
dipeptide analogues. a.-tert-Butyl-N-benzyloxycarbonyl-D-glutamate, which was required
for the synthesis of the D-D and D-L stereoisomers of (1), was synthesised via a route
previously described for the preparation of the L-enantiomer. 6

H3C
HN~~:~~Zxcoo"
..4-..
N
T~D
R1 14
0 L • nH
0 D
Rs

a. R 1 = H, X= H, R5 = CH2CH2COOH d. R 1 =Me, X= F, R5 = CH2CH 2COOH

b. R 1 = Me,X = H, R5 = CH2CH2COOH e. R 1 = H, X= H, R5 = CH3
c. R 1 = H, X= F, R5 = CH2CH2COOH f. R 1 = H, X= H, R5 = CH2Ph

When quinazoline L-D dipeptides were injected into mice no breakdown products were
observed. Moreover each analogue exhibited approximately the same TS inhibition as its L-L
counterpart. The D-Glu-L-Glu and D-Glu-D-Glu analogues were less potent inhibitors of TS
compared with the parent L-Glu-L-Glu dipeptide (1), with the former being unstable in vivo
and the latter resistant to enzymatic hydrolysis (see accompanying paper A.L Jackman et al.).

REFERENCES
1. A.L. Jackman, G.A. Taylor, W. Gibson, R. Kimbell, M. Brown, A. H. Calvert, I. R.
Judson and L.R. Hughes, Cancer Res., 1991, 51, 5579-5586.
2 A.L. Jackman, D.R. Newell, W. Gibson, D.I.Jodrell, G.A. Taylor, J.A. Bishop, L.R.
Hughes and A.H. Calvert, Biochem. Pharmacol., 1991,4 2, 1885-1895.
3. G.M.F. Bisset, K. Pawelczak, A.L. Jackman, A.H. Calvert and L.R. Hughes, J. Med.
Chern., 1992, 35, 859-866.
4. J. Meinhofer, The Peptides, Analysis, Synthesis, Biology, E. Gross and J. Meinhofer
Eds, Academic Press, New York, 1979, 1, pp 264-309.
5. A. Rosowsky, R. Forsch, J. Uren and M. Wick, J. Med. Chern., 1981,24, 1450-1455.
6. K. Pawelczak, I. Krzyzanowski and B. Rzeszotarska, Org. Prep. Proced. Int., 1985,
17, 416-419 and references cited therein.

596
TilE DURATION OF TilE INHIBITION OF THYMIDYLATE
SYNTHASE IN INTACT L1210 CELLS EXPOSED TO 1WO
DIFFERENT CLASSES OF QUINAZOLINE ANALOGUES

Rosemary Kimbell, Ann L. Jackman, F. Thomas Boyle*, Anthea

Hardcastle and Wynne Aherne

Drug Development Section, The Institute of Cancer Research, Sutton,

Surrey, U.K. *ZENECA Pharmaceuticals (ICI), Alderley Park,
Macclesfield, Cheshire, U.K.

INTRODUCTION

ICI D1694 (Fig.l) is a quinazoline anti-folate that inhibits thymidylate synthase

(TS), currently in Phase II clinical trials1•2•

Figure 1. ICI 01694

It is actively transported into cells via the MTX/reduced folate carrier (RFC)
and rapidly polyglutamated by folylpolyglutamate synthetase (FPGS). The intracellular
drug species in mouse and human cell lines have been shown to be higher chain length
polyglutamates, principally tetra- and penta-glutamates. These are substantially more
potent as TS inhibitors (e.g. ICI D1694 tetraglutamate Ki=1nM) than the parent
molecule (ICI D1694 Ki=60nM). These high chain length polyglutamates are retained
intracellularly due to their increased net negative charge.
To overcome some potential disadvantages of drugs subject to polyglutamation
(see Jackman et al, accompanying paper), a second class of TS inhibitors, which do not
undergo polyglutamation, was designed. Novel quinazolines were synthesised and based
on 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) and its
diglutamate (ICI 198583-y-L-glu) because they are both intrinsically better inhibitors
of TS (Ki = lOnM and -0.5nM respectively) than ICI D16943• They are polyglutamated
intracellularly, but to a lesser extent than ICI D1694.

Chemistry fJ1Id Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 597
 Intracellular polyglutamation can be prevented (Moran et al, personal
communication), and TS inhibitory activity improved, by the introduction of a methyl
group on the 7 position of the quinazoline moiety. Another approach is to modify the
second glutamate of ICI 198583-y-L-glu by replacing it with another amino acid e.g.
alanine. However such dipeptides are unstable when injected into mice, with ICI
198583 being detected in the plasma and liver as a hydrolysis product (Jackman et al,
accompanying paper). This problem was overcome by changing the stereochemistry of
the terminal amino acid to the 0-enantiomer. ICI 198583-y-0-glu is only two-fold less
active than the L-enantiomer as an inhibitor of isolated TS or the growth of L1210
cells. Introduction of a 2'F to the benzoyl ring of these analogues increases TS
inhibition at least two-fold.

R = C7 = H or CH 3
X = 2' = H or F
Y = OH or L-glu or D-glu

Figure 2. Structural analogues of ICI 198583

These structural analogues are not active through polyglutamate formation (see
below). It is postulated that compounds such as these, that are prevented from forming
intracellular polyglutamates, will only inhibit TS whilst drug is present in the
extracellular medium. Therefore, a selected group of compounds has been tested by
measuring TS activity in intact L1210 cells, both during drug exposure, and after
removal of drug from the extracellular medium. The method used is the rate of the
release of 3H 20 from 5-3H dUrd and based on that of Yalowich and Kalman4•5•
Intracellular nucleotide levels (TIP and dUMP) were also measured by radio-
immunoassay (Aherne et al; manuscript in preparation).

RESULTS AND DISCUSSION

Transport into the cell by the reduced-folate carrier (RFC) was confirmed using
the Ll210:1565 cell line which has a greatly impaired RFCi. Polyglutamation was
confirmed or otherwise using the L1210:MB3 cell line, which has an altered FPGS that
does not polyglutamate anti-folates 7• A small cross-resistance factor of up to five is
seen with compounds that use the RFC, due to a small transport defect in this line.
Results in Table 1 demonstrate that 7-CH3, 2'F ICI 198583, ICI 198583-y-0-glu
and 7-CH3, 2'F ICI 198583-y-0-glu retain good activity in the L1210:MB3 cell line
which suggests that they are not active through polyglutamation.
After 4hr exposure at equitoxic (10 X IC50) doses, all the compounds inhibited
the rate of 3H release from 5-3H dUrd by > 90% (Table 2). However, after 4hr or 24hr
exposure followed by 4hr in drug-free medium some differences were seen. TS
inhibition was maintained by those compounds which are polyglutamated (ICI 01694,
ICI 198583 and ICI 198583-y-L-glu). The less marked effect of ICI 198583 after only
4hrs incubation (79% of control) reflects its relatively slow formation of polyglutamates
compared with ICI 01694. However the higher dose of 5J,£M (50 x IC50) resulted in this
drug-retentive effect (17% of control; data not shown). With the three non-
polyglutamated compounds, activity returned to control levels during the drug-free
period, after an initial 4hr incubation. However, increasing the exposure time to 24hr

598
 Table 1. In vitro activity of quinazoline TS inhibitors

Inhibition of cell growth, IC50 (11M)

L1210 TS (resistance factors in parentheses)
IC50 , nM
L1210 L1210:1565 L1210:MB3

ICI 01694 600 0.0090 0.92 (102) >100 (> 10,000}

ICI 198583 50 0.13 8.2 (63} 2.7 (21)

7-CH 3,2'F ICI 198583 9.0 0.080 5.9 (74) 0.47 (6}

ICI 198583-y-L-glu 1.8 0.16 4.6 (29) 2.8 (18)

ICI 198583-y-0-glu 4.8 0.33 8.0 (24) 0.87 (3)

7-CH 3 ,2'F ICI 198583-y-0-glu 0.92 0.20 6.8 (34) 0.41 (2)
1210:1565- im paired Rf(.;; L1210:MB3- 1mpaired po1yglutamat1on

Table 2. In situ TS assay as a comparative measure of the recovery of TS activity

after resuspension of L1210 cells in drug-free medium (DFM)

Release of 3 H from 5-3 H dUrd (% control)

drug
con& 4hr 4hr+4hr 24hr+4hr 24hr+24hr
OFM OFM DFM

ICI 01694 0.1011M 8.±.2 21 .±. 16 3.±.2 26 .±. 3

ICI 198583 1.3j1M 3.±.1 79, 79 8, 2 15 .±. 10

7-CH 3 ,2'F ICI 198583 0.8011M 3.±.1 144 .±. 17 92 .±. 4 153, 99

ICI 198583-y-L-glu 1.61-1M 2.±.1 32 .:t 9 <1 15 .±. 7

ICI 198583-y-D-glu 3.3j1M 3.±.1 108 .±. 19 52.±. 22 135, 100

7-CH 3 ,2'F ICI 198583-y-D-glu 2.011M 3.±.2 94 .±. 8 66 .±. 15 96 .±. 18

L1210 cells were exposed to equitoxic concentrations (10 x IC50 at 48hrs) of the compounds. All24hr
incubations included 1011M dThd to prevent cell death

resulted in incomplete recovery of activity, suggesting slow drug efflux. Indeed,

increasing the drug-free period to 24hr allowed activity to return to control levels.
The in situ TS assay gives a comparative measure of flux through the enzyme
and indicates whether recovery of activity is a rapid or slow process once the
extracellular drug has been removed. In this way an indication of drug-retention
(usually as a result of polyglutamation) is obtained. However quantitation of the effect
this may have on suppression of TIP synthesis is required. The rise in the dUMP pool
following TS inhibition can result in a decrease in the specific activity of the

599
intracellular 3H dUMP pool. After 4hrs incubation with the above compounds the
dUMP pool increased 3-fold (Table 3). This may have contributed to the degree of
"flux" inhibition (92%) being greater than that of the TTP pool (-50%) in the cells
treated with ICI D1694 for 4hrs and actual specific activity measurements of the dUMP
pool are therefore required. The dipeptide analogue inhibited TTP more effectively
than ICI D1694 after 4hrs (equitoxic growth inhibitory concentrations by continuous
exposure) and probably reflects the lack of drug metabolism (polyglutamation) required
for this agent. However a 5-fold higher ICI D1694 concentration resulted in an 88%
inhibition of TTP which did not recover after resuspension of the cells in drug-free
medium. The recovery of the TTP pool after 7-CH3,2'F ICI 198583-y-D-glu removal
was in contrast to the lack of recovery seen with ICI D1694 and in agreement with the
results of the in situ TS assay.

Table 3. TTP and dUMP measurements in L1210 cells exposed to equitoxic

concentrations of ICI D1694 and 7-CH3,2'F ICI 198583-y-D-glu

TTP level (% control) dUMP level (%

control)

drug concn 4hrs 4hrs + 4hrs 4hrs +

4hrs OFM 4hrs OFM

ICI 01694 0.111M 44, 50 50, 31 250 180

ICI 01694 0.611M 12 7 300 280
7-CH 3 ,2'F ICI 198583-y-0-glu 2.011M 11 113 350 130
7-CH 3 ,2'F ICI 198583-y-0-glu 10.011M 13 98 290 190

TTP in controls = 20pmoles/106 cells; dUMP in controls = 20pmoles/106 cells

SUMMARY

We conclude that the structural changes described above, aimed at preventing

polyglutamation, result in TS inhibition and reduction in TTP pools being dependent
on the presence of extracellular drug.

REFERENCES
1. A.L. Jackman, G.A. Taylor, W. Gibson, R. Kimbell, M. Brown, A.H. Calvert, I.R. Judson, and L.R.
Hughes. Cancer Res. 51: 5529-5586, 1991.
2. S.J. Clarke, A.L. Jackman and I.R. Judson. In:"Novel approaches to selective treatment of human
tumours: laboratory and clinical correlations". (Y. Rustum. Ed.) Plenum Press, In Press.
3. A.L. Jackman, D.R. Newell, W. Gibson, D.l. Jodrell, G.A. Taylor, J.A. Bishop, L.R. Hughes, and A.H.
Calvert. Biochem. Pharmacal. 42: 1885-1895, 1991.
4. J.C. Yalowich and T.l. Kalman. Biochem. Pharmacal. 34: 2319-2324, 1985.
5. G.A. Taylor, A.L. Jackman, K. Balmanno, L.R. Hughes, and A.H. Calvert. In: "Purine Metabolism in
Man", Vol.6 (K. Mikanagi eta!, Eds.) pp. 383-388, Plenum Publishing Corp. New York, 1989.
6. D. Fry, J.A. Besserer, and TJ. Boritzki. Cancer Res. 44: 3366-3370, 1984.
7. A.L. Jackman, L.R. Kelland, M. Brown, W. Gibson, R. Kimbell, W. Aherne, and I.R. Judson. Proc.
Amer. Assoc. Cancer Res. 33: 406, 1992.

600
THE TOXICITY OF ICI D1694 IN MAN AND MOUSE

Stephen J. Clarke, Ann L. Jackman and Ian R. Judson

Section of Drug Development

Institute of Cancer Research
Sutton, Surrey, UK SM2 SNG

INTRODUCTION

ICI Dl694 is a water soluble, folate-based, thymidylate synthase (TS) inhibitor

which has been developed as the result of a collaboration between ICI Pharmaceuticals
(now Zeneca) and the Institute of Cancer Research and is the successor to CB3717,
the first antifolate TS inhibitor to reach clinical triall. CB3717 showed promising anti-
tumour activity in breast2, ovarian (A. H. Calvert-personal communication) and
hepatocellular3 cancers, but further development was not undertaken because of
nephrotoxicity I. Other toxicities seen were malaisel, manifest by lack of well being
and a rapidly decreasing performance status, and alterations in hepatic biochernistryl
The nephrotoxicity was thought to be due to poor aqueous solubility of the compound
and unrelated to the locus ofaction4,5. Alterations to the 2-position6,7 ofCB3717
produced more water-soluble compounds which did not cause nephrotoxicity in
murine modeJs8 A full structural analysis resulted in the development of a large
number of compounds the most promising of which was ICI D 16949' 10, 11. This
compound proved more active than CB3717 against tumour cells in vitro and in
murine in vivo models because of its use of the reduced folate carrier (RFC) and
avidity for folylpolyglutamate synthetase (FPGS). Use of the RFC facilitates
intracellular drug influx and polyglutamation results in high intracellular drug
accumulation and retention. Furthermore, the polyglutamated forms of ICI D 1694
(especially the triglutamate) are -60 fold better inhibitors of isolated TS than the
monoglutamatell. Animal toxicology suggested the dose limiting toxicities would be
gastrointestinal and haematological. There was no evidence of renal toxicity 12, 13.
The European phase I trial of ICI D1694 commenced on 27/2/91. Patients
were accrued at the Royal Marsden Hospital, London and Sutton, Surrey and at the
Rotterdam Cancer Institute, Rotterdam, the Netherlands. Inclusion criteria were ;1)
age between 18 and 75 years, 2) progressive malignancy not amenable to cure by

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 601
conventional chemotherapy, and 3) a life expectancy of at least 15 weeks. Exclusion
criteria were; I) chemotherapy within the previous 4 weeks, 2) extensive radiotherapy
within the previous 8 weeks, 3) prior chemotherapy for> 6 months with chlorambucil,
mitomycin C or nitrosoureas, 4) irradiation of> 30% of bone marrow, 5) unresolved
toxicity from prior therapy, 6) intra-cerebral metastases, 7) corticosteroids used for
anything other than anti-emetic or replacement purpose, 8) WBC < 3 x I o9 /litre,
platelet count< 100 x 1Q12flitre or Hb < 10 gm/dl, 9) 51cr-EDTA clearance < 70
mls/min, 10) ALT > 50IU/l, alkaline phosphatase >200 IU/1 or bilirubin > 25j..tmol/l
unless known to be due to liver secondaries, 11) WHO performance state > 2, 12)
other major medical illness or pregnancy.
Treatment was administered by constant intravenous infusion over 15 minutes,
repeated 3 weekly to a maximum of 6 courses, or longer in the presence of continuing
response. Disease reassessment was performed after every 2 courses. The starting
dose was 0.1 mg!m2 and dose escalation was via a modified Fibonacci schema with
subsequent dose levels being 0.2, 0.4, 0.6, 1.0, 1.6, 2.6, 3.0 and 3.5 mg!m2. At least 3
patients were treated at each level with 10, 23 and 6 patients treated at 2.6, 3.0 and 3.5
mg!m2 respectively. Table 1 summarises the patient characteristics.

Table 1. Patient Characteristics

Characteristic Patient No. % Characteristic Patient No. %

Centre Tumour types
RMH 49 80 Colon I6 26
RCI 12 20 Ovary 11 I8
Sex Head and neck 5 8
Male 34 56 Mesothelioma 5 8
Female 27 44 Melanoma 4 7
PS Sarcoma 4 7
0 22 36 Breast 3 5
I 30 49 ACUP 2 3
2 9 I5 Pancreas 2 3
Prior therapy Lung 2 3
Nil 3 5 Gastric I 2
Surgery (S) alone I 2 Gall bladder 1 2
Radiotherapy (R) alone 1 2 Undifferentiated 1 2
Chemotherapy (C) alone 7 11 Thyroid 1 2
S+R 1 2 Carcinoid 1 2
S+C 25 41 Oesophagus 1 2
C+R 3 5 TOTAL 61
S+R+C 20 33
5-fluorouracil (5-FU) 25 41

Sixty one patients received 160 courses ofiCI D1694. Forty nine patients (80%) were
treated in the UK and 12 (20%) in Holland. There was a slight male predominance and
the mean age was 53 years. Fifty two patients (85%) had a performance status ofO or
1. A wide variety of tumour types were treated with a slight predominance of colon
(26%) and ovarian (18%) cancers. Fifty five patients (90%) had received prior
chemotherapy and of these 25 (41%) had received 5-FU. The median number of
courses received was 2, although of 23 patients treated at 3.0mg!m2, 7 received 5 or
more courses.

602
TOXICITIES

Toxicity was first seen at 1.6 mg!m2 where two patients (50%) developed mild
elevation of liver function tests after a second course of treatment. By this time
treatment had commenced at 2.6 mg!m2 and similar abnormalities were seen. The
changes were mainly in transaminases (ALT/AST) with lesser and later elevations in
serum alkaline phosphatase (SAP) and gamma glutamyl transferase (yGT). These
changes were also seen at 3.0 and 3.5 mg!m2. The abnormalities did not delay
treatment and settled with repeated dosing and cessation of treatment. No patient
developed progressive liver impairment or jaundice. Three of four patients who
experienced malaise at 3.5 mg!m2 also had WHO grade 3 or 4liver dysfunction, but all
other patients with abnormal liver function tests were asymptomatic.
Myelosuppression was first seen in 5 patients (50%-3 WHO grade 1 and 2
grade 2) at 2.6 mg!m2 and the incidence increased with increasing dose. At 3.0
mg!m2, 14 patients (61%) had myelosuppression with 5 patients (22%) developing
grade 3 or 4 toxicity. Two patients (33%) had grade 3 and 4 neutropenia at 3.5
mg!m2. The mean time to nadir was 8 days (range 7-21 days) with a mean time to
recovery of 10 days (range 2-22). Toxicity was cumulative, but completely reversible
on cessation of treatment and appeared to be less severe in those treated with
concomitant corticosteroids. Anaemia and thrombocytopenia occurred less commonly
than myelosuppression. The incidence of gastrointestinal toxicities also increased with
increasing dose. Fourteen patients (60%) experienced diarrhoea at 3.0 mg!m2. Six of
these and one other patient at 3. 5 mg!m2 had severe diarrhoea (5 WHO grade 3 and 2
grade 4). Five of the 7 patients required hospitalisation and cessation of further
treatment and in 3 the diarrhoea may have contributed to their demise. The diarrhoea
was characterised by profuse, protracted fluid loss with rapid onset of dehydration and
hypoalbuminaemia in those unable to maintain adequate fluid intake. Nausea and
vomiting were commonly seen (14/23 or 61% at 3.0 mg!m2) and in all except 3 cases
the symptoms were readily managed by oral emetics, usually dexamethasone and
metoclopramide. In the other 3 patients, all treated at 3.0 mg!m2, hospitalisation was
required to manage severe symptoms. Mucositis was seen in 11 patients (48%) at 3.0
mg!m2, but did not effect oral intake.
Other toxicities seen were rash, fever, arthralgia and elevated erythrocyte
sedimentation rate. The highest dose level investigated was 3.5 mg!m2 at which six
patients received only nine courses oftreatment because of the occurrence in 4 (67%)
of debilitating malaise. In one patient the symptoms were related to WHO grade 4
myelosuppression. In the other 3 there were no significant anti-proliferative toxicities
although, as mentioned above, in each there were grade 3 or 4 abnormalities in hepatic
function which may have contributed to the malaise This poor tolerance of3.5 mg!m2
prompted expansion of accrual at 3.0 mg!m2, the dose which has been recommended
for phase II trials.
Anitumour activity was seen in 4 patients at 3.0 mg!m2 and 1 at 2.6 mg!m2.
One patient with breast cancer achieved a complete response and other responses were
seen in ovarian cancer (2 minor responses), nasopharyngeal cancer (minor response)
and adenocarcinoma ofunknown primary (partial response).
The toxicity which was most difficult to manage was diarrhoea. We have
recently reported the finding of diarrhoea in Balb C mice treated with ICI D 1694 and
this may provide a model with which to explain and treat the unpredictable symptoms
seen in man12.

603
MURINE MODEL

On a daily times five schedule of intraperitoneal (i. p) injections the maximally tolerated
dose (that dose at which no deaths occurs) in Balb C mice is 5 mglkg. The mice lose
20-25% of body weight with nadir weight loss occurring on day 7. Diarrhoea
commences on day 4 and continues for several days. Histopathology of small bowel
shows ulceration and villous atrophy. In DBA2 mice a dose of 50-100 mglkg is
required to induce 14-22% weight loss, which is maximal on day 9, although the mice
can tolerate >500 mglkg without the occurrence of diarrhoea. The diarrhoea and most
of the weight loss is abolished by the co-administration of thymidine in a dose of 500
mglkg given three times a day for 8 days. Folinic acid co-administration, 20mglkg
daily times five, completely abolishes weight loss and diarrhoea. Full blood count with
differential on days 1, 5, 8 and 12 was performed in both strains after the following
doses ofiCI D1694: 5 mglkg for Balb C and 100 mglkg for DBA2 on a daily x 5 i.p
schedule. Severe neutropenia was seen in the DBA2 mice on day 8, but not on any
day in the Balb C mice. Serum thymidine levels appear to be similar in both strains at a
level of 1-21J.M. Further experiments may provide information as to the cause of this
differential toxicity and its applicability to the clinical situation.

REFERENCES

1. A.H. Calvert, D.L. Alison, S.J. Harland, B.A. Robinson, A.L. Jackman, T.R Jones, D.R Newell,
Z.H. Ziddik, E: Wiltshaw, T.J. McElwain, I.E. Smith, and K.R Harrap, J C/in Onco/. 4:
1245-1252, (1986)
2. B.M.J. Cantwell, V. Macaulay, A.L. Harris, S.B. Kaye, I.E. Smith, RA.V. Milstead, and A.H.
Calvert, Eur J Cancer C/in Oncol. 24: 733-736, (1988)
3. M.F. Bassendine, N.J. Curtin, H. Loose, A.L. Harris, and O.F.W. James, J Hepato/. 4: 349-356,
(1987)
4. D.L. Alison, D.R Newell, C. Sessa, S.J. Harland, L.I. Hart, K.R. Harrap, and A.H. Calvert, Cancer
Chemother Pharmacal. 14: 265-271, (1985)
5. D.R. Newell, D.L. Alison, A.H. Calvert, K.R. Harrap, M Jarman, T.R Jones, M. Manteuffel-
Cyrnborowska, and P. O'Connor, Cancer Treat Rep. 70:971-979, (1986)
6. T.R Jones, T.J. Thornton, A. Flinn, A.L Jackman, D.R Newell, and A.H. Calvert, J Med Chem.
32:847-852,(1989)
7. L.R. Hughes, A.L. Jackman, J. Oldfield, R.C. Smith, K.D. Burrows, P.R Marsham, J.A.M. Bishop,
T.R. Jones, B.M. O'Connor, and A.H. Calvert, J Med Chem. 33: 3060-3067, (1990)
8. A.L. Jackman, G.A. Taylor, B.M. O'Connor, J.A. Bishop, RG. Moran, and A.H. Calvert, Cancer
Res. 50: 5212-5218, (1990)
9. P.R. Marsham, L.R. Hughes, A.L. Jackman, A.J. Hayter, J. Oldfield, J.M. Wardleworth, J.A.M.
Bishop, B.M. O'Connor, and A.H. Calvert, J Med Chem. 34: 1594-1605, (1991)
10. A.L. Jackman, D.R Newell, W. Gibson, D.I. Jodrell, G.A. Taylor, J.A.M. Bishop, L.R. Hughes,
and A.H. Calvert, Biochem Pharmacal. 42: 1885-1895, (1991)
11. A.L. Jackman, G.A. Taylor, W. Gibson, R. Kimbell, M. Brown, A.H. Calvert, I.R. Judson, and
L.R Hughes, Cancer Res. 51: 5579-5586, (1991)
12. D.I. Jodrell, D.R. Newell, S.E. Morgan, S. Clinton, J.P.M. Bensted, L.R. Hughes and A.H.
Calvert, Br J Cancer. 64: 833-838, (1991).
13. A.L. Jackman, D.I. Jodrell, W. Gibson and T.C. Stephens, In R.A. Harkness, G. Elion and N.
Zollner (eds.), Purine and Pyrimidine Metabolism in Man VII, pp. 19-23, Plenum press, New
York, (1991)
14. S.J. Clarke and A.L. Jackman, Proc Amer Assoc Cancer Res. (1993) In press

604
EVALUATION OF IMMUNOHISTOCHEMICAL STAINING AND ACTIVITY

OF THYMIDYLATE SYNTHASE IN CELL LINES

Clasina L. Vander Wilt, Kees Smid, G. Wynne Aheme2 , Herbert M.

Pinedo 1, Godefridus J. Peters

Dept. Oncology, Free University Hospital, PO BOX 7057, 1007MB

Amsterdam
1 Netherlands Cancer Institute, Amsterdam the Netherlands
2 Institute of Cancer Research Sutton, Surrey SM2 5NG, United

Kingdom

INTRODUCTION

Levels of the enzyme thymidylate synthase (TS) in tumour tissue play an

important role in the outcome of chemotherapy with dmgs directed against TS. High TS
levels may be predictive for resistance against these drugs 1• It has been shown, both in
vitro and in vivo that high intrinsic TS levels correlate with resistance to 5-fluorouracil
(5FU) 2•3 • Moreover exposure of several neoplastic cells and tissues to 5FU cause a
marked increase of TS levels4 •5 . The common methods to measure TS in cells and
tissues are enzyme assays on crude extracts6 • The FdUMP binding assay determines the
number of free FdUMP binding sites, while the tritium release assay, based on the
conversion of [VH]-dUMP into dTMP and 3H20, provides data about the catalytic
activity of TS. These assays require about 2.107 cells. With a polyclonal antibody,
raised against recombinant human TS (ahTS)7 the detection of TS protein levels in
smaller numbers of cells is possible, either with ELISA or immunohistochemical (IHC)
staining methods

METHODS

A panel of 13 cell lines (Table 1) was used for IHC staining and measure-
ments of FdUMP binding and catalytic activity including 3 murine, 1 rat and 4 human
colon cancer cell lines; 3 human cell lines derived from squamous cell carcinoma of the
head and neck and 2 human lympoblastoid cell lines, the parent Wl-L2 and the
resistant, TS overproducing Wl-L2:Cl 8 • We applied standard IHC staining methods.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 605
Cytospins were flxed in 100% acetone. They were incubated with the flrst antibody,
ahTS (1:300) for 1 h, and after washing, for 1 h with the second antibody (swine-anti-
rabbit immunoglobulins conjugated to peroxidase, 1: 100). The staining was completed
by addition of 3,3'-diaminobenzidine (0.5 mg/ml) and H 20 2 (3% solution). Two
individuals scored independently the intensity of lliC staining of the cytospins from low
(+)to high(+++++).
Enzyme assays for FdUMP binding and catalytic activity of TS were per-
formed as described by Peters et at'.

RESULTS

The lympoblastoid cells had an intense staining at the rim of the cell, while
the cytoplasm showed minimal staining (Fig. la-b). Two of the human colon cancer cell
lines also stained stronger at the rim of the cell than in the cytoplasm, but all the other
cells had a homogeneous staining of the cytoplasm. The ahTS also recognized rat and
murine TS. The murine and rat cells showed a staining comparable to that of the human
colon cancer cells.
Comparison of the intensity of the staining with the known catalytic activity
or FdUMP binding of the cell lines revealed a good correlation of high intensity and
high TS activity and high capacity of FdUMP binding to TS within the murine colon
cancer cells and the human head and neck cancer cells (Table 1). For the head and neck
cancer cell lines UM-SCC-14C with 379 pmol/h/mg protein (catalytic activity) and 158
fmol/mg protein (FdUMP binding) had a staining scored as ( +), while those values for
UM-SCC-llB were 2526, 864 and(++++), respectively (Fig. le-e). The staining
of the resistant lympoblastoid cells ( + + +) was stronger than that of the parent ( +)
(Fig la-b). Within the panel of the human colon cancer cell lines no clear relation could
be detected. Interestingly two colon cancer cell lines (1 murine, 1 human), which have
been adapted to low folate levels showed an increase in TS catalytic activity (1.5x and
2.5x, resp.) and intensity of lliC staining (1.25x and 2x, resp.) as compared to their
parental line.

DISCUSSION

rnc staining of cytospins revealed two types of staining, cytoplasmic and cell
rim. In most of the cell lines grouped according to origin a good relation between TS
levels measured with classic enzyme assays and intensity of lliC staining was observed.
A similar trend has been observed for results obtained by Western blotting with a
monoclonal antibody against human TS and FdUMP binding9 •
rnc staining for TS can be an additional method to indicate TS protein
levels, but quantification of TS levels was disputable with this method. Quantification
may be performed by densitometric measurements of Western blots or EUSN. It has
been shown for the parent and resistant lympoblastoid cells that the difference in TS
protein levels could also be detected by EUSA methods, with the polyclonal antibody7 .
The rnc detection of TS in tumor samples could be of prognostic value for patients
which receive (adjuvant) FU therapy, since high TS levels seem to be correlated to
resistance against FU.

606
 A

Figure 1. Immunohistochemical staining with ahTS (1:300) of 2 lymphoblastoid

cell lines W1-L2 (A)
and W1-L2:C1 (B) and 3 human squamous cell carcinoma cell lines of the head
and neck UM-SCC-14C
(C) UM-SCC-22B (D) and UM-SCC-llB (E). Bar is 10 p.m.

607
 Table 1
Comparison of intensity of lliC staining of TS
with FdUMP Binding to TS an TS Catalytic activity

IHC cytospins TS catalytic activity FdUMP binding

(pmollh/mg protein) (fmol/mg protein)

Human colon cell lines

SW620 ++ 358 ± 97 161 ± 17
WiDr +++ 675 ± 257 218 ± 98
HT29 +++ 700 ± 281 206 ± 61
WiDr/F* +++++ 1556 ± 22 222 ± 93

Rat colon cell line

CC531 ++ 1242 ± 87 712 ± 93

Murine colon cell lines

C38-2 ++ 109 ± 27 289 ± 64
C26-10 +++ 7100 ± 1300 3113 ± 512
C26-10/F* ++++ 9503 ± 1178 1756 ± 136

Human squamous cell carcinoma cell lines of the head and neck
UM-SCC-14C + 379 ± 177 158 ± 39
UM-SCC-22B +++ 969 ± 119 150 ± 19
UM-SCC-llB ++++ 2526 ± 1498 864 ± 339

Human lymphoblastoid cell lines

W1-L2 + 5130 ± 203 152 ± 49
W1-L2:C1 +++ > 16206 5337 ± 24

• Cell lines derived from WiDr and C26-10, respectively, but adapted to grow in folate depleted medium
(1 nM folinic acid). Values are means of 2 independent scores (IHC) or at least 3 enzyme assays

ACKNOWLEDGEMENT

The lymphoblastoid cell lines were a gift from Dr. A.L. Jackman. This research was supported by grant
92-88 of the Dutch Cancer Society

REFERENCES

l. G.J. Peters, C.J. Van Groeningen. Ann. Oncol. 2: 469-480 (1991)

2. S.H. Berger, J. Chung-Her, L.F. Johnson, F.G. Berger. Mol. Pharmacal. 28: 461-467 (1987)
3. C.P. Spears, A.H. Shahinian, R.G. Moran, C. Heidelberger, T.H. Corbett. Cancer Res 42: 450-456
(1982)
4. E. Chu, S. Zinn, D. Boarman, C.J. Allegra. Cancer Res. 50: 5834-5840 (1990)
5. C.L. van der Wilt, H.M. Pinedo, K. Smid, G.J. Peters. Cancer Res. 52: 4922-4929 (1992)
6. G.J. Peters, C.J. van Groeningen, E.J. Laurensse, H.M. Pinedo. Eur. J. Cancer 27: 263-267 (1991)
7. G.W. Aherne, A. Hardcastle, R. Newton. Ann. Oncol. 3 (suppl. 1): 77 (abstr. 79), (1992)
8. B.M. O'Connor, A.L. Jackman, P.H. Crossley, S.E. Freemantle, J. Lunec, A.H. Calvert. Cancer
Res. 52: 1137-1143 (1992)
9. P.G. Johnston, C.M. Liang, S. Henry, B.A. Chabner, C.J. Allegra. Cancer Res. 51: 6668-6676
(1991)

608
THE INTERVAL BETWEEN METHOTREXATE AND LEUCOVORIN

DETERMINES THE EFFICACY OF 5-FLUOROURACIL MODULATION

IN VITRO AND IN VIVO

Clasina L. van der Wilt!, Marjon de Jong 1, Boudewijn J.M. Braakhuis2 ,

Herbert M. Pinedo 1'3, Godefridus J. Peters'

'Dept. Medical Oncology Free University Hospital, PO BOX 7057, 1007

MB Amsterdam, The Netherlands
2 Dept. Otolaryngology and Head and Neck Surgery, Free University

Hospital, Amsterdam
3 Netherlands Cancer Institute, Amsterdam

INTRODUCTION

The toxic effect of methotrexate (MTX) due to a depletion of reduced folates

can be rescued by leucovorin (LV) 1' 2 • In contrast LV can potentiate the activity of 5-
Fluorouracil (FU) due to an increase of the reduced folate cofactor 5,10-methylene-
tetrahydrofolate3'4'5. The modulation of FU by MTX is mediated by an elevation of
phosphoribosyl pyrophosphate (PRPP) after inhibition of the purine de novo synthesis by
MTX, leading to increased formation of active FU nucleotides6 • However the addition of
LV can abrogate the effect of MTX by enhancement of reduced folates and recovory of the
purine de novo synthesis. Thus dosing and scheduling of LV should be very careful since
the potentiating effect of MTX may be negligible when the drugs are given simultaneously
or with a short time interval7 • We studied the time interval of addition of LV and/or FU
after MTX treatment in vitro and in vivo.

METHODS

Two colon cancer cell lines, SW948 (human) and C26-10 (murine), and one
head and neck cancer cell line, UM-SCC-14C were used for growth inhibition tests with
MTX, FU and LV. Cells were seeded in 96 well plates in different densities, depending
or their doubling time. MTX was added at final concentrations of 0, 0.05, 0.1 and 1 p,M,
after 24 h (SW948, UM-SCC-14C) or 48 h (C26-10). Subsequently FU and/or LV was
added after different time intervals: 2, 4, 6, 24 h. Fixed fmal concentrations of LV and FU
were used, 5 p,M and 1 p,M, respectively. The FU concentration corresponded to the IC 50

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 609
of the different cell lines at the drug exposure time applied in these experiments (48 h for
C26-10, 72 h for UM-SCC-14C and 144 h for SW948). Results were evaluated by the
sulforhodamine B (SRB) assay and expressed relative to control growth without drugs,
which was set at 100% 8 •
In vivo experiments were performed with Balb/c mice bearing Colon 26 and
nude mice bearing UM-SCC-14C. Dose and time interval between the drugs were varied.
Antitumor effects were evaluated after two treatment courses and expressed as the growth
delay factor (GDF). GDF indicates the tumor doubling time (TD) gained by treatment and
was calculated as follows: GDF == (TDtreated - TDcontro1)/ TDcontro14

100

.,e. 80
.s::.
i0 60
...Cl

.. 40
Q)
>
!II
20
...
Q)

0
0 0.05 0.1
concentration MTX (~M)

- MTX ~ MTX-> c::J MTX-> CJ MTX->

FU LV FU+LV

Figure 1 Growth inhibitory effects of MTX/FU/LV on the human colon cancer cell line SW948. Time
interval between addition of various concentrations of MTX and FU (I ~M) and/or LV (5 ~M) was 2 h.
Values are means ± SEM of three different experiments.

RESULTS

Additive growth inhibitory effects of MTX and FU (without LV) were observed
in SW948 at all time intervals (2 (Fig. 1), 4, 6 and 24 h), in C26-10 only after 24 hand
in UM-SCC-14C not at all. LV reversed the MTX (without FU) effects completely in all
cell lines, up to the 6 h interval between MTX and LV. At 24 h interval the 80% growth
inhibition of the high concentration of MTX could not be reversed anymore. Potentiation
of the FU effect (without MTX) by LV was seen exclusively in SW948 (Fig. 1, left panel
of bars). Consequently, additive antiproliferative effects of FU, LV and MTX compared
to FU/MTX occurred only in this cell line, but those effects were restricted to the 2 hr
interval between MTX and LV/FU and a relatively low MTX concentration (0.05 ~M)
(Fig. 1). At all other time intervals and higher MTX concentrations (0 .1 and I ~M) a
reversal of the MTX/FU induced growth inhibition was seen in SW948. Similar reversal
effects of LV on the combination with MTX/FU were observed in C26-10, but in UM-
SCC-14C those effects were absent.

610
 MTX potentiated the in vivo antitumor effect of FU in nude mice bearing UM-
SCC-14C tumors (Table 1), at a time interval of 8 h compared to 1 hand 4 h. Addition
of LV to this combination reduced the antitumor effect of MTX/FU when a time interval
of 4 h was used. MTX/FU showed a good antitumor activity compared to FU alone in
mice bearing Colon 26 tumors, but it was very toxic too. A time interval of 17 h between
MTX and FU was more effective than a 4 h interval. The antitumor effect of FU as a
single agent could be enhanced by LV in this tumor with a two-fold increase of and was
comparable to MTX/FU, but a much lower toxicity was observed (8.9% weight loss for
MTX/FU compared to 4.3 % for LV/FU). The addition of LV to MTX/FU treatment did
not increase the antitumor effect and even a tendency to reversal of MTX/FU antitumor
effect was observed.

Table 1 Antitumor effect of MTX/FU/LV combinations on UM-SCC-14C and Colon 26

UM-SCC-14C

<0
0.4

MTX 100 --1 h--> FU 100 1.4

MTX 100 --8 h--> FU 100 2.0
MTX 100 --1 h--> FU 50 --4 h--> FU50 0.2

MTX 100 +LV 50--1 h--> FU 50 +LV 50--4 h--> FU 50 + LV 50 0.2

Colon 26

O.Q7
1.2

MTX 100 -- 4 h--> FU 50 1.1

MTX 100 --17 h--> FU 100 2.1

LV 50--1 h--> FU 50 + LVso 1.5

LV 50--1 h--> FU 100 + LV 50 2.5

MTX 100 --4 h--> LV 50--1 h -->LV 50 0.13

MTX 100 --16 h--> LV 50 --1 h -->LV50 0.06

MTX, 00 --4 h--> LV 50 --1 h--> FUso +LV 50 0.9

MTX, 00 --17 h--> LV 50+ FU,oo 1.6
MTX 100 --16 h--> LV 50 --1 h--> FU 100 +LV50 1.7

The dose of the drugs is indicated by the subscript is mg/kg

DISCUSSION

These experiments showed that the time dependent modulation of FU by MTX

can be influenced negatively by the addition of LV. In vitro we only observed additive
effects of MTX/FU for the two colon cancer cell lines; for C26-10 only after a 24 h

611
interval. Benz and Cadman9 described synergistic effects of MTX/FU for HCT-8, colon
cancer cells when time intervals of 12-24 h were used; for L1210 and CCRF-CEM cells,
which have a very short doubling time, time intervals of 3-4 h were sufficient to obtain
synergystic effects7 •10 • The absence of additive or synergystic effects of sequential MTX/FU
for UM-SCC-14C cells might be related to a low FPGS activity 11 and low accumulation of
MTX-polyglutamates 12 • Optimal sequencing of MTX, FU and LV appeared to be very
difficult. We achieved a very small additive effect of MTX/FU/LV at one MTX
concentration, in one cell line, one time interval of the 36 combinations tested ( 3 cell lines
x 3 MTX concentrations x 4 time intervals, respectively). In an other in vitro study,
Danhauser et a/ 13 also reported no increase of growth inhibition for MTX/FU/LV compared
to MTX/FU.
Preclinical in vivo experiments on sequential MTX/FU described superior
antitumor effects when intervals of 24 h were used instead of 6-12 h14 • Our data showed
a similar trend although different doses of FU were used at 4 and 17 h interval of drug
administration. The addition of LV to this combination seemed to reverse antitumor
activity compared to MTX/FU.
Trials using the combination of MTX/FU/LV are often reported as MTX/FU
trials although varying doses and schedules were used7•15 • Martin 15 postulated that many
trials on the MTX/FU combination were negative because of the too short interval between
MTX and FU. Clinical evidence from a randomized trial was obtained by Browman et a/16 ,
who observed a better anticancer effect at the long interval compared to the short interval.
In a randomized study Glimelius et aP 1 observed a better antitumor activity of MTX/FU
(with LV) compared toFU alone, although in all arms low response rates were observed.
It might however very well be possible that the increased response rate are just due to a
modulating effect of LV on FU, even despite the low dose of LV. Preclinical data,
including this report, do not support the addition of LV to MTX/FU therapy, due to
possible reversal effect of LV.

REFERENCES

1. S. Bernard, M.C. Etienne, j,L. Fischel, P. Formento, G. Milano, Br. J. Cancer 63: 303-307 (1991)
2. F.M. Sirotnak, R.C. Donsbach, D.M. Moccio, D.M. Dorick, Cancer Res. 36: 4679-4686 (1976)
3. K. Keyomarsi, R.G. Moran. J. Biol. Chern. 263: 14402-14409 (1988)
4. J.C. Nadal, C.J. van Groeningen, H.M. Pinedo, G.J. Peters, Invest. New Drugs 7: 163-172 (1989)
5. C.L. van der Wilt, H.M. Pinedo, K. Smid, G.J. Peters, Cancer Res. 52: 4922-4929 (1992)
6. E. Cadman, R. Heimer, L. Davis, Science 205: 1135-1137 (1979)
7. E. Mini, J.R. Bertino, In: Synergism and antagonism in chemotherapy, Academic Press, San Diego
pp. 449-490 (1991)
8. Y. P. Keepers, P. E. Pizao, G.J. Peters, J. van Ark-Otte, B. Winograd, H.M Pinedo, Eur. J. Cancer
27: 897-900 (1991)
9. C. Benz, E. Cadman, Cancer Res. 41: 994-999 (1981)
10. D.J. Fernandes, J.R. Bertino, Proc. Natl. Acad. Sci. U.S.A. 77: 5663-5667 (1980)
11. G.J. Peters, C.L. van der Wilt, J. Cloos, H.M. Pinedo, these proceedings (1993)
12. B.J.M. Braakhuis, G. Jansen, G.J. Peters, these proceedings (1993)
13. L.L. Danhauser, R. Heimer, E. Cadman, Cancer Chemother. Pharmacal. 15: 214-219 (1985)
14. I. Brown, H.W.C. Ward, Cancer Letters 5: 291-297 (1978)
15. D.S. Martin, In: New avenues in developmental cancer chemotherapy, K. Harrap, T. Connors, ed.,
Academic Press, London, pp 113-161 (1987)
16. G.P. Browman, M.N. Levine, M.D. Goodyear, R. Russell, S.D. Archibald, B.S. Jackson, J.E.M.
Young, V. Basrur, C. Johanson, J. Clin. Oncol. 6: 963-968 (1988)
17. B. Glimelius, C. Ginman, S. Graffman, L. Pahlman, E. Stahle, Eur. J. Cancer Clin. Oncol. 22:295-
300 (1986)

612
POTENTIATION OF 5-FLUOROURACIL INDUCED INHIBITION OF

THYMIDYLATE SYNTHASE IN HUMAN COLON TUMORS BY

LEUCOVORIN IS DOSE DEPENDENT.

Godefridus J. Peters*, Klaas Hoekman*, Cees J. van Groeningen*,

Clasina L. van der Wilt*, Kees Smid*, Sybren Meijer@,
Herbert M. Pinedo*,**

Dept. Oncology* and Dept. Surgery@, Free University Hospital,

PO Box 7057, 1007 MB Amsterdam, and
**Netherlands Cancer Institute, Amsterdam, the Netherlands

INTRODUCTION

The schedule and dose dependency of leucovorin (LV) administration in

order to potentiate the antitumor activity of 5-fluorouracil {5FU) is still a unclear
issue. 1•2•3 Traditionally two schedules for single agent of 5FU were used; weekly
administration at 500 mg/m2 and daily (x 5} administration at 300-500 mg/m 2,
which was repeated every 4 weeks. These two schedules of 5FU have been
combined with a variety of LV schedules, which can roughly be divided in high
dose (2 hr infusion of LV at 500 mg/m 2 with 5FU injected mid-infusion}, 4
intermediate dose (2 hr infusion of LV at 200 mg/m 2) 5 •6 and low-dose LV (bolus
injection of LV at 20-25 mg/m2 injected just before 5FU). 6 •7 LV has also been
administered at similar doses but in different schedules. The response rates at
the high and intermediate doses was almost always higher (about 30%} than for
single agent 5FU, but for the low dose (as well as for oral LV) a larger variation
in response rates was observed. 6•7 At a recent symposium on modulation of
5FU 8 a concensus was achieved that exposure to LV should be 2 hrs or longer
in order to facilitate accumulation of polyglutamates of the reduced folate co-
factor. The dose of LV remains un unresolved issue, although it was clear from
preclinical studies that at least a concentration of 1 J.LM should be present in the
culture medium in order to achieve modulation of 5FU. 9 Although both the
intermediate and the high dose of LV result in plasma concentrations of /-LV
above this levels 10 this does not provide information on the intra-tumoral
concentration of LV and the reduced folate cofactor 5,1 0-methylene-tetra-
hydrofolate (CH 2-THF). Only limited information on the CH 2-THF concentration in

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 613
the tumor after administration of a high-dose of LV is available. 11 No information
is available on intratumoral concentration after administration of a low dose.
Recently we completed a study on the inhibition of thymidylate synthase (TS) in
tumors of patients after administration of 5FU or 5FU in combination with high-
dose LV. 11 •13 From this study it was concluded that inhibition of TS was
maintained for at least 48 hr after 5FU administration, while high-dose LV clearly
potentiated this inhibition. This study was extended to the question whether low-
dose LV would also be able to potentiate the inhibition of TS.

PATIENTS AND METHODOLOGY

Biopsy specimens of primary tumors or metastases were obtained during

laparotomy from patients with histologically proven colorectal cancer. Patients
received one i.v. bolus injection of 5FU {500 mg/m2) or LV/5FU prior to surgery,
as described previously. 12"14 All tumor samples were taken between 40 and 50 hr
after drug administration. Sixteen patients received 5FU alone; 11 patients
received high-dose LV (HD-LV; 500 mg/m2 as a 2-hr i.v. infusion; with the i.v.
bolus injection of 5FU midway infusion); and 8 patients received low-dose LV
(LD-LV; 25 mg/m2 as a bolus just before 5FU). Biopsy specimens were
immediately frozen in liquid nitrogen and subsequently stored at -80 oc.
Frozen tissue samples were pulverized using a micro-dismembrator and
processed as described previously. 11 •13•14 In the tissue extract we measured the
activity of TS with both the FdUMP binding assay (with [6- 3H]-FdUMP giving the
number of FdUMP binding sites) and the tritium release assay (with [5-3 H]-dUMP
giving the conversion of dUMP to dTMP) at a substrate concentration of 1 1-LM
dUMP. The inhibition of TS was evaluated by comparison of the FdUMP binding
and the catalytic activity in tumor samples with (total activity; TS-tot and TS-tota/,
respectively) and without dissociation (inhibited activity; i.e. the number of free
binding sites and the residual activity; TS-free and TS-res, respectively) of
FdUMP from the ternary complex between FdUMP-TS-CH 2 -THF. The number of
FdUMP binding sites and the catalytic activity was measured as described
previously. 11 •14

RESULTS

For all patients a large variation was found in both the number of total
FdUMP binding sites (0-383 fmol/mg protein, median 70; 35 patients) and the
total catalytic activity (0-178 pmol/hr/mg protein, median 35; 35 patients). A
significant inhibition of TS was observed in tumor samples of patients who
received 5FU as a bolus injection (Table 1). In patients receiving HD-LV a clear
potentiation of the inhibition of TS was observed, as could be evaluated using
both assays. However, in patients receiving the LD-LV the inhibition of TS was
clearly less than with HD-LV, although the inhibition as evaluated using the
FdUMP binding assay was more pronounced than with 5FU alone. However,
when using the catalytic assay for evaluation of the results, the effect of LD-LV is
lower than that of HD-LV and an even slightly higher TS activity was observed
than for 5FU alone. It should be noted that the TS activity was measured at a low
non-saturating dUMP concentraiton of 1 1-LM; however, at a substrate concentra-
tion of 10 1-LM dUMP, which is saturating, a similar relative inhibition was obser-
ved as at 1 1-LM dUMP. The inhibition of TS was selective for the tumor tissue,

614
since the inhibition of TS in normal liver and normal mucosa in these patients
was not affected by LV (both LD-LV and HD-LV; data not shown). The number of
free FdUMP binding sites in normal liver was 70-80% for all three protocols. For
the catalytic activity (at 1 J.LM) these values ranged from 69 to 106%.

Table 1. Inhibition of TS in biopsy specimens of colon tumors after

administration of a test dose of 5FU
or 5FU combined with HD-LV or LD-LV

Treatment Inhibition of TS evaluated with:

the FdUMP binding assay the catalytic assay

5-FU alone 49.0 {14} 74.0 {18}

HD-LV + 5-FU 24.0 {12}* 49.0 {12}*
LD-LV + 5-FU 32.0 {9) 94.7 (9)

Values (calculated as ratios x 100%, between TS-free[f'S-tot or TS-res{f'S-tota/, respectively)

represent means from the number of samples indicated within parentheses. The means were
calculated from the separate samples.
Significantly different from 5FU alone at the level; significantly different from 5FU at the level; *,
0.002 < p < 0.02; (Mann Whitney U test).

DISCUSSION

In this paper we demonstrate that the 5FU induced inhibition of TS in

patients can be enhanced significantly by HD-LV, but not or only to a minor
extent by LD-LV. The inhibition by 5FU was selective, only a minor inhibition of
TS was observed in normal tissues.
Dosing of LV is a major clinical problem. From preclinical studies8 •9 it is
clear that a prolonged exposure at concentrations of at least 0.1 J.LM LV is
required to achieve the desired potentiating effect. However, for most cell lines a
higher LV concentration is required, even higher than 1 J.LM. Other cell lines can
however even not be modulated at these high LV concentrations. 15 Since the
requirement for each tumor is not known and can not be determined in a routine
clinical setting, the most optimal LV schedule and dose should be used.
Besides the concentration dependence the in vitro studies also
demonstrated a schedule dependence of LV modulation. After intracellular
uptake LV has to metabolized to CH 2-THF, the cofactor for TS. The binding of
FdUMP to TS will be enhanced after folate polyglutamylation, 16 thus it is of
advantage that accumulation of CH 2-THF-polyglutamates is maximal. It has been
observed that polyglutamylation is a time-dependent process, and
polyglutamates accumulate in time. Thus, a longer exposure at higher
concentrations will result in an optimal polyglutamylation. Also in vivo data
indicate that a prolonged exposure to LV is advantageous compared to a short
exposure. 14' 17
Up to now no clinical biochemical data were available supporting either a
LD-LV or a HD-LV. The clinical observations on the antitumor activity of LD-LV +
5FU were contradictory for colon cancer, 6•7 although this difference might also be
related to scheduling. However, when using the 25 mg/m2 dose of LV the
potentiation of 5FU induced TS inhibition was lower than for the HD-LV. This

615
effect was even more pronounced for the catalytic assay. It should be noted that
the catalytic assay represents the actual biochemical proces to be inhibited in
the tumor. Although up to now a limited number of patients could be entered in
the LD-LV arm, these preliminary data suggest that LD-LV is not sufficient for
most patients to achieve the desired potentiation of TS inhibition. Pending
confirmation of this pattern in more patients it seems desirable to treat patients at
a relatively high dose of LV.

REFERENCES
1. G.J. Peters and C.J. van Groeningen. Ann Onco/2: 469-480 (1991)
2. C.-H. Kohne-Wompner, H.-J. Schmoll, A. Harstrick, Y.M. Rustum. Sem Onco/19 (sup):
105-125 (1992}
3. P. Piedbois, M. Buyse, Y. Rustum, D. Machover, C. Erlichman, R.W. Carlson, F. Valone,
R. Labianca, J.H. Doroshow, N. Petrelli J Clin Onco/10: 896-903 (1992}
4. N. Petrelli, L. Herrara, Y. Rustum, P. Burke, P. Creaven, J. Stulc, L.J. Emrich, A.
Mittelman. J Clin Onco/ 5: 1559-1565 (1987)
5. C. Erlichman, S.W. Fine, Wong et al. J Clin Oncol 6: 469-475 (1988}
6. M.J. O'Connell. Cancer 63: 1022-1025 (1989}
7. N. Petrelli, H.O. Douglas Jr, L. Herrera, et al. J Clin Onco/7: 1419-1426 (1989)
8. G.J. Peters, Y.M. Rustum. Ann Onco/4: in press (1993)
9. E. Mini and J.R. Bertino. In: Synergism and Antagonism in Chemotherapy (Acad. Press),
449-506 (1991)
10. P. Trave, Y.M. Rustum, N.J. Petrelli, L. Herrera, A. Mittelman, C. Frank, P. Creaven.
J Clin Oncol 6: 1184-1191 (1988}
11. G.J. Peters, C.J. van Groeningen, C.L. van der Wilt, S. Meijer, K. Smid, E. Laurensse,
H.M. Pinedo. Sem Onco/19 (Sup): 26-35 (1992)
12. G.J. Peters, C.J. van Groeningen, C.L. van der Wilt, K. Smid, S. Meijer, H.M. Pinedo HM.
Adv Exp Med Bio/309A: 131-134 (1991)
13. G.J. Peters, C.L. van der Wilt, C.J. van Groeningen, K. Smid, S. Meijer, H.M. Pinedo.
submitted for publication
14. C.L. van der Wilt, H.M. Pinedo, K. Smid, G.J. Peters. Cancer Res 52: 4922-4928 (1992)
15. C.L. van der Wilt, M. de Jong, B.J.M. Braakhuis, H.M. Pinedo, G.J. Peters.
These proceedings
16. S. Radparvar, P.J. Houghton, J.A. Houghton. Arch Biochem Biophys 260: 342-350
(1988)
17. J.C. Nadal, C.J. van Groeningen, H.M. Pinedo, G.J. Peters. Invest New Drugs 7: 163-172
(1989}

616
INTERACTION WITH 2(4)-THI0-5-FLUORO-dUMP
OF THYMIDYLATE SYNTHASES WITII DIFFERING
SENSITIVITIES TO 5-FLUORO-dUMP

Jolanta M. Dzik, 1 Zbigniew Ziefulski, 1 Joanna CieSla, 1 Wojciech Rode, 1

Maria Bretner, 2 Tadeusz Kulikowski, 2 and David Shugar2

1Nencki Institute of Experimental Biology

Polish Academy of Sciences, 3 Pasteur St.
02-093 Warszawa, Poland
2Jnstitute of Biochemistry and Biophysics
Polish Academy of Sciences, 36 Rakowiecka St.
02-532 Warszawa, Poland

INTRODUCTION

To determine how modifications of 5-fluoro-dUMP (FdUMP), a thymidylate synthase

(EC 2.1.1.45) chemotherapeutically active inhibitor, may affect its specificity, a study was
conducted on inhibition of the enzyme from parental (Ll210P) and 5-fluoro-dUrd-resistant
(L1210R) mouse leukemia L1210 cells, the tapeworm Hymenolepzs diminuta (H.d.) and
regenerating rat liver (RRL) by FdUMP and its 2-thio- and 4-thio- congeners. The enzymes
differed in sensitivity to time- and methylenetetrahydrofolate- dependent inactivation by
FdUMP, with Ki values ranging from 10-9M for the Ll210P and 10-8 M for the Ll210R1
and RRL enzymes,2 to 1o-7 M for the H. d enzyme. 2

MATERIALS AND METHODS

2-Thio-5-fluoro-dUMP (2-thio-FdUMP) and 4-thio-5-fluoro-dUMP (4-thio-FdUMP)

were synthesized as previously described.3,4 Highly purified preparations of thymidylate
synthase from Ll210P, L1210R, RRL and H.d (specific activities of0.26, 0.35, 1.5 and 0.9
!J.mo1inin/mg, respectively), were described elsewhere.2,5 Enzyme assays and identification
of the type of inhibition involved were as previously described. 6 Quantitative analyses of
thymidylate synthase inhibition, leading to time-dependent inactivation of the enzyme, were
performed as earlier reported.1

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 617
RESULTS

Inhibition of the enzymes from Ll210P, Ll210R, RRL and H. d., by competition vs
dUMP, of 2-thio- and 4-thio- analogues of FdUMP, was examined by varying the dUMP
concentration with different concentrations of inhibitor, added simultaneously to the reaction
mixture. Competitive inhibition was indicated by intersection at the ordinate of
Lineweaver-Burk plots (not shown), described by the apparent Ki values presented in Table
1.

Table 1. Apparent Ki values for inhibition of thymidylate synthases

from L1210P, Ll210R, RRL and H.d by FdUMP, 2-thio-FdlJMP
and 4-thio-FdUMP.
Ki(j.lM)
Analogue
Ll210P Ll210R RRL H. d.

FdUMP 0.02 0.03 O.o2 0.02

2-Thio-FdUMP 0.07 0.14 0.22 0.30
4-Thio-FdUMP 0.57a 0.44a 0.58 0.22

aMean result of two experiments differing not more than by l 0%

Regenerating rat liver T S

100 Regenerating rat liver TS

0.33
[2-thio-5-fluoro-dUMP] (pM)

:;::
·;;
=fi
~
• 0.07
:;:: 40
:2:
<1:
~
~
~0.11
u
<1:
~2.00
~
20
~ 0.27
0.34
20

100 6 8 10 10
2 4 0 2 4 6 8 10

100 Tapeworm TS Tapeworm TS

~o•
60

:;:: ~0.14 :;:: 40

·;;
• 0.67
:~
u ~0.20 n
<1: ~0.34 <1:
1.00
~ 0.51 ;l<
0
A 1.33
20 20

10o:---=2-~4:---6!--~8-----:1'::-0- -
Preincubation time (min) Preincubation time (min)

Fig. 1. Slow-binding inhibition of RRL (upper panels) and H. d. (lower panels) thymidylate synthase (TS) by
2-thio-FdUMP (left panels) and 4-thio FdUMP (right panels).

618
 When preincubated with each of the enzymes studied, in the presence of N5, 10-meth-
ylenetetrahydrofoJate, the FdUMP analogues caused time-dependent inactivation (Fig. 1),
consistent with the behaviour of each as a slow-binding inhibitor. 7 The inactivation rate
usually decreased after about 2 min preincubation. reflected by a biphasic plot of log (re-
maining activity) vs time (Fig. 1), consistent with differing interactions of each inhibitor with
the two binding sites on the thymidylate synthase molecule. Consequently, inhibition con-
stants and inactivation rate constants were calculated with the use of apparent inactivation
rate constants during the initial (0.0-1.5 min) and later (4-10 min) periods of preincubation
v1rith a given inhibitor at various concentrations. The corresponding inhibition constants and
inactivation rate constants are then .Ki' and k2', and .Ki" and k2", respectively (Tables 2 and
3). Only for inactivation of the RRL enzyme by 4-thio-FdUMP did the inactivation rate not
change during preincubation (Fig. 1, Table 3).

Table 2. Parameters for inactivation by FdUMP, 2-thio-FdUMP and 4-thio-FdUMP of

thymidylate synthase from L1210P and L1210R
Enzyme source ~· (nM) ~" (nM) k2' (nrin-1) k2" (min-1)

FdUMPa
L1210P 1.8 ± 0.4 (6)b 20 ± 5 (4) 0.17 ± 0.02 (6) 0.12± 0.04 (5)
L1210R 12.2 ± 1.4 (6) 14±3(4) 0.25 ± 0.04 (6) 0.06 ± 0.02 (4)

2-thio-FdUMP
Ll210P 41 ± 9(3) 46± 25 (3) 0.12 ± 0.02 (3) 0.02 ± 0.01 (3)
Ll2lOR 297 ± 93 (3) 93 ± 31 (3) 0.40 ± 0.04 (3) 0.04 ± 0.01 (3)'

4-thio-FdUMP
L1210P 102 ± 36 (3) 202± 36 (3) 0.21 ± 0.04 (3) 0.05 ± 0.00 (3)
Ll210R 14 ± 4(5) 34± 9 (5) 0.11 ± 0.03 (5) 0.03 ± 0.01 (5)

aFromrefl
bResults are presented as means ± SEM, followed by the number of separate experiments in parentheses.

Table 3. Parameters for inactivation by FdUMP, 2-thio-FdUMP and 4-thio-FdUMP of

thymidylate synthases from RRL and H. d.
Enzyme source Ki'(nM) K((nM) kz' (min-I) k2" (min-I)

FdUMP
RRL 10± 1 (3) 15± 5 (3) 0.29 ± O.o7 (3) 0.14±0.03(3)
H. d. 146 ± 32 (3)a 25± 3 (3) 1.22 ± 0.17 (3) 0.09 ± 0.03 (3)

2-Thio-FdUMP
RRL 64± 10(3) 14± 4(3) 0.30 ± 0.08 (3) 0.03 ± 0.01 (3)
H. d. 632 ± 179 (3) 52± 7 (3) 0.82 ± 0.09 (3) 0.22 ± 0.02 (3)

4-Thio-F-dUMP
RRL 850 ± 40 (4) 0.10 ± 0.03 (4)
H. d. 910 ± 290 (3) 1,600 ± 610 (3) 0.22 ± 0.01 (3) 0.11 ± 0.02 (3)

3 Results are presented as means ± SEM, followed by the number of separate experiments in parentheses.

619
 Relative to FdUMP, inactivation by 2-thio-FdUMP was about 5-fold weaker for the
H.d and RRL enzymes, and about 20-fold weaker vs the L1210P and L1210R enzymes. By
contrast, although 4-thio-FdUMP was not a better inactivator of the H.d enzyme than the 2-
thio analogue, it was significantly less active vs RRL (Ki ~w-6 M) and L1210P (Ki ~w-1
M) enzymes, but more potently inactivated the L1210R enzyme (Ki ~to-8 M) lhan 2-thio-
FdUMP.
Hence 4-thio-FdUMP was more specific vs the Ll210R, relative to the L1210P (the
reverse of that observed with FdUMP) or RRL (the same but less pronounced relation found
with FdUMP) enzyme forms. Furthermore, while both FdUMP and 2-thio-FdUMP were
distinctly more effective inhibitors ofRRL than of H. d. thymidylate synthase, 4-thio-FdUMP
lacked their specificity. These findings, in conjunction with those describing similar specific-
ity variations concerning inactivation of different enzyme forms by N4-hydroxy-5-fluoro-
dCMP, relative to FdUMP, 1 suggest that modification of the C(4)=0 of FdUMP may pro-
vide some control over selective inactivation of thymidylate synthase from different sources.

ACK...""l'OWLEDGMENTS

Supported by the State Committee for Scientific Research (Grant No. 0071/P2/92/03).

REFERENCES

I. W. Rode, Z. Zieliilski, J.M. Dzik, T. Kulikowski, M. Bremer, B. Kierdaszuk, J. Ciesla, and

D. Shugar, Biochemistry 29: I 0835 (1990)
2. J. Ciesla, W. Rode, M. Kernpny, K. Pawelczak, and B. Rzeszotarska, in: "Chemistry and
Biology ofPteridines 1989. Pteridines and Folic Acid Derivatives," H.-Ch. Curtius,
S. Ghisla, and N. Blau, eds., Walter de Gruyter, Berlin (1990) p.829.
3. J. M. Dzik, T. Kulikowski, Z. Zieliilski, J. Ciesla, J., W. Rode, and D. Shugar,Biochem. Biophys. Res.
Commun. 149:1200 (1987).
4. M. Bretner, T. Kulikowski, J.M. Dzik, W. Rode, and D. Shugar, Collect. Czech. Chem. Commun. 55, Sp.
Iss. I: I09 (1990).
5. Z. Zieliilski, J.M. Dzik, W. Rode, T.Kulikowski, M. Bretner, B. Kierdaszuk., D. Shugar, in:" Chemistry and
Biology ofPteridines 1989. Pteridines and Folic Acid Derivatives," H.-Ch. Curtius, S. Ghisla, and
N. Blau, eds., Walter de Gruyter (1990) p. 817.
6. W. Rode, T. Kulikowski, M. Jastreboff; and D. Shugar, Biochem. Pharmacal. 33:2699 (1984).
7. J.F. Morrison, Trends Biochem. Sci. 7:102 (1982).

620
MECHANISM OF THYMIDYLATE SYNTHASE
INHIBITION BY N4-HYDROXY -(N4-HYDROXY-
5-FLUORO)-dCMP IN VIEW OF THE STRUCTURE
AND CONFORMATION OF N4-HYDROXY-
(N4-HYDROXY -5-FLUORO)-CYTOSINE CALCULATED
BY THE AB INI110 QUANTUM MECHANICAL METHODS

Andrzej Les,1,2 Ludwik Adamowicz,2 and Wojciech Rode3

1Department of Chemistry, University of Warsaw,

1 Pasteur St., 02-093 Warsaw, Poland
2Department of Chemistry, University of Arizona,
Tucson, Arizona 85721, U.S.A.
3Nencki Institute of Experimental Biology, Polish Academy
of Sciences, 3 Pasteur St., 02-093 Warsaw, Poland

INTRODUCTION

Thymidylate synthase (EC 2.1.1.45) slow-binding inhibition by N4-hydroxy-dC:MP

(oh4dCMP) was shown to depend on confonnation of the exocyclic N4-0H group, with the
anti rotamer, relative to the ring N(3), indicated as an active species.1 Potentiation of inhibi-
tion by the 5-fluoro substituent, observed for N4-hydroxy-5-fluoro-dCMP (oh4f.idCMP), was
explained in terms of hydrogen bonding between the N4-0H and C(5)-F groups, influencing
an assumed syn-anti equilibrium by stabilization of the anti rotamer.l In order to test the latter
hypothesis two cytosine analogues, N4-hydroxy-cytosine (oh4C) and N4-hydroxy-5-fluoro-
cytosine (oh4f5C), were theoretically studied, and their molecular structures determined, by ab
imtio quantum mechanical methods.

THEORETICAL CALCULATIONS

Structural and energetic properties of amino and imino tautomers, and syn and anti ro-
tamers of oh4c and oh¥C were calculated. Optimal molecular geometries and molecular
energies were obtained within the Self Consistent Field (SCF) method corrected for the
electron correlation effects by the second-order Many Body Perturbation Theory (SCF +
MBPT(2)). Molecular structures were optimized by the SCF method with the standard 3-21G
basis set andre-optimized with the use of the SCF procedure and the 6-31 G** Gaussian ba-
sis set. The calculations were perfonned with the use of the GAUSSIA...""J 90 program.2 While
geometries of the imino fonns were assumed planar, in the amino forms where significant

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 621
non-planarity, especially in the N4-0H region, was expected, no geometrical constraints were
imposed.

RESULTS AND DISCUSSION

The results of the ab initio calculations are presented in Tables 1 and 2. The relative total
en~es of various tautomers and rotamers are also shown on Fig. 1. For each oh4C and
oh4f.'C the most stable is the imino-syn and the next stable the imino-anti form, with the 5-
fluoro substituent raising the energy difference between the syn and anti rotamers over 3-fold.
Such an unexpected effect of the 5-fluoro substituent can be rationalized based on the atomi-
zation energies, defined as the diference between the total energy of a molecule (Tables 1 and
2, SCF/6-31G** values) and the sum of the atomic energies (values from ref.3). A compari-
son of atomization energies shows that the intetnal bonding effect in oh4c is weakened by
some 200 kJ mot·l when the fluorine atom replaces the hydrogen atom at the C( 5) position.
As a consequence the imino-anti form of oh4f5C with the internal hydrogen bond is signifi-
cantly less favourable than the non-substituted oh4C molecule (Fig. 1).

Table 1. Theoretical ab initio calculations for oh4c in Tl, T3, T5 and T7 tautomeric forms
(see Fig. 1)
Tl T3 T5 T7

Energy (a.u.)
SCFI3-21Ga -464.813924 -464.810601 -464.790623 -464.791823
SCF/6-31G**b -467.437426 -467.433300 -467.421148 -467.423744
MBPT(2)c -1.353234 -1.352349 -1.353498 -1.351360
ZPEd 0.100885 0.100839 0.100503 0.101134
Total energy e -468.689774 -468.684910 -468.674143 -468.673970
Relative f total
energy (kJ/mol) 0.00 12.77 41.05 41.49

a SCF/3-21G//3-21G calculations.
b SCF/6-31G**//6-31G** calculations.
c MBPT(2)/6-31G**//6-31G** calculations.
d ZPE is the zero-point nuclear vibration energy. A common factor of0.9 was used to scale down all
hannonic frequencies obtained with the SCF/6-31G**//6-3IG** method.
e SCF(6-31G**)+MBPT(2)(6-31G**)+0.9*ZPE(6-31G**).
f 1 a.u. of energy= 2625.5 kJ/mol.

Table 2. Theoretical ab initio calculations for oh4f.SC in T2, T4, T6 and T8 tautomeric forms
(see Fig. 1)
T2 T4 T6 T8

Energy (a.u.)
SCF/3-21Ga -563.122552 -563.105322 -563.102126 -563.099148
SCF/6-3IG**b -566.271677 -566.254868 -566.256150 -566.254941
MBPT(2)c -1.515117 -1.516875 -1.516380 -1.515006
ZPEd 0.093133 0.092743 0.092573 0.093352
Total energy e -567.693661 -567.679000 -567.679957 -567.676595
Relative f total
energy (kJ /mol) 0.00 38.49 35.98 44.81

a-f See footnotes to Table l.

622
 H
.o.E I
H/O'N/H H,N/0

j):JJ:
kJ mor1

50

I I
H H
40

H,
/0
30 N

" ' NJ " I

20 /H
o,N O~NI H
41.0 41.5

"':)" _t
H
10 N I
O.(N H
I
H
0
r, T3 Ts Tl

.o.E H,N/O'IIH
kJ mor1
N
/o,H
.
/'N/H
N~'
~ I
50 H,NJF : )F 0
.N::?' N
I N
H,
N
/0 O~N I H ~ IH H
I 0 N
40 H I
H'NJF H

30
O.(N IH
I
H

/H 44.8
20 o, 38.5

"'J'
N 36.0

10 N I 25.3

O.(N H
I
H
0
T1 Tn T4 Ts Ta

Figure 1. Total energy differences for various tautomeric forms of oh4c (upper panel) and oh4f 5c (lower
panel). Energies ofTl and T2 forms are taken as the reference values (see Tables 1 and 2).

In order to estimate the barrier height for the rotation of the hydroxyl about the C( 4)=N4
double bond, oh4C feometry was optimized with the SCF/3-21G method assuming the tetra-
hedral N(3)-C(4)-N -0 angle fixed to 90 degrees. One of the harmonic frequencies, calcu-
lated after the optimiztion process had been completed, was imaginary indicating a transition
state structure (Fig. 2). Single-point SCF + :tvmPT(2) calculations, performed with the 6-
31G** basis set on the transition state structure, showed the energy of the latter to be by
184.5 kJ/mol higher than the energy of the non-rotated syn-imino form. Thus the rotation bar-
rier appears to be high enough to prevent the syn-anti flip of the imino N4(0H) group at any
temperatures relevant to biochemical processes. In consideration of a possibility of syn-imino-

623
Figure l. Stereo-view of the oh4c transition state. The chemical bonds are marked by the thick solid lines. Thin
lines are added to make the transirion structure more transparent. Circles ofincreasing diameter denote
hydrogen, carbon, nitrogen, and oxygen atoms.

oh4c and syn-amino-oh4c enerf difference reduction, due to the C(5)-C(6) bond saturation
in the thymidylate synthase - oh dCMP - methylenetetrahydrofolate ternary complex, 4 the to-
tal energy difference between amino and imino tautomers of syn-oh4c with saturated C(5)-
C(6) bond was calculated. Since it was found to be increased up to 74.4 kJ/moL then the
C(5)-C(6) bond saturation should not facilitate an imino --. amino transformation. Moreover,
the rotation of the amino-like N4(0H)(H) group must be also strongly hindered, since the
apparently single C(4)-N4 bond becomes conjugated with the ring system. The latter
statement is based on the calculated values of the Mulliken overlap populations, a by-product
of the SCF calculations, indicating that the "single" C(4)-N4 bond in syn-amino-oh4c is
stronger than N(l)-C(2) or N(l)-C(6) bonds in the aromatic ring. In agreement with the
foregoing, studies of 1-methyl-N4-hydroxycytosine hydrochloride crystals showed the C(4)-
N4 bond (1.30 A)5, in the protonated oh4c residue, to be longer than the double bond in the
imino form (1.29 A)6 but shorter than the single bond in cytosine (1.32-1.33 A)7,8.
The theoretical ab initio results suggest that (i) the 5-fluoro substituent in oh4f5dCMP
potentiates thymidylate synthase inhibition by a mechanism different than the intramolecular
hydrogen bond formation and (ii) the syn-imino and anti-imino forms of oh4(oh4f5)dCMP, at
temperatures relevant to biochemical conditions, can be treated as two structural isomers that
do not interconvert, and thus are not in thermodynamic equilibrium as formerly assumed. Fur-
thermore, it appears that obtaining the anti rotamer of oh"(oh4f5)dCMP in pure form should
be possible via either separation from the syn rotamer or stereospecific synthesis.

ACKNOWLEDGMENTS

A. Les and W. Rode were supported by the State Committee for Scientific Research
(Grant Nos KBN-CHEM-BST-412/23 and 0071/P2/92/03, respectively), and L. Adamowicz
by the American Cancer Society Junior Faculty Research Award.

REFERENCES
1. W. Rode, Z. Zieliil.ski, J.M. Dzik, T. Kulikowski, M. Bretner, B. Kierdaszuk. J. Ciesla, and D. Shugar,
Biochemistry 29: I 0835 (1990).
2. GAUSSIAN 90, Revision I, M.J. Frisch, M. Head-Gordon, G.W. Trucks, J.B. Foresman, H.B. Schlegel,
K. Raghavachari, M. Robb, J.S. Binkley, C. Gonzalez, D.J. Defrees, D.J. Fox, R.A Whiteside,
R. Seeger, C.F. Melius, J. Baker, R.L. Martin, L.R. Kahn, J.J.P. Steward, S. Topiol, and J.A
Pople, Gaussian, Inc., Pittsburgh PA (1990).
3. J. Leszczyitski,J Phys. Chern. 96:1649 (1992).
4. S. Goldstein, A.L. Pogolotti, Jr., E.P. Garvey, and D. Santi,J. Med. Chern. 27:1259 (1984).
5. G.I. Birnbaum, T. Kulikowski, and D. Shugar, Can. J Biochem. 57:308 (1979).
6. D. Shugar, C.P. Huber, and G.I. Birnbaum, Biochim. Biophys. Acta 447:274 (1976).
7. D.L. Barker, and R.E. Marsh, Acta Cryst. 17:1581 (1964).
8. R.J. McClure, and BM. Craven, Acta Cryst. 26:20 (1973).

624
SULPHONAMIDE ANTIFOLATES INHffiiTING
THYMIDYLATE SYNTHASE: SYNTHESIS, ENZYME
INHffiiTION AND CYTOTOXICITY

K. Pawelczak, 1 M. Makowski, 1 M. Kempny, 1

J. M. Dzik, 2 M. Baliriska, 2 W. Rode2

1Institute of Chemistry, Pedagogical University of Opole

48 Oleska St., 45-052 Opole, Poland
2Nencki Institute of Experimental Biology,
Polish Academy of Sciences,
3 Pasteur St., 02-093 Warszawa, Poland

INTRODUCTION

Two quinazoline-based antifolates possessing a wide spectrum of antitumor activity,

10-propargyl-5,8-dideazafolic acid 1 and its 2-desamino-2-methyl congener 2 are strong
thymidylate synthase (TS) inhibitors1. The present study concerns the effect of
replacements of (i) the p-aminobenzoyl with p-aminobenzenesulphonyl moiety in parent
compounds 1 and 2 and (ii) glutamyl residue with glicine, alanine, valine, norvaline, or
phenylglycine in sulphonamide analogue of 2 (compound 4). Seven new analogues 3-9 of
1 and 2 were synthesized and their activities as inhibitors of Ehrlich carcinoma thymidylate
synthase and mammalian tumor cell growth tested.

RESULTS AND DISCUSSION

The sulphonamide antifolates 3-9 were synthesized as in Fig.l. General synthetic

approach was similar to that used in synthesis of 1 and 21•2 . 4-nitrobenzenesulphonyl
chloride was coupled with appropriate tert-butyl ester of amino acid H2NR" to give
nitroderivative (I) which after reduction by catalytic hydrogenation gave tert-butyl ester of
N-(4-aminobenzenesulphonyl)amino acid (II). Alkylation with propargyl bromide gave
secondary amine (III). Further alkylation with 2-amino- or 2-methyl-(6-bromomethyl)-4-
hydroxyquinazoline gave antifolate esters (IV). Removal of the tert-butyl groups in the last
step was accomplished with trifluoroacetic acid (TFA) to give appropriate antifolates (V),
3-9. The structure and purity of all compounds were estabilished by elemental analysis,
1HNMR spectroscopy and the final products were additionaly characterized by FAB-mass
spectrometry.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 625
 C:CH
I
CH 2
H~~S02 - NHR" H2N - o S 02 - NHR"

( Ill )
( II )

c=cH
I
0 CH R = NH2 , CH 3

~m-CH2-~-oSO,- NHR' R" = tert-butyl esters

R' = free acids

R (IV)

l TFA
c=cH
I
0 CH

H~)~CH2-~-oS02 - NHR'
AN
R (V)

Figure 1. Synthesis of sulphonamide antifolates

Inhibition of highly purified Ehrlich carcinoma TS 3 was examined by varying the

5, 10-methylenetetrahydrofolate concentration with different concentrations of inhibitor,
added simultaneously to the reaction mixture4. A mixed type inhibition by each analog
studied was indicated by Lineweaver-Burk plots. It was described by the apparent ~
values presented in Table 1. L5178Y cells were plated with density 105 cells per ml,
grown for 4h and then exposed to different concentrations of inhibitor for 48h. Direct cell
counting was conducted with inverted light microscope, following staining with 0. 02%
trypan blue. Each experiment was conducted 3 times in triplicates. The IC50 values
describing cell growth inhibition are shown in Table 1.
While the replacement of CONH by S02NH in the parental compound 1 resulted in
10-fold weaker inhibition of both the enzyme and cell growth (Table 1, compound 3), the
same modification of the compound 2 caused 19-fold loss in TS inhibition potency but over
800-fo1d loss in cell growth inhibition potency (Table 1, compound 4). Substitution of the
glutamyl residue in the compound 4 with norvaline resulted in 5-fold stronger TS inhibition
and unchanged cell growth inhibition (Table 1, compound 9), whereas similar substitutions

626
Table 1. 10-Propargyl-5,8-dideazafolic acid 1, 2-desamino-2-methyl-10-propargyl-5,8-di-
deazafolic acid 2 and their analogs 3-9. Structures and parameters of Ehrlich carcinoma
thymidylate synthase and L5178Y cell growth inhibition.

C::CH
I
0 CH

:m-CH,-~-o-X-NHR'
Ki,JlM TC50, JlM

No. R X NH-R' Ehrlich carcinoma TS L5178Y cells

COOH

1 H 2N co HN~COOH 0.008 ± 0.002 2

COOH

2 CH3 co HN~COOH o.o1oa 0.06

COOH

3 H 2N so2 HN~COOH 0.082 ± 0.008 20

COOH

4 CH3 so2 HN~COOH 0.192 ± 0.010 50

COOH
5 CH3 502 HN_/ 0.303 ± 0.061 300

COOH
6 CH3 502 HNi- 0.386 ± 0.074 300

COOH
7 CH3 502 HN-< 0.184 ± 0.022 250

COOH
8 CH3 so2 HN-<o 0.150 ± 0.020 80

COOH
9 CH3 502 HN~ 0.048 ± 0.001 60

a From ref. 1

with glycine, alanine, valine and phenylglycine were either without a distinct effect
(Table 1, compound 8) or lowered inhibitory potency (Table 1, compounds 5-7).

627
ACKNOWLEDGMENTS

Supported by the State Committee for Scientific Research Grant no. 6 6254 92 03.

REFERENCES
1. L.R. Hughes, A.L Jackman, J. Oldfield, R.C. Smith, K.D. Burrows, P.R. Marsham, J.A.M. Bishop,
T.R. Jones, B.M O'Connor and A.H. Calvert, J. Med. Chern. 33:3060 (1990).
2. T.R. Jones, A.H. Calvert, A.L. Jackman, S.J. Brown, M Jones and K.R. Harrap, Eur. J. Cancer 17:11
(1981).
3. M. Jastreboff, B. K~dzierska and W. Rode, Biachem. Pharmacal. 31:217 (1982).
4. W. Rode, T. Kulikowski, M. Jastreboff and D. Shugar, Biachem. Pharmacal. 31:2299 (1984).

628
FOLYLPOLY-y-GLUTAMATE SYNTHETASE

Barry Shane, Tim Garrow, Alfred Brenner, Linda Chen, Yun-Jung Choi,
Juei-Chuan Hsu and Patrick Stover

Department of Nutritional Sciences

University of California
Berkeley, California 94720

INTRODUCTION

Folylpolyglutamate synthetase (FPGS) catalyzes the addition of glutamate residues to

folates and antifolates to form the physiological active coenzymatic forms of the vitamin and
more potent anti-folate agents (reviewed in reference 1). Studies on FPGS have been
hampered by the low abundance and instability of the protein. We previously reported the
purification of the Corynebacterium, Lactobacillus casei, Escherichia coli and pig liver
proteins2-5, and the L. casei and E. coli FPGS genes have been cloned and the proteins
expressed at high levels4,6,7. Although the bacterial proteins are useful for studying the
mechanism of the FPGS reaction, their specificities for folate substrates are markedly
different from the mammalian proteins. Consequently, they are not good models for studying
how substrates interact with the mammalian protein. To aid in the further characterization of
the eukaryotic protein, we have cloned yeast and human genes for FPGS and have
overexpressed and purified the gene products. This has allowed studies on the mechanism of
FPGS and on the regulation of its expression in mammalian cells.

RESULTS AND DISCUSSION

Cloning of Folylpolyglutamate Synthetase Genes and Properties of the Gene

Products

Three distinct yeast genes were isolated that complemented an E. coli FPGS- mutant.
The genes were over-expressed in E. coli and the gene products were characterized. The first
gene encoded a dihydrofolate synthetase (DHFS) which was purified to homogeneity. The
properties of this enzyme are compared to bacterial FPGS/DHFS enzymes in Table 1. Yeast
DHFS lacks any FPGS activity and pteroylmonoglutamates have little, if any, affinity for the
protein. This is in sharp contrast to the Corynebacterium and E. coli proteins which possess
both DHFS and FPGS activities. Yeast DHFS shares limited (approx. 30 percent) amino acid

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 629
identity with the L. casei FPGS, although the latter lacks DHFS activity, and slightly less
identity with the E. coli protein. All these proteins are monomeric and are of similar size.
Their catalytic turnover numbers with their optimal pteroyl substrates, which differ for the
different enzymes, are also similar and are quite slow, with the yeast DHFS being the least
efficient.

Table 1. Properties of dihydrofolate and folylpolyglutamate synthetases.

Corynebacterium Lactobacillus Escherichia coli S. cerevesiae DHFS

Purification (fold) 7,000 200,000 15 18

DHFS activity YES NO YES YES
FPGS activity YES YES YES NO
FPGS substrate THF methylene-THF 10-fotmyl-THF
pH optimum 9.5 9.5 9.4 9.4
monoval. cation 200mMK+ 200mMK+ 200mMK+ 200mMK+
divalent cation Mg2+ Mg2+ Mg2+ Mg2+
Mr 53,000 44,000 45,000 48,000
kcat (sec- 1) 0.6 0.8 0.7 0.09

The second yeast gene encoded a protein which possessed FPGS activity but lacked
DHFS activity. The third yeast gene encodes a protein of unknown function and its deduced
amino acid sequence has no similarility to any DHFS or FPGS protein.
A human eDNA for FPGS was isolated by expression cloning in E. coli and the eDNA
characterized and its gene product over-expresssed and purified&. The human and yeast
FPGS proteins share the highest degree of amino acid identity (36 percent) and these proteins
share only 6 percent amino acid identity with the E. coli, Lactobacillus and yeast (DHFS)
proteins. The major sequence conservation among these proteins are in the regions of the A
and B nucleotide binding sites.

Table 2. Properties of eukaryotic folylpolyglutamate synthetases.

S. cerevesiae FPGS pig liver human

Purification (fold) ND1 180,000 610

DHFS activity NO NO NO
FPGS substrate THF,DHF THF,DHF THF, DHF
pH optimum 9.5 9.5 9.4
monovalent cation 20mMK+ 20mMK+ 20mMK+
divalent cation Mg2+ Mg2+ Mg2+
Mr 62,000 (54,000)2 59,000 60,000
kcat (sec- 1) ND 2.1 0.8

1. ND not done
2. mature protein

630
 The properties of yeast, human and pig FPGS are compared in Table 2. The properties
of the proteins are similar and differ from the bacterial proteins and yeast DHFS (Table 1) in
their larger molecular size, their lower requirement forK+, and their preference for DHF and
THF as the optimal pteroylmonoglutamate substrates. The eukaryotic proteins will also
metabolize folate substrates to long chain polyglutamates while the bacterial FPGS proteins
extend the polyglutamate chain primarily to the tri- and tetraglutamate derivatives.

Expression of Folylpolyglutamate Synthetase in Mammalian Cells

We have shown that FPGS is located in the mitochondria and cytosol of eukaryotic cells
and mitochondrial FPGS activity is required for mitochondrial one carbon metabolism and
for a normal one carbon flux in the cytosol (unpublished data). Expression of the human
FPGS eDNA in AUXB 1 cells, Chinese hamster ovary (CHO) cell mutants that lack FPGS
activity9, restored cytosolic FPGS activity in these cells and overcame the cell's requirement
for thymidine and purines but the cells remained auxotrophic for glycine, reflecting the
absence of a folate pool in the mitochondria (Table 3). The 5' region of the human eDNA
prior to the first ATG codon would code for an amino acid sequence with homology to a
mitochondrial leader sequence (Figure 1), but lacking a start methionine. When an ATG
codon was introduced at the start of this region and the modified eDNA transfected into
AUXB 1 cells, transfectants expressed FPGS activity in the mitochondria and contained
normal mitochondrial folate pools (Table 3).

Table 3. Folate and FPGS distribution and glycine requirement in CHO AUXBl
transfectants

FPGS DNA FPGS activity in folate in glycine

transfected cytosol mitochondria cytosol mitochondria auxotrophy

None No No No No Yes
human eDNA Yes No Yes No Yes
human cDNA(ATG) Nol Yes Yes Yes No
yeast gene Yes Yes Yes Yes No

1. proportion of cellular FPGS activity in cytosol similar to that of a mitochondrial matrix marker

+1
ATG .... G CGC GGC ATA ACG ACC CAG GTC GCG GCG CGG CGG GGC TTG
Met ..... Arg Gly Ile Thr Thr Gln Val Ala Ala Arg Arg Gly Leu

+71
AGC GCG TGG CCG GTG CCG CAG GAG CCG AGC ATG GAG TAC CAG GAT

Ser Ala Trp Pro Val Pro Gln Glu Pro Ser MET Glu Tyr Gln Asp

Figure 1. 5' Region of Human FPGS eDNA. The first ATG in the eDNA is shown in bold. The amino
acid sequence of the putative mitochondrial leader sequence is shown italicized.

631
 Transfection of AUXB 1 cells with the yeast FPGS gene also restored mitochondrial and
cytosolic folate pools and FPGS activity (Table 3). Translation of the yeast gene from its first
ATG would generate a protein with an N terminal resembling a mitochondrial leader
sequence. Translation from a downstream ATG, preceded by concensus transcription and
translation signals, would result in a mature protein (Table 2) lacking a leader sequence.

Role of Folylpolyglutamate Synthetase in Anti-folate Cytotoxicity

Table 4 shows the effect of FPGS activity on the cellular accumulation of folinate and
methotrexate. When CHO cell transfectants were incubated with physiological levels of
folinate (2 nM), there was little effect of FPGS activity on folate accumulation in cells
expressing low to very high levels of human FPGS. At the lowest level of FPGS activity,
folinate accumulation was depressed somewhat. Under these conditions, folinate
accumulation is limited by influx and essentially all transported folate is metabolized to
polyglutamates and retained by the cell.

Table 4. Folate and methotrexate accumulation by CHO cell transfectants

folate or analog accumulation

cell FPGS activity 5 nM folinate 2 J.1M folinate 5J.LMMTX

pmoles/h/106 cells pmoles/106 cells

WIT2 90 2.3 22.9 4.5
AUX-human-21 <2 0.4 5.5 3.7
AUX-human-7 6 1.3 11 3.9
AUX-human-21 19 1.7 19 4.4
AUX.-human-79 71 2.2 44 9.7
CHO-human-14x 1400 3.7 430 135

1. AUX-human-x are CHO cells expressing human FPGS activity, x representing percent activity relative
to wild type CHO cells.

At pharmacological levels of folinate and with methotrexate, cellular folate or analog

accumulation was influenced by FPGS activity (Table 4). Increasing the medium folinate
concentration 400-fold to 2 J.iM increased folinate accumulation by CHO wild type cells about
ten-fold and about 95 percent of folinate transported was not retained due to an inability to
metabolize it to the retainable triglutamate derivative. Cells expressing very high levels of
human FPGS accumulated very high levels of folate. Methotrexate accumulation was also
proportional to FPGS activity. Little or no polyglutamates were detected in cells expressing
low levels of human FPGS while longer chain polyglutamates (pentaglutamates) accumulated
in cells expressing higher levels of enzyme.
Although methotrexate does not require polyglutamylation to inhibit its target enzyme,
polyglutamylation does aid in the retention of the drug by cells. The effect of FPGS activity
levels on the cytotoxicity of methotrexate are shown in Table 5. There was no effect of FPGS
levels on the cytotoxicity of methotrexate when cells were continuously exposed to MTX (72
hours). However, when cells were exposed to the drug for 4 hours, and then cultured in
drug-free medium for 72 hours, cells expressing higher levels of FPGS activity were more
sensitive to methotrexate. It has been demonstrated that a decrease in FPGS activity is a
mechanism by which mammalian cells can become resistant to methotrexate10.

632
 Table 5. Effect of FPGS activity on sensitivity of cells to methotrexate

time of exposure to methotrexate

Cells 72hour 4hour

ED so
nM J1M
CHO--WT 3-10 10-33
HT1080 3-10 0.1-0.3
AUX-human-2 1-3 33-100
AUX-human-7 3-10 10-33
AUX-human-21 3-10 3-10
AUX-human-79 3-10 1-3

Kinetic Mechanism of Folylpolyglutamate Synthetase

The catalytic mechanism and substrate binding specificity of E. coli DHFS•FPGS has
been investigated by affinity labelling of active site residues, stopped flow fluorescence,
rapid quench kinetics, isolation of reaction intermediates and by site-directed mutagenesis.

12000 r - - - - - - - - - - - - - - - - - w - - r - - - - - - ,
• H2Pte
--o-- H2Pte + Glu

ATP

8000

~a.
(.)
Pi
acyi-P

4000

H2Pte

~
0
0 10 20 30 40

fraction no.
Figure 2. HPLC separation of reaction intermediates. E. coli DHFS•FPGS (2iJ.M) was incubated with
dihydropteroate (10 j..lM) and [y_32p]ATP (10 j..lM) for 5 seconds and products were separated on a SAX
column.

633
 The E. coli enzyme uses an unusual mechanism for increasing the affinity of the
dihydropteroate substrate for the DHFS reaction. The enzyme catalyzes the rapid formation
of a dihydropteroate quinonoid species. This species is highly fluorescent which has allowed
a rapid reaction kinetic analysis. The enzyme catalyzes the formation of an acyl-phosphate
intermediate. This intermediate slowly forms in the absence of the glutamate substrate but
glutamate binding, which causes a conformational change in the protein, accelerates acyl-
phosphate formation. The rate limiting steps in the overall DHFS reaction (kcat 0.35 sec-1 at
250) are acyl-phosphate formation (0.8 sec-1) and a conformational change in the protein
required before products can be released (1.2 sec-1).
The acyl phosphate intermediate, which forms slowly in the absence of glutamate, can be
isolated by HPLC using an anionic exchanger (Figure 2). The slow formation of this
intermediate, and its even slower conversion back to substrates, explains previous
observations that dihydropteroate is a non-competitive inhibitor of the FPGS reaction. These
studies were usually carried out with non-saturating levels of glutamate. The DHFS and
FPGS reactions are ordered, with ATP binding as the first substrate. Folates and
dihydropteroate do bind to free enzyme in a nonproductive fashion but have to be displaced
by ATP. This binding to free enzyme, and consequent competition with ATP, explains the
substrate inhibition observed with higher levels of some folate substrates.

ACKNOWLEDGEMENTS

Supported in part by PHS grants CA-41991 and DK-42033 from the Department of
Health and Human Services.

REFERENCES

1. B. Shane, Vit. Horm. 45:263 (1989).

2. B. Shane, J. Bioi. Chern. 255:5655 (1980).
3. A.L. Bognar, and B. Shane, J. Bioi. Chern. 258: 12574 (1983).
4. A.L. Bognar, C. Osborne, B. Shane, S.C. Singer, and R. Ferone, J. Bioi. Chern. 260:5625 (1985).
5. D.J. Cichowicz, and B. Shane, Biochemistry 27:504 (1987).
6. A.L. Bognar, C. Osborne, and B. Shane, J. Bioi. Chern. 262:12337 (1987).
7. J. Toy, and A.L. Bognar, J. Bioi. Chern. 265:2492 (1990).
8. T.A. Garrow, A. Admon, and B. Shane, Proc. Nat!. Acad. Sci. USA 89:9151 (1992).
9 M.W. McBurney, and G.F. Whiunore, Cell 2:173 (1974).
10. D.E. McCloskey, J.J. McGuire, C.A. Russell, B.G. Rowan, J.R.Bertiuo, G. Pizzorno, and E. Mini, J.
Bioi. Chern. 266:6181 (1991).

634
INCREASED ACTIVITY OF 1-GLUTAMYL HYDROLASE IN HUMAN SARCOMA CELL. LINES:
A NOVEL MECHANISM OF INTRINSIC RESISTANCE TO METHOTREXATE (MTX)

Wei Wei Li, Mark Waltham, William Tong, Barry I. Schweitzer,

and Joseph R. Bertino
Program of Molecular Pharmacology and Therapeutics
Sloan-Kettering Institute for Cancer Research
New York, NY 10021

INTRODUCTION
Intracellular levels of folate or methotrexate (MTX) polyglutamates
are regulated at least in part by the enzyme, folylpolyglutamate synthase
(FPGS), which is responsible for synthesis, and 1 -glutamate hydrolase
(GGH), which catalyzes the hydrolysis of polyglutamates to monoglutamate
forms. 1 Decreased accumulation of long chain polyglutamates of methotrexate
(MTX) may result from decreased FPGS activity or increased GGH activity.
Previous studies showed that natural resistance to MTX in soft tissue
sarcomas is associated with the inability of these cells to accumulate long
chain MTX polyglutamates after exposure to this drug. 2•3 As no appreciable
difference of FPGS activity was observed between MTX-resistant and
sensitive cells, we measured GGH activity in these cell lines and found
increased levels of this enzyme in MTX resistant soft tissue sarcoma cell
lines.

METHODS
Cell Lines
Human lymphoblastic leukemia cell line, CCRF-CEM, human lympho-
blastoid cell line, RPMI-1788, and 3 human soft tissue sarcoma cell lines,
HT-1080, HS-16, and HS-42, were studied. The cell lines were maintained in
RPMI-1640 medium containing 10% fetal bovine serum (FBS) as a cell
suspension (CEM and RPMI-1788) or monolayer culture (HT-1080, HS-16, and
HS-42). HS-16 and HS-42 cells are 7-, 13-, and 30-40-fold more resistant
to MTX (24h exposure) as compared to the MTX-sensitive lines, RPMI-1788 and
HT -1080. 3• 4
Analysis of Polyglutamate Formation
Cells were incubated in complete medium containing 10 ~M [3H] MTX at
37·C either for 24h or for 24h followed by 4-12h efflux in fresh drug-free
medium. Cells were harvested and suspended in 500 ~1 of boiling 50 mM
sodium phosphate, pH 5.5, boiled for 5 min., and centrifuged at 20,000 x
g for 10 min. Supernatant was analyzed by HPLC. MTX polyglutamate standards
were added to certify the radiolabeled peaks.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993
635
FPGS Activity Assay
FPGS activity in cell extracts was measured as described previously. 3
The reaction mixture contained, in a final volume of 250 ~1, 100 mM tris-
HCl (P.H 8.85}, 10 mM ATP, 20 mM MgC1 2 20 mM KCl, 100 mM 2-mercaptoethanol,
4 mM [3H] glutamate, enzyme and substrate (FH 4 , MTX). Polyglutamate products
in reaction were separated from glutamate by DE-52 mini-column
chromatography.
GGH Activity Assay
GGH activity in cell extracts was determined using a modification of
the method of Samuels at al. 5 Cell-free enzyme preparations were incubated
at 37•C in MTEN buffer (pH 4.5 and 7.2). Fifty ~M MTX tetraglutamate (final
concentration) was added to initiate the reaction. Aliquots were removed
at various times and the reaction stopped by placing the reaction tube in
boiling water for 10 min. Substrate and products were analyzed by HPLC.
Enzyme activity was expressed as nmol of product formed per mg of protein
per min.

RESULTS
The accumulation of long chain MTX polyglutamates (3-6) of HS-16 and
HS-42 cells after exposure to MTX for 24h was much less compared to the
other 3 MTX-sensitive cell lines (Figure 1). After cells were resuspended
in drug-free medium for 4-12h, the level of polyglutamates in HT-1080 cells
decreased rapidly. MTX polyglutamates decreased in GEM cells but remained
high even after 12h of efflux, while the levels of long chain MTX
polyglutamates in the 1788 cell line did not decrease appreciably. The low
levels of MTX polyglutamates persisted in HS-16 and HS-42 cells and after
12h of efflux still had the lowest level of MTX polyglutamates as compared
to the other 3 cell lines. Increased GGH activity was observed at pH 4.5
in HS-16 cells than that in HT-1080, GEM, and RPMI-1788 cells (Figure 2).
There is a significant correlation (p < 0.05) between the increase of GGH
activity and the decreased retention of long chain polyglutamates of MTX
when cells {excluding HS-42 cells) were re-incubated in MTX-free medium for
4-8h. GGH activity measured in HS-42 cells was similar to that in MTX-
sensitive cells. Thus, other factors in addition to hydrolase activity are
important in regulating the level of intracellular polyglutamates. GGH from
all 5 cell lines appears to be an exopeptidase and is inhibited completely
by PHMB in vitro (data not shown).

DISCUSSION
In present study, increased GGH activity was observed in HS-16 cells
which is naturally resistant to MTX due to low levels of MTX polyglutamates
that accumulate in the cells. No significant increase of GGH activity was
observed in HS-42 cells although this cell line is also naturally resistant
to MTX. However, it is possible that GGH activity increases when cells are
exposed to MTX (unpublished data, references 6,7). Results from McCloskey
et al. 8 showed that long chain MTX polyglutamates were not accumulated in
a MTX-resistant cell line with too low FPGS activity. In contrast we
observed that tri-, tetra-, and pentaglutamates are still present in small
amounts in HS-16 and HS-42 cells. This result suggests that the reduced
intracellular levels of MTX polyglutamates that are formed in HS-16 and HS-
42 cells are most probably due to increased hydrolysis rather than
decreased synthesis since GGH exopeptidase can breakdown long chain
polyglutamates stepwise.

636
 120 r-----------------------------~
wash

·~
100
·~
80
0~~
60
~CEM
-
E
....
0
01788
0
E
Q. 40

20
~0---L). 1080

.t. . t . - - -.t.- - · HS-16

o---o
- - - D I - - - 1 0 HS-42
0 ~--~---~--~---L---~
0 24 4 8 12 14
time(h)

Figure 1. Long chain MTX polyglutamates (3-6) retained in cells after a 24h incubation with 10 jt)4 3 H- MTX.
MTX polyglutamates were determined by HPLC (see Materials and Methods).

1 .4 .----------------------------------------------~

1. 2 D pH 4.5 1111 pH 7.2

'2 1.0
.E
C;
:S 0 .8
0
E
.s
~
0 .6
·s:
·~
<G 0.4

0.2

0 .0

RPMI - 1788 C EM HT - 1080 HS- 16 HS-4 2

Figure Z. GGH activity in extracts from 5 cell lines measured at pH 4.5 and 7.2.

637
 Increased FPGH activity may be a novel mechanism of natural
resistance to MTX. We are testing other tumors in addition to human soft
tissue sarcoma cells intrinsically resistant to MTX for evidence of this
phenotype.

REFERENCES
1. J.R. Barruceco and F.M. Sirotnak, J. Biol. Chern. 266:11732-11737 (1991).
2. W.W. Li, J.T. Lin, W.P. Tong, T.M. Trippett, M.F. Brennan, and J.R.
Bertino, Cancer Res. 52:1434-1438 (1992).
3. W.W. Li, J.T. Lin, B.I. Schweitzer, W.P. Tong, D. Niedzwiecki, and J.R.
Bertino, Cancer Res. 52:3908-3913 (1992).
4. W.W. Li and J.R. Bertino, Cancer Res. 52:6866-6870 (1992).
5. L.L. Samuels, L.J. Goutas, D.G. Priest, J.R. Piper, and F.M. Sirotnak,
Cancer Res. 46:2230-2235 (1986).
6. E. Sikora, B. Kaninska, and B. Grzelakowska-Sztabert, Cell Biol. Intl.
Rep. 16:369-375 (1992).
7. P. Sur, D.G. Priest, and M.T. Doig, Biochem. Cell Biol. 64:363-367
(1986).
8. D.E. McCloskey, J.J. McGuire, C.A. Russell, B.G. Rowan, and J.R.
Bertino, J. Biol. Chern. 266:6181-6187 (1991).

638
MECHANISM-BASED APPROACHES TO INHffiiTION
OF THE SYNTHESIS AND DEGRADATION OF
FOLATE AND ANTIFOLATE POLYGLUTAMATES

Thomas I. Kalman

Departments of Medicinal Chemistry and Biochemical Pharmacology

State University of New York
Buffalo, NY 14260

INTRODUCTION

Tetrahydrofolate and its one-carbon coenzyme derivatives undergo in the cell poly-
glutamylation, which is essential for their cellular retention and optimal enzymatic
activity. 1•2 The biolegical activity of glutamate-containing "classical" antifolates also
depends on the extent of their intracellular poly-y-glutamylation. Cells unable to
polyglutamylate these antifolates are resistant to the cytotoxicity of these drugs. While the
importance of polyglutamate chain length has been clearly demonstrated, the regulatory
role of the enzymes responsible for the synthesis and breakdown of the poly-y-glutamyl
chain, folylpolyglutamate synthetase (FPGS) and y-glutamyl hydrolase (GGH, conjugase),
respectively, is poorly understood. Complicating factors are the low rate of chain
elongation and shortening compared to other coenzyme metabolizing activities and the
tissue-to-tissue, cell-to-cell and subcellular variation of GGH activities associated with
different proteins. 1•2
0 COOH H
0 ~N~N~COOH
H,N~N"Y'~~ H 8"" COOH
A ..Jl) H
HN N N PteGlu
2 n+ 1

FPGS
PteGlu0 + ATP + Glu ----~ PteGlu0 +1 + ADP +Pi
GGH
PteGlun+ 1 + H20 ----~ PteGlllu + Glu

Selective inhibitors of FPGS and GGH can serve as biochemical tools helping to
elucidate the mechanism of the regulation of cellular polyglutamate turnover and may have

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 639
potential therapeutic applications. We have chosen mechanism-based approaches to design
selective inhibitors of these enzymes. A critical evaluation of our results together with
some of those from other laboratories permits an analysis of the structural requirements
of enzyme inhibitory activity. A difficulty shared by mechanism-based inhibitors of both
enzymes is the polar nature of these compounds which hampers effective cellular uptake.
These and other challenging problems demand the refinement of our design strategies and
the development of new approaches.
This paper presents a brief review of our present understanding of the molecular
basis of enzyme inhibitory activity of selected compounds targeted against FPGS or GGH.
In addition, it presents a critical discussion of past and present mechanistic rationales as
a foundation for future development of more effective inhibitors.

RESULTS AND DISCUSSION

Design of Folylpolyglutamate Synthetase (FPGS) Inhibitors

Our design strategy is based on the mechanistic similarity between FPGS and two
glutamate metabolizing enzymes: glutamine synthetase and y-glutamylcysteine synthetase. 3
The common mechanism involves ATP-mediated phosphorylation of the y-COOH group
to form a carboxylic-phosphoric mixed anhydride, which reacts with the nucleophilic N-
atom of the second substrate NH2R. The resulting tetrahedral intermediate collapses to
product and inorganic phosphate.
Many mechanism-based inhibitors of glutamine synthetase and y-glutamyl cysteine
synthetase are known. The extensively studied natural products methionine sulfoximine and
phosphinothricin and their derivatives are prototype inhibitors, which undergo enzymic
phophorylation resulting in tightly bound tetrahedral intermediate analogs. These
y-substituted glutamate analogs exert their inhibitory effect as "enzyme generated transition
state analogs" 4 or "reaction coordinate analogs". 5 Following these leads, we have
synthesized derivatives of folate, aminopterin and MTX, in which the glutamate is replaced
by S-alkylhomocysteine sulfoximines3 ' 6 and L-phosphinothricin, 7 structures 1 and 2,
respective! y:

While analog 1 had no effect on FPGS activity, analog 2 showed weak competitive
inhibition. There was no evidence for phosphorylation - preincubation of the enzyme with
ATP and 1 or 2 did not change the results. The inactivity of the sulfoximines 1 suggested
that no binding of these analogs to the active site of FPGS can occur. The inhibitory
activity of 2 was attributed to the presence of the negative charge on the y-substituent. This

640
charge effect is a general property of folate analogs, e.g., phosphonates and sulfonates can
be effective inhibitors of FPGS, 8 and even a negatively charged ring at this position, like
tetrazolate, binds to the enzyme. 9
It is surprising that no analog, in which the y-COOH has been replaced by another
functionality, could serve as an acceptor of phosphate from ATP in the FPGS reaction.
Coward's y-F-glutamate derivatives 10 having the y-COOH group intact, do serve as
substrates, but create obstacles to chain elongation. The planar tetrazole ring is both acidic
and nucleophilic, yet is incapable of attacking the P-atom. These observations indicate that
very significant differences must exist between the architecture of the active site of FPGS
and its mechanistic relatives glutamate and y-glutamylcysteine synthetase.
It is of interest to examine the geometric changes associated with the enzyme
catalyzed reaction. As outlined in Figure 1., there is no change in the sp2 hybridization at
the C-atom of the y-COOH during the phosphorylation step - the trigonal (planar)
geometry remains unchanged until the nucleophilic attack involved in amide bond
formation takes place.

0 0
I I
P-0 P-0
o/ 'o
0 ~ '0 RNH2
~0
ATP
0- ~0
NHR
tngonal tngonal tetrahedral

0
I
.P-0
0 NH ATP NIH 'o
I
yo
-.......;-s-R
'o
---\-· -.......;-s-R
'o
F 0
I
0 0 /P;-0
N-N ATP I 0
JN
N'
I
-.......;-P;-Me
0
---\-· -.......;-P;-~e

Figure 1. Geometric changes at the cS-carbon atom of the glutamate of folates leading to the tetrahedral
intermediate in the FPGS catalyzed reaction. The corresponding trigonal (planar) and tetrahedral
geometries of representative y-substituted folate analogs are represented for comparison.

There may be greater restrictions at the active site of FPGS than for the other two
enzymes and the tetrahedral geometry of some of the substrate analogs may not permit
productive attack on the y-phosphate of ATP during the phosphorylation step. Faithful
analogs of the tetrahedral intermediate, while potential tight-binding inhibitors, must
contain phosphates, phosphonates and related highly charged functionalities with the known
handicap of inefficient cellular penetration. 8

Design of y-Glutamyl Hydrolase (GGH) Inhibitors

The inability to cross the cell membrane is also a serious problem with inhibitors of
GGH. There is a large variety of conjugase enzymes capable of hydrolyzing folate and
antifolate polyglutamates. The development of GGH inhibitors in our laboratory was based
on the assumption that an intracellular regulatory enzyme would show preference to
sequentially remove one glutamate at a time. Thus, we were targeting a C-terminal
glutamate-specific metallo(Zn)-carboxypeptidase. Based on the generally accepted
mechanism of the carboxypetidase catalyzed reaction, 5 a prototype inhibitor, 2-mercapto-
methylglutaric acid (MMGA) was designed. Although it is active against isolated enzymes
at submicromolar concentrations, it requires ~ 100 11-M for cellular activity .U Interaction

641
of its SH-group with the zinc at the active site of GGH is primarily responsible for the
inhibitory activity of MMGA, while the glutarate backbone provides selectivity for C-
terminal glutamate specific carboxypeptidases.
As an alternate approach, N-(benzylthiocarbamoyl)-L-glutamic acid and (BTGA) and
N-(benzylcarbamoyl)-L-glutamic acid (BCGA) were designed as potential reaction
coordinate analogs. Both inhibited carboxypeptidase G2 strongly - Ki-values for BTGA
and BCGA are 4.5 J1.M (competitive) and 0.12 J1.M (noncompetitive), respectively. The
inhibitory pattern of BCGA is consistent with the action of a reaction coordinate analog.
It is interesting that the sulfur-containing BTGA is less potent than the urea derivative
(BCGA). This may reflect a water promotion role for the zinc ion in carboxypeptidase G,
rather than carbonyl activation of the substrate by direct coordination to the carbonyl
oxygen of the scissile bond.
Whitehead et al. 11 reported previously on the effects of MMGA on the accumulation
and retention of methotrexate (MTX) polyglutamates in leukemia cells isolated from ALL
and AML patients. Since that time, cells of more patients were examined and the observed
characteristic difference between the two cell types was confirmed. Myeloblasts, which
accumulate shorter chain length MTX polyglutamates (triglutamate predominating) are
more effected by MMGA exposure than ALL cells, which tend to accumulate longer chains
(pentaglutamate predominating). In the presence of MMGA, AML cells change their
pattern of MTX polyglutamate distribution to resemble that of the ALL cells - greatly
increasing the MTX pentaglutamates at the expense of the MTX triglutamates. Figure 2.
illustrates this phenomenon by comparing the effects of MMGA on MTX polyglutamate
distribution during cellular uptake and during efflux. It should be pointed out that there are
considerable individual variations in these effects, therefore, it is important that the average
differences between the penta- vs. triglutamate values are statistically significant.
The data suggest that if the intrinsic resistance of AML to MTX is related to an
inability to accumulate longer chain MTX polyglutamates, than this resistance should be
reversible by pharmacologic modulation using GGH inhibitors in most of those cases which
are not the result of an FPGS defect.

NS <.02

n= 1 2 3 4 5 6 n= 1 2 3 4 5 6

MTX Glu ( n)

Figure 2. The effects of MMGA (100 J.JM) on the chain length distribution of MTX-polyglutamates in
myeloblasts of patients with acute myeloblastic leukemia (AML). The accumulation and retention of
I J.JM 3H-MTX expressed as the percent difference in the presence and absence of MMGA. Uptake
(8 patients) of 3H-MTX is during 0 - 24 hours, efflux (5 patients) represents values at the end of an
additional 24 hours incubation in the absence of 3H-MTX. (Data provided by V.M. Whitehead, Montreal
Children's Hospital, Montreal, Quebec).

642
 It is important to note that natural resistance of human acute nonlymphocytic leukemia
associated with impaired MTX polyglutamylation was indeed described recently by Bertino
and coworkers. 12

ACKNOWLEDGMENT

This work was supported in part by grant CA35212 from the National Cancer
Institute, NIH, USPHS.

REFERENCES

1. J.J. McGuire and J.K. Coward, in: "Folates and Pterins," R.L. Blakley and S.J. Benkovic, Wiley,
New York (1984).
2. B. Shane, Vitamins Hormones 45:263 (1989).
3. R.G. Moran, P.D. Colman, P.J. Harvison and T.I. Kalman, Biochem. Pharmacal. 37:1997 (1988).
4. T.I. Kalman, Acta Pharm. Suecica, (Suppl. 2):297 (1985).
5. D.W. Christianson, W.N. Lipscomb, Accounts Chern. Res. 22:62 (1989).
6. P.J. Harvison and T.I.Kalman, J. Med. Chern. 35:1227 (1992).
7. T.I. Kalman, C.S. Jones and R.G. Moran, Proc. Amer. Assoc. Cancer Res. 31:337 (1990).
8. A. Rosowsky, R.A. Forsch, V.E. Reich, J.H. Freisheim and R.G. Moran, J. Med. Chern. 35:1578
(1992).
9. J.J. McGuire, C.A. Russel, W.A. Bolanowska, C.M. Freitag, C.S. Jones and T.I. Kalman, Cancer
Res. 50:1726 (1990).
10. J.J. McGuire and J.K. Coward, J. Biol. Chern. 260:6747 (1985).
11. V.M. Whitehead, T.I. Kalman, D.S. Rosenblatt, M.-J. Vuchich and C. Payment, Proc. Amer.
Assoc. Cancer Res. 29:287 (1988).
12. J.T. Lin, W.P. Tong, T.M. Trippett, D. Niedzwiecki, Y. Tao, C. Tan, P. Steinherz, B.I. Schweizer
and J.R. Bertino, Leuk. Res. 15:1191 (1991).

643
POLYGLUTAMATE PRODUCT FORMATION BY LACTOBACILLUS CASE/
FOLYLPOLYGLUTAMATE SYNTHETASE IN VITRO AND IN VIVO IN
RECOMBINANT ESCHERICHIA COLI

Jeffrey Toy and Andrew L. Bognar

Department of Microbiology
University of Toronto
Toronto, Ontario, Canada M5S lAS

INTRODUCTION

Lactobacillus casei has the longest chain folate polyglutamates, predominantly

H 4PteGlu8_9, found in a prokaryote. Previous studies have shown only shorter chain
polyglutamates, up to H 4PteGlu4, could be synthesized by L. casei extracts or the
purified folylpolyglutamate synthetase (FPGS)(l). We have cloned the gene encoding
the L. casei FPGS in Escherichia coli (2) and have overexpressed the enzyme. We
wished to purify the enzyme in large amounts to investigate whether the chain length
of the polyglutamate products depended on the effective enzyme concentration and
whether long chain polyglutamates could be produced in vitro by using very high
enzyme concentrations. An alternate approach is to express the gene encoding the L.
casei FPGS in a strain deficient in FPGS activity and see whether a single gene
product leads to the appearance of long chain polyglutamates in vivo. In this study we
report the results of both approaches, which show that the L. casei FPGS was able to
catalyze the synthesis of long chain folate polyglutamates in vivo in recombinant E.
coli, although only pentaglutamate products could be detected in vitro.

MATERIALS AND METHODS

The L. casei FPGS gene was expressed from the lac promoter of plasmid pGT3-
8.1 (2) in E. coli strain SF4, which is deficient in dihydrofolate synthetase-
folylpolyglutamate syntetase (3,4). The L. casei FPGS was purified from recombinant
E. coli as described previously (5) FPGS was assayed as described previously (6,7)
using 5, 10-methylene-H 4PteGlu 1_6 derivatives as substrates. F alate polyglutamates were
purchased from Schirks Laboratories and enzymatically reduced to tetrahydrofolate
derivatives using L. casei dihydrofolate reductase (provided by B. Shane). Folates were
labelled with 14C-glutamate (Amersham) in the in vitro reaction. Folate pools in
control and recombinant E. coli SF4 were labelled by growth in folate-free medium
supplemented with 14C-p-aminobenzoic acid (p-aba)(ICN). Cellular folates were
extracted and polyglutamate chain lengths were determined by the HPLC method of
Shane (8) using Partisil 10 SAX columns (Whatman). To determine y-glutamate
chain lengths, 14C-p-abaglu products were eluted from the HPLC, purified with C 18 Sep-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et at., Plenum Press, New York, 1993 645
Pak cartridges (Waters), cleaved with carboxypeptidase Y as described by Ferone (9)
and rechromatographed by HPLC.

RESULTS

The Lactobacillus casei FPGS was overproduced in recombinant Escherichia coli

to about 5% of soluble protein. The enzyme was purified in high yield (53%) giving
96 mg of purified enzyme from 10 litres of cell culture. Incorporation of 14C-glutamate
into 5, 10-CH2-H4PteGlu1_6 catalyzed by the purified enzyme was measured and the
results are shown in Table 1.

Table 1. Utilization of foiate polyglutamates by L. casei FPGS.

Substrate Activitl (nmol)

5,10-methylene- H 4PteGlu 1 20.3

5,10-methylene- H4PteGlu2 19.2
5,10-methylene- H 4PteGlu3 7.1
5,10-methylene- H 4PteGlu4 0.23
5,10-methylene- H 4PteGlu5 0.005
5,10-methylene- H 4PteGlu6 0.012

1 The assay mixture contained 240 ng of purified enzyme

The amount of purified enzyme used was 10-fold higher than in the previously
reported in vitro experiments (1). The apparent activity with 5,10-methylene-H4PteGlu4
was 1% that of the monoglutamate, while activity with pentaglutamate and
hexaglutamate were 0.02% and 0.04% of monoglutamate values, respectively. To verify
that the products of the in vitro reaction were the H4 PteGlun+l derivatives, the products
were converted to p-abaGlun and subjected to HPLC analysis. Figure 1 shows that
the products with the tetraglutamate substrate gave equal-sized peaks which eluted
with the retention times corresponding to those of tetraglutamate and pentaglutamate.
The profiles obtained with the products of the reactions with pentaglutamate and
hexaglutamate substrates showed no H4PteGlun+l peaks but only shorter chain length
products corresponding to tri-, tetra- and pentaglutamates (not shown).
4,---------------------------------,

"' 3
I~
><
E pABA-5
C.2
(,)

..~\ .-....--v·
obL~~==~~--~~~~~~~~-L--~
~
0 10 20 30 40 50 60 70 80
Fractions

Figure 1. HPLC elution profile of p-abaGlu products of FPGS with H 4PteGlu4 as

substrate.

646
 2000
A 56 7 9

1500-

~ 1000
u

~
500

0
__..,..!• \_
0 10 20 30 40 50 60 70 60 90
Fractions

500
B 7 8 9
400

300
E
Q.
u

ro w ~ ~ ~ ~ ro ~ ~

Fractions

4oo~c--------------~6~7~B~9~--------~

300

E
Q.
2oo
u

100

ro w ~ ~ ~ ~ ro ~ ~

Fractions

Figure 2. HPLC profiles of pabaGlun extracted from pGT3-8.1/SF4 cells. A, whole cell
extracts; B, p-abaGlu products eluted and purified with Sep-Pak C18; C, eluted
products from B treated with carboxypeptidase Y.

647
 1400
A 3 4 5 6 7
1200

1000

800
E 800
Q
()

400

200

0
0 10 20 30 40 50 eo 70 80 90
Fractions

300
B 7 8
250

200

E
Q 150
()

10 20 30 40 50 60 70 80 90
Fractions

350.-c----------~~4
.-----------------.

300

250

200
E
c. 150
()

100

50

w w ~ ~ ~ ~ ro ~ ~

Fractions

Figure 3. HPLC profiles of pabaGlun extracted from SF4 cells. A, whole cell extracts;
B, p-abaGlu products eluted and purified with Sep-Pak Cl8; C, eluted products from
B treated with carboxypeptidase Y.

648
 An in vivo system was developed to determine if the L. casei FPGS expressed
in E. coli was capable of generating long chain folate polyglutamates. The plasmid
pGT3-8.1 (2), which encodes the FPGS gene but not the downstream gene was
transformed into E. coli SF4. The transformants and control cells were grown with
radiolabelled p-aba, folates were extracted and polyglutamate chain lengths
determined by HPLC (Figures 2A and 3A). Radioactive peaks with retention times
from 40 to 72 min were observed with extracts from the pGT3-8.1/SF4 transformant.
Peaks eluting after 52 min are assumed to be longer chain lengths than
theheptaglutamate standard, suggesting that folate polyglutamates up to H 4PteGlu12
were synthesized with nonaglutamate and decaglutamate predominant (Figure 2A).
However long chain polyglutamates were also observed in the untransformed host
strain, although these products were of slightly shorter chain length, with
heptaglutamate predominant (Figure 3A). The amount of labelled folates in both
strains were comparable despite the high FPGS activity from the L. casei enzyme,
since this depended on the dihydrofolate synthetase activity of the mutant host strain.
These folates in E. coli were likely to be H 4Pte-y-Glu3-a-Glun as described by Ferone
et al. (9). Our ion exchange columns do not distinguish between y- and a-
polyglutamates, as do the reverse phase columns of Ferone et al. but the o:-linked
polyglutamates are suceptible to cleavage by carboxypeptidase Y (9). The longest
chain major p-abaGlu derivatives from the SF4 cells, hepta and octaglutamates, were
eluted, purified and digested with carboxypeptidase Y. The retention times were
shifted to those of tri- and tetraglutamates as a result of the carboxypeptidase Y
treatment (Figure 3C). In contrast, similarly treated derivatives from the transformant
containing the L. casei enzyme were resistant to carboxypeptidase Y treatment (Figure
2C), suggesting that they were y-linked polyglutamates. There was an increase in the
amount of hexaglutamate present after carboxypeptidase Y treatment, indicating that
the E. coli o:- FPGS did contribute to thelength of the polyglutamates and that that
enzyme can use y-glutamates longer than triglutamate as substrates.

DISCUSSION

The L. casei FPGS was shown to produce pentaglutamate products in vitro but
we found no evidence for longer chain products. This is in contrast to the E. coli
enzyme, which normally makes triglutamate products in vivo in cells containing normal
concentrations of enzyme but can produce hexaglutamates in vitro when high
concentrations of enzyme are used (R. Ferone, personal communication). Penta- and
hexa-y-glutamates were identified following extraction and carboxypeptidase Y
cleavage from E. coli cells transformed with pAC5, which overproduces the E. coli
FPGS 15-100-fold (10) (not shown). We have tried different reaction conditions,
including those which favour the E. coli o:-FPGS (9) but have produced no longer
chain product. The shorter chain products observed with the polyglutamate substrates
may be the result of elongation of shorter chain length polyglutamates present as
impurities in the substrate or produced by a hydrolase activity of the L. casei enzyme
or by a hydrolase contaminant in the extract. The amounts of these shorter chain
products found are < 0.5% of the substrate conversion observed with the
monoglutamate, suggesting that any putative hydrolysis is minor.
The in vivo experiments in recombinant E. coli show that the L. casei FPGS gene
alone is capable of generating the long chain polyglutamates observed in L. casei
extracts. If any accessory factor is required, which is not present in the in vitro
reaction, then the factor is also present in E. coli cells and is not specific to L. casei.
The product of the downstream gene found adjacent to the L. casei FPGS gene is not

649
involved in this elongation, since transformants containing plasmids which coexpress
both genes had no longer chain polyglutamates than transformants expressing only the
FPGS gene (not shown). The putative accessory factors may simply be other folate-
binding enzymes in the cell, although addition of crude extracts to purified enryme did
not increase the chain length of products formed in vitro.
Although its total folate pools are depleted, SF4 contains long chain
polyglutamates similar to those found in wild type E. coli cells (9). The y-linked
glutamates may be longer chain than in the wild type cells, since tetraglutamates were
observed in SF4 cells after carboxxypeptidase Y digestion, whereas only y-
triglutamates were observed in wild type cells (3,9). This may be due to the low
intracellular concentrations of folate monoglutamates, which would normally compete
with polyglutamates as substrates for the enzyme. The defect in the dihydrofolate
synthetase activity in this mutant appears to be more severe than in the FPGS activity
(11) allowing sufficient FPGS activity to produce longer chain products from the lower
folate pools. Alternatively, the carboxypeptidase cleavage may have been incomplete.

REFERENCES

1. A.L. Bognar and B. Shane, J. Biol. Chern. 258:1574-1581 (1983).

2. J. Toy and A.L. Bognar, J. Biol. Chern. 265:2492-2499 (1990).
3. R. Perone, S. C. Singer, M. H. Hanlon, and S. Roland, p. 585-589, in "Chemistry
and Biology of Pteridines", J. A. Blair ed., de Gruyter, Berlin.
4. A.L. Bognar, C. Osborne, B. Shane, S. Singer, and R. Perone, J. Biol. Chern.
260:5625- 5630 (1985).
5. V. Cody, J.R. Luft, W. Pangborn, J. Toy and A.L. Bognar, J. Mol. Biol. 224:1179-
1180 (1992).
6. J.J. McGuire, P. Hsieh, J.K. Coward and J.R. Bertino, J. Biol. Chern. 255:5776-5788
(1980).
7. B. Shane, J. Biol. Chern. 255: 5655-5662 (1980).
8. S.K. Foo, D.J. Cichowicz and B. Shane, Anal. Biochem. 107:109-115 (1980).
9. Perone, M.H. Hanlon, S.C. Singer and D.F. Hunt, J. Biol. Chern. 261:16356-
16362 (1986).
10. A.L. Bognar, C. Osborne, B. Shane, J. Biol. Chern. 262:12337-12343 (1987)
11. K. Keshavjee, C. Pyne and A.L. Bognar, J. Biol. Chern. 266:19925-19929 (1991).

650
DEVELOPMENT OF A SIMPLE FOLYLPOLYGLUTAMATE SYNTHETASE

ASSAY IN TISSUES AND CELL LINES.

Godefridus J. Peters*, Clasina L. van der Wilt*,

Jacqueline Cloos@, Herbert M. Pinedo*,**

Dept. Oncology* and Dept. Otolaryngology@,

Free University Hospital, PO Box 7057, 1007 MB Amsterdam, and
**Netherlands Cancer Institute, Amsterdam, the Netherlands

INTRODUCTION

Polyglutamylation of natural folates and anti-folates, such as methotrexate

(MTX), is an essential feature for their intracellular retention and subsequent
activity. 1 It has been demonstrated that polyglutamates of MTX accumulate
specifically in leukemic cells compared to normal tissues such as bone marrow
and gut mucosa. 2 Polyglutamates of normal folates are better substrates for
most enzymes than the monoglutamates, while polyglutamates of the antifolates
are usually better inhibitors. 2 •3 The binding of the active metabolite of 5-
fluorouracil (5FU), 5-fluoro-2' -deoxyuridine-5' -monophosphate (FdUMP) to
thymidylate synthase (TS} is enhanced in the presence of 5,1 0-
methylenetetrahydrofolate (CH 2- THF) 4 ; this binding is even more enhanced in the
presence of polyglutamates of CH 2-THF, 5 leading to an increased inhibition of
TS. This feature is an important issue in the treatment of colon cancer with the
combination of 5FU with leucovorin (LV). 6 It has been concluded from preclinical
studies that a prolonged exposure to LV seems to be essential for the desired
potentiation of the antitumor effect of 5FU. Such a long exposure favors
polyglutamylation.
Polyglutamylation is catalyzed by the enzyme folylpolyglutamate
synthetase (FPGS). A number of different assays have been described for this
enzyme, 7•8 •9•10 but basically all assays use a natural folate or an antifolate as one
substrate and glutamate as the other substrate. Depending on the assay either
the (anti)folate or the glutamate is labeled. ATP is an essential cosubstrate. A
number of these assays are rather laborious due to the use of columns to
separate the polyglutamylated (anti)folate from the substrate; this separation is
based on the difference in charge between the product and the substrate. This

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 651
and several other attractive features of reported assays were combined to
develop an FPGS assay employing thin-layer chromatography (TLC) enabling a
rapid separation of multiple samples in one run.

MATERIALS AND METHODS

As radiolabeled substrate for FPGS we used L-[2,3-3 H]-glutamate (New

England Nuclear). Aminopterin (AMT) was used as the folate substrate. The
assay was optimized using a source known to be rich in FPGS, mouse liver.
Livers were removed, washed with physiological salt solution and frozen in liquid
nitrogen. The frozen livers (and other tissues in a later part of the study) were
pulverized using a micro dismembrator. 11 The frozen powder was suspended in
Tris buffer (pH 8.5}, supplemented with 30 mM NaHC03 and 45 mM
mercaptoethanol for enzyme stabilization. Cell lines were suspended in this
buffer; cells were lysed using sanification. After sanification (cells) or pulverization
(frozen tissues), the suspensions were centrifuged at 600g and subsequently at
12,000g (cells) or 160,000g (tissues). FPGS was assayed with 1 mM 3 H-L-
glutamate, 5 mM ATP, and 500 11M AMT as the folate precursor, in the presence
of 10 mM MgCI 2 (final concentrations). This mixture (total volume 60 11L) was
incubated for 1-3 h at 37°C, and the reaction terminated by denaturation of the
enzyme by heating at 95°C for 3 min. Products (polyglutamates) were separated
from the substrate by spotting 5-1 0 11 L of the reaction mixture onto
polyethyleneimine (PEI)-cellulose TLC sheets which were developed using a
mixture of 0.5% NH 4CI and 0.5% mercaptoethanol in water. Using this procedure
the radiolabeled product of the reaction (the diglutamate) remained at the origin
(Rf 0}; the substrate glutamate had a Rf of 0.6. Spots were identified under u.v.
light, cut out, radioactivity was eluted and counted. Various blanks were
included, without enzyme, without ATP and without AMT. The formation of
polyglutamates was confirmed by separation of the mono- and diglutamates with
anion-exchange HPLC. 12

RESULTS

The assay as described in Materials and Methods is the final optimized

assay, which appeared to be linear with time and protein. In order to optimize
this assay, several variables, enzyme preparation, reaction conditions and
quantification of the product, were tested. For the enzyme preparation we tested
several purification procedures. The first step included a centrifugation step;
centrifugation at 160,000g yielded the best results. The 160,000g supernatant
was used to partially purify the enzyme using ammonium sulfate precipitation;
excess of ammonium sulfate was removed by either ultrafiltration or the use of
Sephadex G25 columns. However, these purifications steps did not lead to
better results than the use of the 160,000g supernatants. Similar or even lower
enzyme activities were observed, possibly due to the instability of the enzyme
leading to degradation during purification.
For termination of the reaction several procedures have been described in
the literature; addition of sodiumacetate, addition of excess of unlabeled
glutamate precipitation with trichloroacetic acid or heating at 95°C. From these
variables heating gave the most reliable, reproducible results in our hands. For
separation of the product from the substrate DEAE columns were compared with

652
PEI-TLC; the use of the columns was however rather laborious and results were
variable. The above-mentioned TLC procedure yielded the best reproducible
results. Qualitative and quantitative verification of the formation of polyglutamate
was achieved by separation of the reaction with anion-exchange HPLC, a
procedure similar to quantify polyglutamates of MTX and 1O-ethyl-1 0-
deazaaminopterin {10-Edam). 12 This revealed a clear formation of the
diglutamate, but not of higher glutamates.
The final assay {as described in Materials and Methods) was subsequently
used to measure the FPGS activity in a number of different cell lines and tissues
{Table 1). As a control several tissues were included known to have either high
or low FPGS activities. Our data confirmed that livers had a higher FPGS activity
than gut mucosa or bone marrow cells. From the other tissues tested all colon
tumors had a high activity of FPGS, even higher than the liver. Various cell lines
with a different histological origin have been tested. A large range of FPGS
activity was observed.

Table 1. Activity of FPGS in normal and tumor tissues and cell lines.
Tissue Cell line Origin FPGS activity
(pmol diglutamate formed/hr/)

mg wet weight 106 cells

Liver Mouse 24 ± 5
Gut mucosa Mouse 7 ± 0.2
Colon 38 Murine colon tumor 28 ± 2
Colon 26 Murine colon tumor 52± 6
Colon 26-10 Murine colon tumor 48 ± 1
Bone marrow Mouse 35 ± 6
C26-10 Murine colon tumor 156 ± 28
C26-10/F** Murine colon tumor 567 ± 140
C38-1 murine colon tumor 82 ± 9
UM-SCC-118 Human SCC* 233 ± 23
UM-SCC-228 Human SCC 141 ± 41
UM-SCC-14C Human SCC 113 ± 7
HT 29 Human colon cancer 424 ± 18
WiDr Human colon cancer 178 ± 54
WiDr/F** Human colon cancer 413 ± 105
SW948 Human colon cancer 272 ± 33
CC531 Rat colon cancer 656 ± 43

Values are means ± SEM of 3-6 separate samples. C26-1 0 and C38-1 are cell lines derived
from the murine colon tumors Colon 26 and Colon 38, respectively. C26-10 was used for
establishment the tumor designated Colon 26-10.
*,sec, squamous cell carcinoma;**, cell lines derived from C26-10 and WiDr, respectively, but
adapted to growth in folate-depleted medium.

DISCUSSION

We developed a simple method to determine FPGS activity in several

cancer cell lines and tissues. The method based on the difference in charge
between the mono- and diglutamate, enabled the separation using anion-

653
exchange TLC. Previously the same principle had been used to separate
nucleotides with a different charge. The data were confirmed using standard
HPLC separation of mono- and diglutamates. The appearance of the radioactivity
in the diglutamate demonstrated that not only a qualitative separation was
achieved with the TLC, but that the data could also be verified quantitatively. A
limitation of TLC assay is the limited loading capacity of the thin layers which we
used. This is especially a problem when samples with a low activity have to be
evaluated. However, the amount of radioactivity determined after elution of the
thin-layers was comparable to that used after quantification with columns. 8 The
TLC method enables to evaluate many samples (18 on one TLC sheet) in one
run.
The pattern of FPGS activity as observed in tissues is comparable to that
reported in literature1•2 with a low activity in bone marrow and gut mucosa and a
high activity in liver. The FPGS activity also agrees excellently with the pattern
reported previousll' 13 and with the accumulation of MTX and 10-Edam
polyglutamates in the squamous cell carcinoma celllines. 12 It should however be
noted that polyglutamate accumulation is also dependent on other factors than
the FPGS activity. These include not only the exposure time but also the
intracellular degradation catalyzed by e.g. folylpolyglutamate hydrolase.
Measurement of FPGS activity will enable to screen tumors for their capacity to
polyglutamylate antifolates. This is of particular interest because of the
development of a number of new antifolates, some being selected for their
polyglutamylation properties.
In conclusion, we established a new assay for FPGS enabling a rapid
measurement of the FPGS activity in cell lines and tissues. The results are
comparable to existing assays, but the analytical procedure is more easily to be
performed.

REFERENCES
1. B.A. Chabner, C.J. Allegra, G.A. Curt, N.J. Clendeninn, J. Baram, S. Koizumi, J.C. Drake, and
J. Jolivet, J. Clin. Invest. 76:907-912 (1985).
2. I. Fabre, G. Fabre, and I.D. Goldman, Cancer Res. 44:3190-3195 (1984).
3. R.G. Moran, P.O. Colman, and A. Rosowsky, NCI monographs 5:133-138 (1987).
4. H.M. Pinedo and G.J. Peters, J. C!in. Oncol. 6:1653-1664 (1988).
5. S. Radparvar, P.J. Houghton, and J.A. Houghton, Arch. Biochem. Biophys. 260:342-350
(1988).
6. G.J. Peters and C. van Groeningen, Ann. Oncol. 2:469-480 (1992).
7. B. Antonsson, J. Barreda, and R.G. Moran, Anal. Biochem. 186:8-13 (1990).
8. G. Jansen, J.H. Schornagel, I. Kathmann, G.R. Westerhof, G.-J. Hordijk, B.F.A.M. van der
Laan, Oncol. Res. 4:299-305 (1992).
9. J. Barreda and R.G. Moran, Mol. Pharmacal. 42:687-694 (1992).
10. D.E. McKioskey, J.J. McGuire, C.A. Russell, B.G. Rowan, J.R. Bertino, G. Pizzorno, and E.
Mini, J. Bioi. Chern. 266:6181-6187 (1991).
11. G.J. Peters, E.J. Laurensse, A. Leyva, and H.M. Pinedo, Clin. Chim. Acta 158:193-198
(1986).
12. B.J.M. Braakhuis, G. Jansen, and G.J. Peters, these proceedings.
13. B.F.A.M. van der Laan, G. Jansen, G.A.M. Kathmann, G.R. Westerhof, J.H. Schornagel, and
G.J. Hordijk, Int. J. Cancer 51:909-914 (1992).

654
TWO NOVEL HPLC METHODS WHICH RAPIDLY DETECT THE
SUBSTRATES AND CLEAVAGE PRODUCTS OF ')'-GLUTAMYL HYDROLASE

Ying Wang, Robert F. Rotundo, Zenia Nimec, Thomas J. Ryan,

and John Galivan

Wadsworth Center for Laboratories and Research

New York State Department of Health
Albany, NY 12201-0509

INTRODUCTION

')'-Glutamyl hydrolase (EC 3.4.22.12) is widely distributed throughout the

phylogenetic spectrum and is thought to play an important role in the cellular homeostasis
and nutritional absorption of folates and antifolates 1•2 • The enzyme catalyzes the endo- or
exopeptidyl hydrolysis of the -y-glutamyl side chains of naturally occurring
polyglutamylated folates and synthetic antifolates2 •
A number of methods have been developed to detect the reaction products of -y-
glutamyl hydrolase. Earliest methods utilized to detect -y-glutamyl hydrolase activity were
based upon the ability of mono- and diglutamate folate reaction products to support the
1

growth of folate-requiring bacteria24 • The synthesis of [3' ,5', 7-3H]PteGlq, madr it possible
to detect 1'-glutamyl hydrolase cleavage products using paper chromatography or high-
voltage electrophoresis by analyzing the pteroyl-p-aminobenzoate portion of the reaction
products2•5 • Priest et al. 2•6 developed a sensitive method that measured -y-glutamyl hydrolase
activity using the product formed with 5, 1O-CH2H4PteGlu" in ternary complex with
[ 3H]FdUMP and l. casei thymidylate synthase. Reversed-phase HPLC was also used to
detect the pterin-containing products of hydrolysis7•8• An alternative way to detect the
cleaved products was to examine the glutamate released during the enzyme reaction.
Radiolabelled glutamate, which had been covalently linked to PteGlu by bacterial or
chemical synthesis, was used to detect free glutamate and poly--y-glutamate2•9-11 • While this
system is sensitive and accurate, the need to prepare specifically labelled folyl-[3H or 14C]-
-y-glutamyl substrates is complex and time-consuming. Ninhydrin or TNB (2,4,6-
trinitrobenzene sulfonic acid) was also used as a detection method for released glutamate2•
All of these assays can detect -y-glutamyl hydrolase cleavage products but, many are
limited by their slowness, expense, lack of sensitivity, or complexity of design2 •
Studies reported previously from this laboratory established for the first time that -y-
glutamyl hydrolase is extensively secreted by primary cultures of non-absorptive cells and
many transformed cell lines. During the purification of -y-glutamyl hydrolase secreted from

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 655
rat H35 hepatoma cells, two rapid, sensitive, and reliable assays were developed to aid in
the detection of substrate and cleavage products of this enzyme. We have developed an
assay to measure -y-glutamyl hydrolase activity that uses dimethylaminophenyl-azo-
benzenesulfonyl-poly--y-glutamate, in which the DABSYL group is linked to the N-
terminus of the poly--y-glutamate. The products of the reaction, DABSGlUy,, are detected
by visible absorption following rapid resolution on HPLC. The second method is based
upon o-phthalaldehyde (OPA) derivatization of free amine groups resulting in fluorescent
derivatives of the various-length poly--y-glutamates and free glutamate released from folate
or other poly--y-glutamate species. The latter method can be used to obtain a complete
picture of the cleavage products of -y-glutamyl hydrolase and will be used in its
characterization.

MATERIALS AND METHODS

-y-Glu 1_5 were purchased from the Dr. B. Schircks Laboratories (Jona, Switzerland),
and all other chemicals were reagent grade. DABSGlun was synthesized by the method of
Chang et alY. -y-Glun (20 mg) in 2.5 mL of 0.2 M sodium bicarbonate buffer (pH 9.0)
was mixed with 4-dimethylamino-azo-benzenesulfonyl chloride (13 mg, Pierce) in 2.5 mL
of acetone. The mixture was incubated at 700C for 30 min, diluted with 7.5 mL water and
lyophilized. The product was purified by reversed-phase HPLC (Bakerbond C18 column,
1.5 x 30 em) using a gradient from 20% to 70 % acetonitrile in 50 mM ammonium acetate
buffer, pH 4.5. The DABSGlun was used as a substrate for -y-glutamyl hydrolase.
Pre-column derivatization using OPA was done by mixing 25 ~tL of reaction mixture,
12.5 ~tL methanol, and 12.5 ~tL OPA derivatization reagent for 1 min. The derivatization
reagent contained 5.0 mg OPA in 0.2 mL methanol, 1.8 mL 0.4 M sodium borate (pH
10.5), and 8 ~tL 2-mercaptoethanol. This reagent was made fresh daily. The sample was
then loaded onto a reversed-phase C18 HPLC column for separation and quantitation.

RESULTS AND DISCUSSION

DABSGlu5 , DABSGlu4 , DABSGlu3 , DABSGlu2 , and DABSGlu were analyzed on a

reversed-phase C18 HPLC column using isocratic elution with 28% acetonitrile in 50 mM
sodium acetate (pH 4.5) and detection at 436 nm. The retention times for DABSGlu5
through DABSGlu were 4.2, 4.7, 5.4, 7.0, and 11.5 min, respectively (Fig. 1). This assay
is not only rapid and reliable owing to the ability to separate the products using an
isocratic gradient, but it is also sensitive. The DABSGlUa absorbs at 436 nm (~:=17,000)
with a sensitivity of less than 200 pmol. Reaction with hepatoma -y-glutamyl hydrolase
causes cleavage at the two innermost bonds with the release of -y-Gl~ and -y-Glu3 , which
are subsequently cleaved to glutamate. The reaction rate is about one-half that of folyl or
methotrexate polyglutamate.
The derivatization of glutamate and poly--y-glutamate with OPA is a modification of
other methods 13•14 • -y-Glu5 , -y-Glu4 , -y-Glu3 , -y-Glu2 and glutamate were run on a reversed-
phase C18 HPLC column after pre-column derivatization. The column conditions included
the use of 0.1 M sodium acetate (pH 5.5) with a flow rate of 1 mL/min and a gradient of
0 to 20% acetonitrile over 20 minutes. The OPA-glutamate products were detected by a
Gilson fluorometer with an excitation wavelength at 365 nm and an emission wavelength
at 420 nm. The retention times for -y-Glu5 through glutamate were 12.0, 13.5, 15.0, 18.0
and 23.0 min., respectively (Fig. 2). The procedure could accurately detect 5 pmol of
product. OPA in the presence of 2-mercaptoethanol or ethanethiol forms highly fluorescent
isoindole derivatives when in the presence of primary amino acids15 • This method can

656
 5 4 3 2

w
u
z
<(
m
0::::
0
(f)
m
<(

0 3 6 12 15

RETENTION TIME (min)

Fig. 1. HPLC profile of DABSG!t~;(5), DABSG!u4(4), DABSGI~(3), DABSGlll:l(2), and DABSG!u(l). The
concentration of each compound injected was 50 nmollmL in a sample volume of 20 p,L.

100

5 4 3 2

80
w
u
z
w 60
u OJ
(f)
w ~
0::::
0 40
::;
_j
LL

20
.............-······················

···················································································"' ........
.................... '- c__ ' - - L___ _ ___j L----~
0

0 5 10 15 20 25 30

RETENTON TIME (min)

Fig. 2. HPLC profile of OPA derivatives of y-Glt~;(5), y-Glui4), y-Glul3), y-Glu2(2), and glutamate (1).
The concentration of each compound injected was 1 nmollmL in a sample volume of 10 1-'L.

657
detect poly--y-glutamates released from any substrate (MTXGlun, PteGlun, DABSGlUn,
PABAGlum -y-Glum etc.) and can be used with UV-absorption-HPLC to give a complete
description of the products of the hydrolytic reaction. In this manner, an accurate analysis
of the catalytic properties of -y-glutamyl hydrolase from any source can be made. Both
methods can be used together to measure DABSGlun and the poly--y-glutamates and
glutamate released from the DABSGlu substrate.

ACKNOWLEDGEMENTS

This work was supported by Grant CA25933 from the NCI/NIH. We acknowledge
the assistance of Dr. Robert A. Waniewski of the Wadsworth Center for Laboratories and
Research for his suggestions in the development of the OPA-derivatization assay.

REFERENCES
1. C.H. Halsted, Intestinal absorption of dietary folates, in: FOLIC ACID METABOLISM IN
HEALTH AND DISEASE, M.F. Picciano, E.L. Stokstad and J.F. Gregory III, eds. Wiley-Liss,
Inc., New York (1990).
2. J.J. McGuire and J.K. Coward, Pteroylpolyglutamates: biosynthesis, degradation, and function, in:
FOLATES AND PTERINS: VOLUME 1 CHEMISTRY AND BIOCHEMISTRY OF
FOLATES, R.L. Blakley and S.J. Benkovic, eds. John Wiley & Sons, Inc., New York (1984).
3. O.D. Bird, S.B. Binkley, E.S. Bloom, A.D. Emmett and J.J. Pfiffner, J.Biol.Chem. 157:413 (1945).
4. V. Mims, M.E. Swenseid and O.D. Bird, J.Biol.Chem. 170:367 (1947).
5. I.H. Rosenberg and H. Neumann, J.Biol.Chem. 249:5126 (1974).
6. D.G. Priest, C.D. Veronee, M. Mangum, J.M. Bednarek and M.T. Doig, Mol.Cell.Biochem.
43:81 (1982).
7. D.W. Fry, J.C. Yalowich and I.D. Goldman, J.Biol.Chem. 257:1890 (1982).
8. Z. Nimec and J. Galivan, Arch.Biochem.Biophys. 226:671 (1983).
9. C.L. Krumdieck and C.M. Baugh, Anal.Biochem. 35:123 (1970).
10. M. Silink, R. Reddel, M. Bethel and P.B. Rowe, J.Biol.Chem. 250:5982 (1975).
11. L.M. Kozloff and M. Lute, J. Virol. 40:645 (1981).
12. J.Y. Chang, R. Knecht and D.G. Brown, Methods Enzymol. 91:41 (1983).
13. D.C. Spink, J.W. Swann, O.C. Snead, R.A. Waniewski and D.L. Martin, Anal.Biochem. 158:79
(1986).
14. M.H. Fernstrom and J.D. Fernstrom, Life Sci. 29:2119 (1981).
15. S.S. Simons and D.F. Johnson, J. Org. Chern. 43:2886 (1978).

658
PURIFICATION AND GENERAL PROPERTIES OF HUMAN
FOLYLPOLYGLUTAMATE SYNTHETASE

Timothy A. Garrow and Barry Shane

Department of Nutritional Sciences, University of California

Berkeley, California 94720

INTRODUCTION

We recently cloned a human eDNA encoding folylpolyglutamate synthetase (FPGS) by

complementation of an E. coli strain (SF4) deficient in FPGS activity 1. The open reading
frame of the eDNA predicted a protein having 545 amino acids, a Mr of 60 kDa, and an
estimated pi of 6.95. In this report we describe tl1e plasmid construct used for the
expression of unfused human FPGS in E. coli. In addition, general characteristics of the
purified enzyme are discussed as well as its specificity for selected folyl and analog
substrates.

RESULTS

Figure 1 shows tl1e construction of pET3A-25, a plasmid designed for the expression of
FPGS in E. coli strain JM109(DE3). Plasmid pET3A-25 has the human FPGS eDNA
inserted between the bacteriophage T7 gene 10 promoter/ribosome binding site and
transcriptional termination sequences. JM109(DE3) is a lambda lysogen which has the
bacteriophage T7 RNA polymerase (gene 1) linked to the inducible lacUV5 promoter.
FPGS was purified by a combination of hydroxylapatite, Affigel blue, phenyl agarose,
heparin agarose, and DEAE cellulose chromatography. The preparation was judged to be
homogeneous by SDS-PAGE and had a Mr of -60,000 consistent with the molecular weight
predicted from tl1e deduced amino acid sequence.
Human FPGS had a pH optima of approximately 9.4, required a monovalent cation for
activity (Figure 2A), and used MgATP2 - as the nucleotide substrate (Figure 2B). Excess
Mg2+ or ATP inhibits enzyme activity. Potassium was tl1e preferred monovalent cation while
ammonium and rubidium stimulated activity to a lesser extent. Sodium or cesium were
unable to stimulate FPGS activity and all of the monovalent cations inhibited activity (45-
70%) when the standard assay mixture (20 mM K+) contained 200 mM of the desired
monovalent cation. Manganese was the only other divalent cation that stimulated FPGS
activity, supporting 13% of the activity obtained with magnesium (1 mM cation and 100 ~M
ATP), while no activity was observed with cobalt, calcium, or zinc. Human FPGS required
the presence of a reducing agent for activity. Half-maximal stimulation occurred at 0.48 mM
DTT or 5.5 mM BME. Maximal stimulation was obtained at 5 mM DTT or 50 mM BME and
the maximum activity obtained with BME was 90% of tl1at obtained with DTT.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 659
 f. coR!

ECORI
pSE936-25 pTZ18U-25
IOkbp 5039bp
lox

/
site-directed
mutagenesis

/
pTZ18U-25-
Ndel pET3A-25
504Ibp 6720bp

Figure 1. Construction of pET3A-25 from pSE936-25 for the expression of human FPGS in E. coli. The
EcoRI insert of pSE936-25 was subcloned into the phagemid pTZ 18U for the production of ssDNA used
in the mutagenesis protocol (Eckstein). The Ndel-BamHI fragment containing the entire ORF of the eDNA
was then subcloned into the expression vector pET3A.

660
 100 100 B

75 75
~ ~
·::;: ·::;:
·.c ·.c
g 50 g 50
lf'
25
* 25

0 0
.1 1 10 1 00 .01 .1 1 10 100
!cation! mM LMg] mM

Figure 2. Effect of monovalent cations (panel A), and magnesium concentration on FPGS activity. Assay
conditions have been previously described 2.

The kinetic constants of folylmonoglutamates and analogs for human FPGS are shown
in Table 1. Enzyme activity was assayed using 1 mM ATP and 2 mM glutamate as the fixed
substrates and various concentrations of folylmonoglutamate or analog substrates. The
catalytic efficiency of the enzyme for dihydrofolate and aminopterin were higher and 10-
formyltetrahydrofolate was lower compared to the hog liver enzyme3,4.

Table 1. Kinetic Constants of Folates and Analogs for Human FPGS.

Substrate Km(~) Vmax* Vmax/Km*

PteGlu 60 59 2
H2PteGlu 0.9 101 225
(6RS)-H4PteGlu 2.0 100 100
(6R)-10-formyl-H4PteGlu 3.7 27 14
(6S)-5-formyl-H4PteGlu 105 99 2
(6S)-5-methyl-H4PteGlu 48 96 4
Aminopterin 4.4 136 62
Methotrexate 81 108 3

* Relative values; 100 equals the values obtained with (6RS)-H4PteGlu.

DISCUSSION

Human FPGS has been overexpressed in E. coli and the purified enzyme has general
and kinetic properties similar to hog liver FPGS2,3. A notable difference between the hog
and human enzymes are the reduced Km values for folic acid, aminopterin, and methotrexate,
and a higher Vmax for aminopterin3,4. The di- and triglutamates of methotrexate are also
better substrates (lower Km and higher Vm/Km) for the human than for the hog liver enzyme
(data not shown). Previous studies regarding the substrate kinetics of (6RS)-5-
formyltetrahydrofolate for human FPGS reported values significantly lower than we report
here (approximately an order of magnitude). This discrepancy may have been due to

661
metabolism of the 5-formyl compound into a derivative which has higher affinity for FPGS
since the former studies used relatively crude enzyme preparations.

ACKNOWLEDGEMENT

This research was supported in part by grant no. CA 41991 from the National Cancer
Institute, Department of Health and Human Services.

REFERENCES

1. T.A. Garrow, A. Admon, and B. Shane. 1992 Proc. Natl. Acail. Sci. USA 89: 9151.
2. D.J. Cichowicz and B. Shane.1977 Biochemistry 26: 504.
3. D.J. Cichowicz and B. Shane. 1987 Biochemistry 26: 513.
4. R.J. Coll, D. Cesar, J.B. Hynes, and B. Shane. 1991 Biochem. Pharmacal. 42: 833.

662
ANTITUMOR EFFICACY OF CLASSICAL NON-POLYGLUTAMYLATABLE
ANTIFOLATES THAT INHffiiT DffiYDROFOLATE REDUCTASE

Ann Abraham*, M.G. Nair*, J.J. McGuire+, J. Galivan+, R.L. Kisliuk§,

B.Rao Vishnuvajjala,.

*university of South Alabama, Mobile, Alabama 36688

+Roswell Park Cancer Institute, Buffalo, New York 14263
+wadsworth Cancer for Laboratories and Research, Albany, New York
12201
§Tufts University, Boston, Massachusetts 02111
1National Cancer Institute, Rockville, MD 20852

INTRODUCTION

The non-polyglutamylatable dihydrofolate reductase (DHFR) inhibitors ,-methylene-10-

deazaaminopterin (MD AM) and ,_ meth ylene-1 0-ethyl-1 0-deazaaminopterin (MED AM) 1•2
exhibited striking activity relative to methotrexate (MTX) against the growth of a number
of human tumor cell lines in culture3 •4 . Since both MDAM and MEDAM are non-
polyglutamylatable and therefore could be cleared more efficiently from tissues than MTX,
it was of interest to undertake a comparative study of the in vivo activity and toxicity of
these compounds and MTX in normal and tumor bearing animals. These investigations
were carried out with MEDAM using normal and ip implanted L1210 and P388 leukemic
mice as the animal model using a number of experimental protocols. MDAM and
MEDAM were previously shown to inhibit recombinant human DHFR at equivalent
magnitude as MTX 1•2 . They were neither substrates nor inhibitors of CCRF-CEM human
leukemia cell folypolyglutamate synthetase (FPGS) and they competed more efficiently for
folinic acid transport than MTX in H35 hepatoma cells. Both MDAM and MEDAM were
excellent inhibitors of the growth of H35 hepatoma, CCRF-CEM human leukemia and
Manca human lymphoma cells in culture. In NCI's human tumor disease oriented in vitro
screen, MDAM and MEDAM exhibited a wide spectrum of sensitivity and their activities
were superior to MTX in a large number of tumor cells (Table I).
To determine whether these non-polyglutamylatable DHFR inhibitors 1 and 2 were
capable of exhibiting cytotoxicity in vivo, the relative efficacy of MEDAM and MTX on
the survival of normal BD2F1 mice was evaluated using different experimental protocols.
MEDAM was found to be non-toxic to these animals at a dose of 30 mg/kg/day on a
QdX5 schedule. In the same protocol, MTX was found to be lethal to all animals (Table
II). By increasing the dose from 30 to 100 mg/kg/day, and administering the drug by
continuous infusion using osmotic pumps, MEDAM was found to be equitoxic as MTX
to the animals. The mean survival time (MST) of the animals receiving MTX and
MEDAM were 10.5 and 10.9 days respectively. Next we investigated the cytotoxicity of
Chemistry and Biology of Pteridines ·and Folates, Edited by
J.E. Ayling et al., Plenum Press, New York, 1993 663
MEDAM relative to MTX by the oral route. Both MTX and MEDAM when given at 100
mg/kg/day by three divided oral dosages resulted in equivalent MST for the animals.
Surprisingly when MEDAM dosage was reduced to 50 mg/kg/day for 7 days under the
same protocol, all animals survived with no sign of toxicity. The same MTX protocol was
lethal to all animals. These results indicated that MEDAM is less toxic and is tolerated
better by the animals, and equivalent cytotoxicity as MTX could be induced with MEDAM
by increasing its dosage and changing the protocol. The exciting possibility of the
modulation of toxicity of a classical antifolate by simple adjustment of its dosage, appeared
to be within reach as judged by the above results with MEDAM and a systematic
examination of its antitumor activity under different experimental protocols was therefore
undertaken.
The first objective was to administer MEDAM to tumor bearing animals at different
dosages while maintaining the plasma level of the drug continuously at the desired amount.
This was attempted by encapsulating MEDAM in time released pellets to permit its
continuous release at different dose levels for a period of 15 days. BD 2F 1 mice bearing
ip implanted L1210 and P388 leukemia were chosen as the antifolate sensitive animal
model. MEDAM doses of 15 mg/kg/day and 30 mg/kg/day were arbitrarily selected for
two experiments each with L1210 and P388leukemia. A dose of 5 mg/kg/day of MTX
was used as a positive control. The results of the L1210 model established that MEDAM
had significant antitumor activity, and additional experiments would be needed to optimize
its therapeutic index in this model. The antitumor activity of MEDAM was clearly dose
dependent. The P388 model was more responsive to MEDAM, and a %ILS increase of
-70 was obtained with a dose of 15 mg/kg/day of MEDAM. At 30 mg/kg/day of
MEDAM when combined with bolus of 100 mg/kg there was a further increase of %ILS
(97). Inorder to evaluate the full therapeutic potential of this novel non-polyglutamylatable
antifolate in cancer chemotherapy, additional in vivo experiments are clearly warranted,
and such studies are in progress.

Table I. Activity of MDAM and MEDAM against

the growth of selected tumor cells in culture3.4

Cell line LogwGiso

MTX MDAM MEDAM

Leukemia
CCRF-CEM -7.57 -9.13 -9.16
HL-60 (TB) -7.48 <-10.0 -9.84
K-562 -7.6 <-10.0 <-10.0
MOLT-4 -7.59 -8.75 -9.03
SR -7.57 -9.6 -9.91
Non-Small Cell Lung Cancer
A549/ATCC -7.52 -8.88 -9.23
NCI-H322M -6.77 -8.14 >-6.0
NCI-H460 -7.54 -9.18
Small Cell Lung Cancer
DMS 114 -7.48 -7.92 -8.18
DMS 273 -7.51 -8.41 -9.19
Colon Cancer
DLD-1 -7.43 -8.88 -9.24
HCC-2998 -7.0 -8.71 -6.3
HCT-116 -7.54 -9.4 -9.61
HCT-15 -7.49 -9.21
HT-29 -7.43 -9.01 -8.89
SW-620 -7.51 -8.95
CNS Cancer

664
 Table I. Continued

SF-268 -7.27 -8.2 -8.04

SF-295 -7.46 -8.15 -8.95
SF-539 -7.48 -7.82 -8.5
SNB-19 -6.47 -7.01 -8.74
Melanoma
LOX IM VI -7.59 -9.29 -9.33
Ovarian Cancer
IGROV1 -7.33 -8.47
OVCAR-8 -7.51 -8.19 -8.19
Renal Cancer
786-0 -7.59 -9.22 <-10.0
ACHN -7.39 -8.24 -8.6

A 48 h continuous drug exposure protocol was used combined with an SR13 protein assay to estimate cell
growth or viability.

Table II. Relative efficacy of MTX and MEDAM on the survival of normal BD2F 1 mice
-------------------------------------------------------------1
Compound Dose/day MST Average wt Dose
(days) (GM) Schedule

MTX 30/mg/kg 11.5 22.1 QdX5

ME DAM 30mg/kg Nontoxic 21.6 QdX5

Table ill. Relative efficacy of MTX and MEDAM on the survival of normal BD2F 1
mice on continuous infusion (SC) using osmotic pumps

Compound Dose/day Average wt MST days

(gm)

MTX lOOmg/kg 22.0 10.5

MEDAM 100mg/kg 22.3 10.9

Table IV. Relative efficacy of MTX and MEDAM on the survival of normal BD2F 1 mice
when administered orally
Compound Dose/day MST (days) Dose Schedule

MTX 100/mg/kg 7.5 3 times a day

MEDAM 50mg/kg non-toxic 3 times a day
MEDAM lOOmg/kg 7 3 times a day

~2/
H

H NJ:C?_JO)-cH2-
I@
R
I
0 -c-~-c--H
CH-
0
II
COOH

2 N N MDAM ,R = H H ) - COOH
MEDAM ,R = Et 2

665
 Table V. Antitumor activity of MTX and MEDAM in mice bearing ip
implanted L1210 and P388 leukemia*

MST(days) %ILS

Compound Dose mg/kg/day L1210 P388 Ll210 P388

Control 14 14.5
MTX 5.0 19 20.5 33 41
MEDAM 15.0 19 25 33 72

MEDAM 30.0 20.5 24.5 43 69

MEDAM 1 30.0 21 28.5 49 97

• Time released pellets of MTX, and MEDAM were prepared by Innovative Research, Inc., Ohio. Each
pellet was designed to uniformly release the drug during a 15 day period. The following amounts of drug
was encapsulated in each pellet: MTX, 1.5 mg; MEDAM 4.5 mg and 9.0 mg. The pellet was surgically
inserted SC in animals 24 hours after tumor implantation.
1 A bolus ip injection of 100 mg/kg was given to all animals in the group 24 hours after tumor implantation.

ACKNOWLEDGEMENT

This investigation was supported by grants CA 27101 (MGN), CA 25933 (JG) and CA 43500 (JJM) from
the National Cancer Institute.

REFERENCES

1. A. Abraham, J.J. McGuire, J. Ga!ivan, Z. Nimec, R.L. Kisliuk, Y. Gaumont, and M.G. Nair, J. Med.
Chem. 34:222. (1991).
2. M.G. Nair, A. Abraham, U.S. Patent 4,996,207, (1991).
3. M.R. Boyd, in "Principles and Practice of Oncology" V.T. DeVita, Jr. S. Hellman, S.A. Rosenberg,
eds., Lippincot, Philadelphia, (1992).
4. R.W. Fuiier, J.H. Cardeilina, II, Y. Kato, L.S. Brinen, J. Clardy, K.M. Snader, and M.R. Boyd, J.
Med. Chem. 35:3007. (1992).

666
STUDIES ON THE CROSS RESISTANCE OF FOLYLPOLYGLUTAMATE
SYNTHETASE-DEFICIENT, METHOTREXATE-RESISTANT CCRF-CEM
HUMAN LEUKEMIA SUBLINES

John J. McGuirel Kathryn J. Heitzmanl, William H. Hailel,

Cynthia A. Russell( Diane E. McCloskey I, and James R. Piper2
lRoswell Park Cancer Institute
Buffalo, NY 14263
2Bio-Organic Chemistry Division
Southern Research Institute
Birmingham, AL 35255

INTRODUCTION

CCRF-CEM human leukemia cell lines resistant to "intermittent", but not

continuous, exposure to methotrexate (MTX) as a result of defective MTX
polyglutamate synthesis were recently described1. This defective synthesis was
traced to decreased folylpolyglutamate synthetase (FPGS) activity; the FPGS
level was inversely related to the degree of resistance2. The responses of CCRF-
CEM sublines, which exhibit this new MTX resistance phenotype, to selected
currently used and experimental anticancer asents and regimens were explored
with particular regard to identifying cross-resistance and collateral sensitivity.

MATERIALS AND METHODS

Actinomycin D, adriamycin, aminopterin (AMT), cytosine arabinoside

(araC), cisplatin, 5-fluorouracil-(5-FU), ana 5-fluorodeoxyuridine (5-FUdR) were
from Sigma (St. Louis, MO). MTX was a gift of Lederle (Pearl River, NY). 5,8-
didezapteroyl-ornithine (Q-Orn) was synthesized as previously described3.
DMPDDF4 was a gift of Dr. M.G. Nair (University of South Alabama, Mobile).
Trimetrexate (TMTX), a lipid-soluble DHFR inhibitor, and PD1297295, a lipid
soluble TS inhibitor (compound 13b), were gifts of Warner-Lambert/Parke-
Davis. (6R,S)-leucovorin (LV), BW1843U89, and 6-mercaptopurine (6-MP)
were gifts of Burroughs-Wellcome (Research Triangle Park, NC). ICI D16946,
N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-
2-thenoyl)-L-glutamic acid was a gift from ICI through Dr. Y. Rustum of this
Center. Etoposide (VP-16; Bristol Meyers, Wallingford, CT) and vincristine (Eli
Lilly) were used. Other chemicals were the highest quality available.
Routine culture and growth inhibition assays with the parental CCRF-CEM
human acute leukemia cell line and the two MTX-resistant sublines, R3/71 and
R30dm2, havins 25% and 1%, respectively, of the parental FPGS activity level,
were as prevwusly described2. Drug exposures were either for 120 h
(continuous) or for 0-6 or 0-14.5 h of a 120 h total growth period with extensive
washing after the desired exposure time. Drug potency is described by the

Chemistry and Biology of Pteridines and Palates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 667
concentration effective at inhibiting cell growth by 50% relative to untreated
cultures (EC 50 ). Cultures were tested (GenProbe; San Diego, CA) for
Mycoplasma at 3 month intervals; all studies were performed with
uncontaminated cell lines. All experiments were repeated at least once.

RESULTS

Response to Continuous Exposure

FPGS-deficient sublines were assessed for response to various agents

under continuous exposure, where MTX resistance is not apparent. R3/7 and
R30dm displayed no cross-resistance to actinomycin D, acfnamycin, cisplatin,
araC, and AMT (data not shown). Observed variations in sensitivity to
etoposide, vincristine, and 5-FU did not correlate with FPGS levels (Table 1).
Marked cross-resistance during continuous exposure that correlated with FPGS
level was noted to the polyglutamylatable thymidylate synthase (TS) inhibitors
ICI D1694 and DMPDDF; only slight cross resistance was noted to another poly-
glutamylatable TS inhibitor, BW1843U89, and to 5-FUdR and 6-MP (Table 1).
Slight collateral sensitivity that apparently correlated with FPGS level was
noted to TMTX, and to the FPGS-specific inhibitor, Q-Orn.
Response to Short-term Exposure.

FPGS-deficient sublines were assessed for response to some agents under

intermittent exposure, where MTX resistance is apparent. For each drug tested
in short-term (6 or 14.5 h) exposure, the same or a higher concentration of drug
relative to continuous exposure was required to inhibit growth by 50% (Table
2). The cell lines still did not display any cross-resistance to actinomycin D and
cisplatin and remained collaterally sensitive to TMTX. Etoposide and
vincristine continued to show cross-resistance that did not correlate with FPGS
level. Interestingly, minor cross-resistance to adriamycin and araC appeared in
these intermittent exposure studies.
Leucovorin Modulation of Fluoropyrimidine-induced Growth Inhibition
The effects of LV on fluoropyrimidine-induced growth inhibition in both
FPGS-deficient cell lines were investigated, since it had previously been shown
that 2 h exposures to fluoropyrimidines could not be modulated in R3/7 7.
Simultaneous addition of 20 JlM LV potentiated the potency of both 5-FU (data
not shown) and 5-FUdR (Table 3) during continuous exposure in each cell line;
the degree of potentiation for each individual drug was similar among the cell
lines (data for FUdR, Table 3). Since the highest degree of potentiation, and
thus that most easily quantitated, was observed with 5-FUdR, the interaction
between LV and 5-FUdR was explored further.
The effect of exposure time to FUdR and LV was assessed (Table 3).
Decreasing FUdR exposure time from 120 h (continuous) to 24 h caused little
change in the potency of FUdR itself in any cell line; the degree of potentiation
by LV was also unaffected by decreased exposure time. Further decrease in
exposure time to 4 h caused about a 10-fold loss in potency in both CCRF-CEM
and R30dm with a concomitant loss in the degree of potentiation. The
potentiation loss was less marked in R30dm, however. Since the slopes of the
concentration-response curves became very flat at 4 h with an attendant
increase in variability of the EC50 (data not shown), shorter exposure times
were not tried. Whether LV was present or not for the remainder of the
outgrowth period following a 24 h exposure (Table 3), the degree of
potentiation was essentially the same for CCRF-CEM and R3/7; there was a
small loss in the degree of potentiation in R30dm, the line with the lowest
FPGS level.
The effect of LV concentration on potentiation was examined during
continuous exposure to FUdR and LV (data not shown). All cell lines were

668
Table 1. Growth inhibition of CCRF-CEM, R3/7, and R30dm cell lines selected
agents during continuous (120 h) exposure.

DRUG EC~Values
CCRF-CEM R3/7 R30dm

MTX(nM) 15 15 18
TMTX(nM)a 2.3 n.d. 0.6
DMPDDF(nM) 17 40 225
ICI D1694 (nM) 3.5 3.9 17
BW1843U89 (nM) 0.6 1.0 1.1
Q-Orn (J..LM) 200 130 86

6-MP (J..LM) 0.20 0.36 0.40

5-FU (J..LM) 2.9 4.3 3.1
5-FUdR (nM) 1.2 2.9 3.8

Etoposide (nM) 82 280 112

Vincristine (nM) 1.4 2.6 0.9
i!Data from McCloskey et al.2. Maximum error in any EC50 value is ±30%; in ~85% of cases,
error was :<=;17%.

Table 2. Growth inhibition of CCRF-CEM, R3/7, and R30dm cell lines by

selected agents during intermittent exposure.

DRUG Exposure EC~Values

Time (h) CCRF-CEM R3/7 R30dm

MTX(nM) 6a 650 2200 >50,000

14.5 110 190 480

TMTX(nM) 14.5 100 n.d. 55

Actinomycin D (nM) 6 17 19 18
Adriamycin (nM) 6 28 44 50
Etoposide (nM) 6 580 2150 750
Vincristine (nM) 6 10 13 7
cisplatin (J..LM) 6 1.2 1.1 1.4

araC (nM) 6 22 56 80
6-MP (J..LM) 6 >100 >100 >100
5-FU (J..LM) 14.5 14 n.d. 12
5-FUdR (nM) 14.5 2.2 n.d. 8.1
i!This is EC70 value. R30dm plateaus in growth inhibition at about 50-60% inhibition

maximally potentiated at 1-20 JlM LV and, as observed above, the maximum

potentiation achieved was also similar. Maximum potentiation in CCRF-CEM
and R3/7 was maintained even at 0.3 JlM; between 0.3 and 0.01JlM there was a
concentration-dependent decrease in the degree of potentiation. For R30dm,
potentiation decreased below 1JlM LV, but was still apparent at 0.03 JlM.
The ability of 20 JlM LV to expand the methylenetetrahydrofolate pool as a
function of time was assessed in all 3 cell lines to determine if expansion was
possible in the FPGS-deficient lines (data not shown). Methylenetetrahydro-
fola te pools in all lines in the absence of 20 JlM LV were similar and did not
vary over 120 h of exponential growth. The pool in each cell line was
significantly (?:3-fold) expanded after 4 h of exposure to 20 JlM LV and reached a

669
 Table 3. Effect of exrosure time on potentiation by 20 11M LV of growth
inhibition by 5-FUdR o parental and FPGS-deficient CCRF-CEM cell lines.

Time _Eso (CCRF-CEM) ECso(R3/7) EC50 (R30 dm)

-LV +LV Pot'n -LV +LV Pot'n -LV +LV Pot'n

120 1.2 0.29 4.1 2.9 0.56 5.2 3.8 0.63 6.0

24 1.9 0.41 4.6 2.5 0.61 4.1 3.7 0.65 5.7

24* 1.3 0.32 4.1 1.6 0.39 4.1 4.0 1.1 3.6

4 10.6 7.4 1.4 n.d. n.d. n.d. 34 11 3.1

*LV was not added back to the cells after the wash at 24 h. This experiment was performed
separately from the other 24 h exposure experiments.

maximum level by 24 h. Maximum pool values were similar for cell lines, but
they appeared to increase slightly as a function of relative FPGS level. Each
pool remained elevated until the last time point (120 h); parental cell pools
were unchanged over this period while there appeared to be a slight decrease in
the pool of each FPGS-deficient line.
In summary, the drug responses of cell lines which are MIX-resistant
because of decreased FPGS activity have been determined. These lines are not
cross-resistant to many standard chemotherapeutic agents and regimens. In
particular, FPGS-deficient cells are sensitive to LV modulation of 5-FU and 5-
FUdR growth inhibition as long as the exposure interval is ;:::4 h. This result
also suggests that folylpolyglutamates may not be essential to promoting this
modulation; further studies of the polyglutamate chain lengths m treated cells
will be required to substantiate th1s suggestion. FPGS-deficient cell lines are
cross-resistant to antifolate agents that are dependent on polyglutamylation to
be "activated", such as ICI D1694. These results have implications for the
testing of these new inhibitors in clinical trials and in general clinical use in
patients who have failed MIX-based therapy. The observation that these lines
are collaterally sensitive to TMTX and an FPGS-specific inhibitor offers a lead to
the development of strategies for the selective eradication of this resistance
phenotype.

REFERENCES

1. G. Pizzorno; Mini, E.; Caronnelo, M.; McGuire, J. J.; Moroson, B. A.; Cashmore, A. R.; Dreyer, R.
N.; Lin, J. T.; Mazzei, T.; Periti, P.; Bertino, J. R. Cancer Res. 1988, 48, 2149.
2. D.E. McCloskey; McGuire, J. J.; Russell, C. A.; Rowan, B. G.; Bertino, J. R.; Pizzorno, G.; Mini, E.
f. Bioi. Chem. 1991, 266, 6181. , -
3. J.J. McGuire; Bolanowska, W. E.; Piper, J. R. Biochem. Pharmacal. 1988, 37, 3931.
4. S.D.Patil; Jones, C.; Nair, M.G.; Galivan, J.; Maley, F.; Kisliuk, R. L.; Gaumont, Y.; Ouch, D.;
Perone, R. J. Med. Chem. 1989, 32, 1284.
5. D.J. McNamara; Berman, E. M.; Fry, D. W.; Werbcl, L. M. f.Med. Chem. 1990,33, 2045.
6. A.L. Jackman; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown, M.; Calvert, A. H.; Judson, I. R.;
Hughes, L. R. Cancer Res. 1991, 51, 5579.
7. A. Romanini; Lin, J. T.; Niedzwiecki, D.; Bunni, M.; Priest, D. G.; Bertino, J. R. Cancer Res.
1991,51, 789.

670
VARIABLE PHARMACODYNAMICS OF ANTIFOLATES IN SQUAMOUS CELL
CARCINOMA OF THE HEAD AND NECK

Boudewijn J. M. Braakhuis, 1 Gerrit Jansen/ and Godefridus J. Peters. 2

1Department of Otolaryngology and Head and Neck Surgery

2Medical Oncology
Free University Hospital
De Boelelaan 1117,
1081 HV Amsterdam
The Netherlands

INTRODUCTION

The effectiveness of surgery or radiotherapy to controllocoregional head and neck squamous

cell carcinoma (HNSCC) is well established, but patients with advanced disease still have
a bad prognosis. Over the last two decades chemotherapy has been integrated into combined
modality treatment for patients with progressive cancer. Despite good initial responses to
chemotherapeutic treatment, the survival of patients is not enhanced. 1 Methotrexate (MTX)
and 10-Ethyl-10-deazaaminopterin (10-EdAM), both antifolate drugs, can be considered as
two of the most active drugs against HNSCC. Responses, mostly partial remissions, are
observed in 15-40% of the patients. 2•3 Mechanisms of resistance to antifolate drugs include
defective membrane transport, increased levels of intracellular dihydrofolate reductase (DHFR)
and a failure to form polyglutamates. 4-6 In this report we aimed to characterize intrinsic resistance
of three HNSCC cell lines to MTX and 10-EdAM. Sensitivity was related to 1) the time
of exposure, 2) the capacity to regrow after a drug free period, and 3) the ability to synthesize
polyglutamate forms.

MATERIALS AND METHODS

MTX and [3' ,5', 7,9-3H]-MTX (10 mCi/p.mol) were purchased from Lederle (Etten-Leur,
the Netherlands) and Amersham (Amersham, UK), respectively. 10-ethyl-10-deaza-aminopterin
(1 0-EdAM, Edatrexate) was a gift from Ciba-Geigy (Arnhem, the Netherlands). fH]-10-EdAM
was synthesized by Moravek Biochemicals (Brea, CA, USA) and was a gift from dr J. Jolivet,
Montreal, Canada. MTX-polyglutamate standards, GlucGlu5 , were purchased from B. Schircks
Laboratories (Jona, Switzerland).
HNSCC cell lines were obtained from dr T.E. Carey, Ann Arbor, MI, USA and are

Chemzstry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 671
described elsewhere. 7 UM-SCC-14C originated from a local recurrence of the floor of the
mouth, UM-SCC-llB from a laryngeal and UM-SCC-22B from a hypopharyngeal tumor.
Cells were routinely cultured in DMEM with 5% Fetal Calf Serum. Cellular doubling times
were 26 hr for UM-SCC-14C, 28 hr for UM-SCC-llB and 34 hr for the UM-SCC-22B cell
line.
Cell growth inhibition ofHNSCC cell lines was determined by applying the "SRB-assay" .8
Cells were cultured in 96-well-plates and growth was measured at 540 nm using a plate reader,
after staining the cellular proteins with Sulforhodamine B (SRB). Cells were plated at a concentration
of 1500 cells/well in 150 t-tl DMEM and 5% FCS and were allowed to attach and grow exponentially
for a period of72 hr. Consequently, MTX and 10-EdAM were added in 50 t-tl medium resulting
in a concentration that varied between 10·5 and 10·9 M. Cell growth was measured after 24,
48, 72 and 96 hr. Exposure for less than 24 hr did not cause any reduction of cell growth.
After the lag-phase, it was found that control (only treated with culture medium) cell growth
was logarithmic for a period up to 96 hr. Regrowth experiments were performed for both
drugs and the three cell lines after an exposure period of 48 hr. Cells were washed 2 times
with fresh medium and were allowed to grow for an additional drug free period of 24 and
48 hr. IC50-values were estimated based on the absorption values and defined as the concentration
that corresponded to a reduction of cellular growth by 50 % when compared to values of
untreated control cells.
MTX and 10-EdAM polyglutamates were measured by HPLC-techniques. 9 Retention
times for MTX and 10-EdAM were similar and the tritiated peaks following MTX exposure
had similar retention times as the commercially available MTX polyglutamate standards.
Since no 10-EdAM polyglutamates were available as standards, their chain length was estimated
on the basis of the pattern seen with the MTX polyglutamate standards.

RESULTS AND DISCUSSION

When the experiments without a drug free period were analyzed, it was found that,
the IC50-values strongly depended on the time of exposure. For example, with respect to
MTX, IC50-values at 24 hr ranged from 2.9 (UM-SCC-14C) to over 10 t-tM (UM-SCC-22B
and UM-SCC-llB), but when exposed for 96 hr, IC50-values varied between 0.039 and 0.1
t-tM (Table 1). The minimal time to achieve significant growth inhibition varied between
the cell lines, less than 24 hr for UM-SCC-14C and over 24 hr and 48 hr for UM-SCC-llB
and UM-SCC-22B, respectively. 10-EdAM followed a similar sensitivity pattern with 5 to
20-fold lower IC50-values, confirming previous results. 10
The cell lines also varied with respect to growth behavior when cultured in drug free
medium for an additional period (Table 1). As for UM-SCC-14C, the MTX IC50-values did
not change after the additional drug free period of 24 or 48 hr, indicating that recovery has
taken place to a significant extent. When the drug remained present during the additional
period, growth inhibition continued: the IC50-values of 72 and 96 hr continuous exposure
were lower than the one corresponding to 48 hr exposure. Cells from the UM-SCC-llB
cell line showed a similar behavior as that of UM-SCC-14C, but UM-SCC-22B showed a
different pattern with a more continuous growth inhibition. With respect to this latter cell
line, after 48 hr the IC50-values decreased dramatically, when the cells were incubated in
drug free medium for an additional period. As for MTX, the IC50-values were almost independent
of the drug being absent or present.
This variable pattern of sensitivity was correlated with the capacity of the cells to form
polyglutamates. Polyglutamylation has been reported being important in the activity of MTX
in HNSCC cells. 5•11 Folylpolyglutamatesynthetase levels were 40.4 (UM-SCC-14C), 135.2
(UM-SCC-11 B) and 39.1 (UM-SCC-22B) pmol glutamate incorporated/hr/ 1ff' cells, as published
previously. 11 In addition, polyglutamated forms of both MTX and 10-EdAM were measured

672
 Table 1. Pharmacodynamic profile of antifolates

Cell line Drug Exposure/drug free period (hr)

48/0 48/24 48/48 72/0 96/0

UM-SCC-14C MTX 0.47 0.3 0.41 0.17 0.06
UM-SCC-14C 10-EdAM 0.05 0.029 0.06 0.031 0.016
UM-SCC-llB MTX 0.53 0.37 0.36 0.15 0.10
UM-SCC-llB 10-EdAM 0.03 0.011 0.011 0.006 0.005
UM-SCC-22B MTX >10 0.35 0.14 0.20 0.039
UM-SCC-22B 10-EdAM >10 0.006 0.005 0.03 0.0036

Data are expressed as IC50 -values (~-tM), means of three separate experiments.

Table 2. MTX and 10-EdAM polyglutamate formation in HNSCC celllines. 1

Cell line Drug Intracellular polyglutamate concentration

(pmol/106cells)
Total Glu 1 Glu2 Glu3 Glu4 Glul
UM-SCC-14C MTX 10.2 5.5 1.3 1.3 1.4 0.7
UM-SCC-14C 10-EdAM 10.3 2.1 1.6 2.7 2.0 1.8
UM-SCC-llB MTX 58.4 11.4 8.4 17.0 13.1 8.6
UM-SCC-llB 10-EdAM 172.8 6.0 9.6 44.1 50.7 62.4
UM-SCC-22B MTX 26.7 5.7 3.6 4.7 6.0 6.7
UM-SCC-22B 10-EdAM 101.1 4.3 5.6 14.0 23.7 53.5

1Drug exposure time was 24 hr, extracellular concentration [3H]MTX: 50 ~-tM (specific activity:
100 cpm/pmol), extracellular concentration of PHJ-1 0-EdAM: 1~-tM (specific activity: 100 cpm/pmol).
Results are expressed as mean from 3 separate experiments. S.D. never exceeded 15% of the mean.
20ur highest glutamate standard during the HPLC analysis was MTX-GilJs. It is reasonable to assume

that the Glu5 fraction may also contain higher glutamate forms.

after 3 hr and 24 hr exposure to 50 and 1 ~tM, respectively, and an additional drug free period
of 24 hr after the three hr exposure period, was included (Table 2 shows the results of the
24 hr continuous exposure). After 24 hr exposure drug accumulation of 10-EdAM and MTX
was higher in the UM-SCC-llB and -22B cell lines than in the -14C cell line. In the UM-SCC-llB
and -22B lines the intracellular retention of 10-EdAM polyglutamates was higher than that
ofMTX, with predominant formation of the higher polyglutamates. This pattern, UM-SCC-14C
being impaired with respect to polyglutamylation, and 10-EdAM being superior with respect
to retention, could already be observed after 3 hr exposure. Additionally, after 3 hr exposure,
predominant polyglutamates (PG) in UM-SCC-llB were PG2 and PG3, and in UM-SCC-22B
more PG4 and PG5 were present (data not shown).
In conclusion, these experiments show that the pharmacodynamic profile varies between
HNSCC cell lines and plays a role in the growth inhibition by antifolates. Both exposure
time and the intrinsic capacity to synthesize polyglutamates are important determinants in
the sensitivity to antifolate drugs.

673
REFERENCES

1. I.F. Tannock and G. Browman, J. Clin. Oncol. 4:1121 (1986).

2. M. Al-Sarraf. Sem. Oncol. 15:70 (1988).
3. J.H. Schomagel, J. Verweij, P.H.M. de Mulder, F. Cognetti, J.B. Vermorken, P. Cappelaere,
P .J. Armand, J. Wildiers, M. Clave!, A. Kirkpatrick and J .L. Lefebvre, Ann. Oncol.
3:223 (1992).
4. A. Rosowsky, J.E. Wright, C.A. Cucchi, J.A. Lippke, R. Tantravahi, T.J. Ervin and
E. Frei III, Cancer Res. 45:6205 (1985).
5. G. Pizzorno, Y-M Chang, J.J. McGuireandJ.R. Bertino. Cancer Res. 49:5275 (1989).
6. B.F.A.M. van der Laan, G. Jansen, I. Kathmann, J.H. Schornagel and G.J. Hordijk,
Eur. J. Cancer 27:1274 (1991).
7. T.E. Carey, G.T. Wolf, S.R. Baker and C.J. Krause, in: Head and Neck Cancer, vol.
2, W.E. Fee, H. Goepfert, M.E. Johns, E.W. StrongandP.H. Ward, eds., B.C. Becker
Inc, Toronto, 77 (1990).
8. Y.P. Keepers, P.E. Pizao, G.J. Peters, J. Van Ark-Otte, B. Winograd and H.M. Pinedo,
Eur. J. Cancer 27:897 (1991)
9. J. Jolivet, R.L. Schilsky, B.D. Bailey, J.C. Drake and B.A. Chabner, J. Clin. Invest.
70:351 (1982).
10 D.H. Brown, B.J.M. Braakhuis, G.A.M.S. van Dongen and G.B. Snow, Otolaryngol.
Head Neck Surg. 102:20 (1990).
11. B.F.A.M. van derLaan, G. Jansen, G.A.M. Kathmann, G.R. Westerhof, J.H. Schornagel
and G.J. Hordijk, Int. J. Cancer 51:909 (1992).

674
METABOLISM OF FOLATE GLUT AMATES
IN ASPERGILLUS NIDULANS

Irmina Lewandowskal, Ewa Sikora2, Irmina Szablewska2,

Malgorzata Balinska2 and Andrzej Paszewskil

!Institute of Biochemistry and Biophysics, Polish Academy of Sciences,

SA Pawinskiego St., 02-105 Warszawa, Poland
2Nencki Institute of Experimental Biology, Polish Academy of Sciences,
3 Pasteur St., 02-093 Warszawa, Poland

INTRODUCTION

Folate polyglutamates of various glutamyl chain lengths represent active folate deri-
vatives involved in methionine, purine and thymidylate synthesis in all organisms. Fungi
possess a vitamin B12-independent methyltetrahydropteroiltriglutamate:homocysteine met-
hyltransferase [EC 2.1.1.14], which utilizes the triglutamate derivative of methyltetrahydro-
folate as a methyl donor. 1•2 Among fungi synthesis of folate polyglutamates has been
studied mostly in Neurospora crassa3A· 5 and yeast6.
We have previously observed that synthesis of a number of folate enzymes in Asper-
gillus nidulans is coordinately regulated. 7 It was therefore of interest to determine if this
is also true in the case of enzymes involved in the metabolism of folate polyglutamates,
namely polyglutamate synthetase (tetrahydrofolate:L-glutamate y-ligase (A TP-forming),
[EC 6.3.2.17]) andy-hydrolase (y-glutamyl hydrolase, [EC 3.4.22.12]), and whether the
levels of these enzymes affect the proportion of various folate polyglutamates in the cell.

RESULTS AND DISCUSSION

The wild type strain and two methionine requiring mutants, methD and methH, of
A. nidulans were used in this study. Both mutants are leaky: the growth of methH is
stimulated only by methionine, while methD also grows on betaine. 9 Interestingly, these
two mutations seem to be allelic 9 (no wild-type recombinants among 4000 progeny scored
in the cross methH x methD). MethD but not methH was found to have an increased level
of methyltetrahydropteroiltriglutamate:homocysteine methyltransferase and betaine:homo-
cysteine methyltransferase as compared with the wild type strain. Both mutants exhibited
lower than wild-type levels of serine hydroxylmethyltransferase [EC 2.1.2.1]. 10 Evidently,
both mutations have pleiotropic metabolic effects.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling eta!., Plenum Press, New York, 1993 675
Table 1. Activity of polyglutamate synthetase in the wild-type and two methionine-
requiring mutants of A. nidulans.

Activity (nmoles/mg protein/h)

Strain
Supplements to the growth medium:
L-methionine (0.2mM) L-methionine (5mM) D,L-homocysteine (3mM)
Wild-typea 3.9±0.2 0.77±0.1 10.0±0.2
methD 1.3±0.1 0.72±0.1 1.7±0.2
methH 2.0±0.1 0.73±0.1 2.5±0.1
aA pyrodoxine requiring mutant was used as a reference wild type strain with respect to sulphur and folate
metabolism.
The strains were grown in minimal medium 8 or supplemented with methionine or homocysteine, for 18h
at 30° in rotary shaker (150rpm). The mycelia were harvested and cell-free extracts were prepared as
described earlier11 using lOmM phosphate buffer pH 7.4. Enzyme assays was performed according to
McGuire et alP
Results are based on 3-6 independent experiments.

The results shown in Table 1 indicate that the mutants possess a lower level of poly-
glutamate synthetase than the wild type. What is more interesting, however, methionine
in the growth medium brings the enzyme to the same repressed level in all three strains.
Besides this, homocysteine added to the growth medium induces (derepresses) the enzyme
over two-fold in the wild-type strain, while it has only a slight effect in the mutant strains.
This implies that synthesis of polyglutamate synthetase is repressed by methionine and
induced by homocysteine as was observed earlier for other folate enzymes,U and that the
latter regulation is impaired in the mutants.

Table 2. Activity of "{-hydrolase in the wild-type and two methionine-requiring mu-

tants of A. nidulans.

Activity (nmoles/mg protein/h)

Strain
Supplements to the growth medium:
L-methionine (0.2mM) L-methionine (5mM)
Wild-type 15 14
methD 21 26
methH 10
anot estimated
Growth conditions as in the footnote to the Table 1. Cell-free extracts were prepared using lOOmM Tris
buffer, pH 6.5 with 0.1 mM zn+2 • The enzyme assay was performed according to Sikora et alY
Results of one representative experiment are shown.

The y-hydrolase activities in the studied strains are given in Table 2. These results
are preliminary, but it should be noted that a high level of the enzyme in the methD mutant
was observed repeatedly. We have found no effect of methionine in the growth medium
on the level of y-hydrolase. To our knowledge, this is the first demonstration of y-hydrolase
in fungi.

676
Table 3. In vivo glutamylation of methotrexate in the wild-type and two methionine-
requiring mutants of A. nidulans.

Intracellular MTX (pmoles of eH]MTX/mg wet weight)

Supplements to the growth medium:

Strain L-methionine (0.2mM) L-methionine (5mM)
MTX MTX-GlUn MTX MTX-GlUn
Wild-type 0.9±0.1 4.1±0.2 3.4±0.1 0.8±0.1
methD 2.7±0.1 2.2±0.2 4.3±0.2 0.7±0.1
methll 2.7±0.2 2.2±0.1 4.5±0.3 0.5±0.1
3
The strains were grown for 18h, then [ H]MTX was added and the culture continued for an additional 18h.
The r.reparation of cell extracts and separation of MTX and MTX--Glun was performed according to Balmska
et a/. 14
Results are based on 3-4 independent experiments.

We used methotrexate for estimation of the in vivo glutamylation reaction in the

studied strains. The results presented in Table 3 clearly show a different proportion of
mono- and polyglutamates of methotrexate in the wild-type and mutant strains: polyglu-
tamates predominate in the former, while momoglutamate in the latter. Monoglutamate is
the main folate fraction in all strains grown in the presence of methionine. These results
correlate very well both with the differences in polyglutamate synthetase levels between
the wild-type and mutant strains and with the repression of this enzyme by methionine.
The data presented here confirm our previous flndings 11 that folate metabolizing en-
zymes are under methionine- and homocysteine-mediated regulatory system(s). This re-
gulation bears also on the proportion of folate derivatives of various glutamyl chain lengths.

Acknowledgments

Supported by the State Commmittee for Scientific Research grant number 2231/4/91.

REFERENCES
1. E.G. Burton, J. Selhub and W. Sakami, Biochem. J. 111:795 (1969).
2. M. Flavin, in: "Metabolic Pathways", D.M. Greenberg, ed., Academic Press, New York, London (1980)
p. 457.
3. P.Y. Chan and E.A. Cossins, Arch. Biochem. Biophys. 200:346 (1980).
4. E.A. Cossins and P.Y. Chan, in: "Folyl- and Antifolyl Polyglutamates", I.D. Goldman, B.A. Chabner and
J.R. Bertino, eds., Plenum Press, New York and London (1985) p.183.
5. P.Y. Chan, I.J. Atkinson, F.E. Nargang and E.A. Cossins, in: "Chemistry and Biology of Pteridines"
H-Ch. Curtius, S. Ghisla and N. Blau, eds., Walter de Gruyter, Berlin, (1990) p.926.
6. R. Bossett, D.G. Weir and J.M. Scott, J. Gen. Microbial. 93:169 (1976).
7. J. Nadolska-Lutyk, M. Balinska and A. Paszewski, Eur. J. Biochem. 181:231 (1989).
8. A. Paszewski and J. Grabski, J. Bacterial. 124:893 (1975).
9. A.J. Clutlerbuck, Fungal Genetics News Letters 34:27 (1987).
10. M. Balinska and A. Paszewski, Biochem. Biophys. Res. Commun. 91:1095 (1979).
11. R. Natorff, M. Balinska and A. Paszewski, Mol. Gen. Genet. (in press) (1993).
12. J.J. McGuire, P. Hsieh, J.K. Coward and J.R. Bertino, J. Bioi. Chern. 255:5776 (1980).
13. E. Sikora, L. Krzyzanowski and B. Rzeszotarska, J. Chromatogr. 422:420 (1988).
14. M. Balinska, J. Galivan and J.K. Coward, Cancer Res. 41:2751 (1981).

677
EVIDENCE THAT 5-FORMYLTETRAHYDROPTEROYLGLUTAMATE HAS
A METABOLIC ROLE IN ONE-CARBON METABOLISM

Patrick Stover, Heidi Kruschwitz and Verne Schirch

Department of Biochemistry and Molecular Biophysics

Box 614, MCV Station
Virginia Commonwealth University
Richmond, VA 23298

INTRODUCTION
The metabolic function of 5-formyltetrahydrofolate and its polyglutamate forms
(If4PteGlun) have not been elucidated. There are no known biosynthetic reactions in which it
serves as the one-carbon donor, and until recently only a single enzyme was known to use
this folate derivative as a substrate. Methenyltetrahydrofolate synthetase catalyzes the
conversion of 5-CHO-H4PteGlun to 5,10-CH+-If4PteGlun as shown in Equation 1.1,2 This
irreversible reaction was considered a salvage pathway for the reincorporation of one-carbon

5-CHO-H4PteGlun + ATP -+ 5,10-CH+-H4PteGlun + ADP + Pi (1)

units into those folate derivatives active in biosynthetic reactions. In 1984, when
methenyltetrahydrofolate synthetase was first purified to homogeneity from eucaryotic and
procaryotic sources, it was not established that 5-CHO-H4PteGlun was a normal metabolite
of the cell. Some believed that 5-CHO-If4PteGlun was an artifact of isolation, but this did
not explain the existence of an enzyme that utilized it as a substrate. Since that time the
presence of 5-CHO-H4PteGlun in cells has been firmly established, and methenyl-
tetrahydrofolate activity has been shown to be present in a wide variety of cells.3,4
The origin of 5-CHO-If4PteGlun in cells could be accounted for by the nonenzymatic
hydrolysis of 5,10-CH+-H4PteGlun to 5-CHO-If4PteGlun.5 However, the preferred
pathway for the hydrolysis of 5,10-CH+If4PteGlun is to form 10-CHO-If4PteGlun. Also, at
the pH of cell, the concentration of 5,10-CH+-H4PteGlun must be very small because it is in
rapid equilibrium with 10-CHO-If4PteGlun. The equilibrium for this reaction lies far toward
10-CHO-H4PteGlun at pH values above 7. If the nonenzymatic formation of 5-CHO-
H4PteGlun is slow and the cell has methenyltetrahydrofolate synthetase activity, it is difficult
to explain why there is any 5-CHO-H4PteGlun in vivo. This enigma suggested that there
was another source of 5-CHO-If4PteGlun in the cell. This view was further supported by
the observations of several investigators that in seeds and spores the predominate folate was
5-CHO-If4PteGlun.3

THE ORIGIN OF S-CHO-H4PTEGLUn IN VIVO

In 1952 Kisliuk and Sakami6 showed that rat liver slices converted [14C]formate to serine
when glucose 6-phosphate was included in the medium. However, when the glucose 6-
phosphate was omitted, it appeared that the formate was converted to 5-CHO-If4PteGlun. In
our study of the conversion of serine to formate by the coupled reactions of serine

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 679
hydroxymethyltransferase (SHMT) and C1-tetrahydrofolate synthase, we observed that when
NADPH was depleted a new folate compound was formed),8 This compound was shown
to be S-CHO-H4PteGlun. If one assumes that in the rat liver studies the role of glucose 6-
phosphate was to generate NADPH, then our coupled enzyme studies explained the
observation made almost 40 years ago. Further study of our coupled enzyme system showed
that the origin of the 5-CHO-H4PteGlun was the result of the SHMT catalyzed hydrolysis of
5,10-CH+-R4PteGlun as shown in Reaction 2. This prompted a study of the mechanism of
the SHMT catalysis of hydrolysis of 5,10-CH+-R4PteGlun (Equ. 2), experimental evidence
that SHMT is the in vivo source of S-CHO-H4PteGlun, and a search for a function for the
SHMT catalysis of this reaction.

5,10-CH+R4PteGlun .. S-CHO-H4PteGlun (2)

The role of SHMT in eucaryotic and procaryotic cells

Eucaryotic cells that have been studied with respect to SHMT have both cytosolic and
mitochondrial isoenzymes.9 The role of the cytosolic enzyme is to supply one-carbon units
for three major biosynthetic pathways. These are purine ring biosynthesis, thymidylate
biosynthesis, and methionine biosynthesis. In addition, methionine is the major methyl
donor for a host of methylation reactions through its derivative S-adenosyl methionine.
Carbon-3 of serine has been shown to supply as much as 70% of these one-carbon units.
The role of the mitochondrial isoenzyme of SHMT is less clear. In this organelle there are
folate enzymes for oxidizing methyl groups to formate and COz. Serine and glycine can pass
through the mitochondrial membrane as can formate. At this time it is not clear whether
mitochondrial SHMT functions in the metabolism of serine by converting it to glycine, or if it
supplies serine for the cytosol by converting glycine to serine. ApplinglO has an extensive
review on this subject.
In procaryotes, SHMT plays a role similar to the one in the cytosol of eucaryotes. The
enzymes involved in the biosynthesis of purines, thymidylate, and methionine are present in
bacteria and appear, for the most part, to be related to the eucaryotic enzymes. 5,10-CHz-
R4PteGlun serves as the principle one-carbon donor in bacteria with SHMT being the major
source of S,lO-CHz-H4PteGlun.

Evidence for SHMT catalysis of Equation 2

The in vitro study of the interconversion of formate and serine was the first evidence that
SHMT might be involved in the cellular origin of S-CHO-H4PteGlun.8 However, as
discussed below, the kcat value for the SHMT catalysis of Equation 2 is very low, and may
simply be an unimportant side reaction catalyzed by this enzyme. In vivo evidence that
SHMT levels and S-CHO-H4PteGlun are related was needed. This was addressed by
comparing levels of S-CHO-H4PteGlun in wild-type E. coli and a strain having a deleted
glyA gene, and thus no SHMT. We found in this study that the wild-type strain contained
0.9 nmol of S-CHO-H4PteGlun per gram of wet cells. However, the glyA negative strain
contained less than the detection level of 0.2 nmol of 5-CHO-R4PteGlun per gram of cells.
To further implicate the role of SHMT the glyA negative strain was transformed with a
plasmid containing the glyA gene and expressing SHMT at a level 25 times the level found in
wild-type E. coli. The level of 5-CHO-R4PteGlun in these bacteria was 22 nmol per gram
of cells, or about 24 times the level found in wild type cells. This provides compelling
evidence that in E. coli the 5-CHO-R4PteGlun is generated by SHMT.
Is there any evidence that in eucaryotic cells SHMT is responsible for the formation of 5-
CHO-H4PteGlun? In reviewing the existing data where strains of organisms had been
shown to be deficient in at least one of the isoenzymes of SHMT, and for which data had
been accumulated on the level of S-CHO-H4PteGlun, we found that Cossins3 had
characterized a formate requiring mutant strain of N. crassa . This strain lacked cytosolic
SHMT. Cossins found that in this organism there was a 75% decrease in the level of 5-

680
CHO-H4PteGlun compared to the wild type strain. Hopefully, future studies will focus on
the problem of the relationship of SHMT and 5-CHO-H4PteGlun levels in mutant strains of
eucaryotic organisms.

Kinetic properties of the SHMT catalysis of Equation 2

The assay for the SHMT catalysis of Reaction 2 took advantage of a previous
observation that SHMT forms a ternary complex with glycine and several folate compounds
that absorb near 500 nm.ll Glycine forms an external aldimine with the enzyme bound
pyridoxal phosphate which is converted to a quinonoid complex when either S-CHO-
H4PteGlun, S-CH3-H4PteGlun, or H4PteGlun are bound. In the quinonoid complex the
glycine has lost it pro-2S proton. The apparent molar absorbtivity coefficient for these
ternary complexes is about 40,000 M-1cm-1. The assay for Reaction 2 is to incubate SHMT
with 50 mM glycine and 5,10-CH+-H4PteGlun and to monitor the increase in absorbance at
502 nm, where the SHMT•Gly•S-CHO-H4PteGlun ternary complex absorbs. In this assay
the concentration of SHMT is in the 11M range and the absorbance changes are in the 0.02 to
0.2 range.
The first observation in this assay was that a burst of absorbance at 502 nm was followed
by a slower steady-state rate. This burst could be evidence of two forms of SHMT, one that
catalyzed a rapid formation of S-CHO-H4PteGlun until it became inhibited by its own
product, and another form of SHMT that catalyzed a slower formation of 5-CHO-
H4PteGlun. By raising the concentration of SHMT in these studies one should observe only
the rapid phase. However, the ratio of the amplitudes of the burst and slow phases remained
essentially constant, even when the SHMT concentration was in excess of the concentration
of 5,10-CH+-H4PteGlun. This suggested that the burst was not due to two forms of SHMT,
but rather two forms of the substrate 5,10-CH+-H4PteGlun. Comparison of the amplitudes
for the burst and slow phases showed that the stock 5,10-CH+-H4PteGlun contained about
20% of a compound which was converted to S-CHO-H4PteGlun much faster than 5,10-
CH+-H4PteGlun. The putative structure of this compound will be described in the following
paragraphs.
As noted above, the assay for SHMT catalysis of the reaction shown in Equ. 2 had
glycine in addition to SHMT. In experiments where the glycine was omitted it was observed
that S,lO-CH+-H4PteGlun was not converted to 5-CHO-H4PteGlun by SHMT. Serine or L-
alanine could not substitute for glycine, but D-alanine could serve as a substitute. Glycine
and D-alanine share the property that both have an alpha proton that is transferred to a base
on the enzyme to form the quinonoid complex. The results suggest that this proton plays
some role in the catalysis of Reaction 2.12 The Km and kcat for the SHMT catalysis of the
monoglutamate form of 5,10-CH+-H4PteGlun to S-CHO-H4PteGlun are 0.14 mM and 1 x
I0-4 s-1, respectively (Table I).

S-CHO-H4PteGiun polyglutamates are slow tight binding inhibitors of SHMT

In the assay of the SHMT catalysis of 5-CHO-H4PteGlun, it became apparent that the
polyglutamate forms of the product were effective inhibitors. Interaction of 5-CHO-
H4PteGlun polyglutamates with SHMT was investigated to characterize this inhibition. The
assay determined the rate of increase in absorbance at 502 nm in the formation of the SHMT•
Gly •S-CHO-H4PteGlun ternary complex. Reaction with the monoglutamate form was
observed to give biphasic kinetics with the slow phase having a t112 of about 2 s. A study of
the relative amplitudes and the rate constants for the fast and slow phases confirmed that the
fast phase was the result of only one of two rotamers binding to SHMT, and the slow phase
was the nonenzymatic interconversion of the two rotamers. The rotamer which binds to
SHMT has the formyl oxygen facing away from the C4 keto group of the pteridine ring
(Figure 1).13
The triglutamate form of the coenzyme was found to bind slowly to SHMT with a t112 of
about 15 s. The off-rate of the triglutamate form was determined and found also to be 15 s.
Kct values for the monoglutamate and triglutamate forms of S-CHO-H4PteGlun are 10 and
0.2 11M, respectively, with the rabbit cytosolic enzyme.

681
THE NONENZYMATIC FORMATION OF S-CHO-H4PTEGLU 0

The mechanisms for the nonenzymatic interconversion of 10-CHO-R4PteGlu0 and S-

CHO-H4PteGlu and their formation from S,lO-CH+-H4PteGlun have been previously
proposed. It has long been observed that hydrolysis of 5,10-CH+-R4PteGlu0 yields initially
10-CHO-R4PteGlu0, but continued incubation at high temperature results in the formation of
5-CHO-H4PteGlu 0. This has been attributed to the breakdown of a key hydrated
intermediate of 5,10-CH+-B4PteGlu0. These reactions are shown as A, B, and C in Figure
2. The intermediate eliminates NS rapidly because of the higher pKa (4.8) to form 1O-CHO-
H4PteGlu0. However, the most stable compound is 5-CHO-H4PteGlu0, which slowly
accumulates. When water adds to en of 5,10-CH+-R4PteGlu0 stereoelectronic arguments
predict that it will form the (llS) isomer of 5,10-CHOH-R4PteGlu0 rather then the (11R)
isomer.

0
II

"'r'ii)~~ . o~
c
I
~H,
CH 2

r~~c
NH·CH
i:.oH
II
0
Enol Form of Rotamer I .. / " 30%
0 H

tt 0
y

'"(11:)~~""0'
I
9H2
CH 2

~II
NH·CH

g i:.oH
c II
0
Enol Form of Rotamer II / " • 70%
H 0

Figure 1. The enol form of the two rotamers of S-CHO-H4PteGlun. The rate of interconversion of the
rotamer exhibits a tl/2 of about 2 s. Only rotamer II binds tightly to SHMT.

As discussed above, we observed in the SHMT catalysis of 5,10-CH+-B4PteGlun to 5-

CHO-H4PteGlun that there was a compound in the substrate solution which was rapidly
converted to S-CHO-H4PteGlun. 8 In 1952 Cosulich et at.l4 described a hydrated compound
formed from solutions of 5,10-CH+-B4PteGlun during boiling at pH 4. This compound was
stable at pH 7, unlike the putative intermediate formed by Reaction A shown in Figure 2.
They called this unknown folate compound anhydroleucovorin B. We investigated the
possibility that anhydroleucovorin B might be the compound which was rapidly converted to
S-CHO-H4PteGlun by SHMT. NMR and proton exchange studies confirmed that the
unknown compound in our solutions of S,lO-CH+-H4PteGlu0 was anhydroleucovorin B,
which we believe is the (llR) isomer of 5,10-CHOH-H4PteGlu0.15 We observed that the
ciLproton of lO-CHO-H4PteGlun exchanged with solvent at a rate which exceeded the rate
of formation of 5-CHO-B4PteGlun at all pH values from 6 to 12. This supports the proposal
that anhydroleucovorin B is formed from the (11 S) isomer by loss of the C 1Lproton to form
an ylid which inverts and is reprotonated to form the (11R) isomer (Reaction D, Fig. 2).
The (11R) 5,10-CHOH-H4PteGlu0 may be more stable than the (11S) isomer because it can
not eliminate NS by an antiperiplanar mechanism. This compound breaks down to form only
5-CHO-H4PteGlun at all pH values above 5 (Reaction E, Fig. 2). Below pH 4 this
compound is converted to 5,10-CH+-H4PteGlu0.

682
THE MECHANISM OF THE SHMT HYDROLYSIS OF CH+-H4PTEGLU 0

The next question addressed was whether the mechanism of the SHMT catalysis of 5,10-
CH+-H4PteGlun to S-CHO-H4PteGlun was related to the nonenzymatic mechanism as
shown by Reactions A, D, and E in Figure 2. A key experiment was to determine if the
SHMT catalysis of Reaction 2 results in exchange of the Cl Lproton, as demanded by this
pathway, as opposed to the previously proposed pathway of Reactions A and C.12 The
experimental results confirmed that the SHMT catalysis of Reaction 2 results in complete loss
of the ClLproton ofCH+-B4PteGlun.
The natural folate substrate for SHMT is S,lO-CH2-H4PteGlun, where the one-
carbon group is at the oxidation level of formaldehyde, while in Equ. 2 the one-carbon group
is at the oxidation level of formate. In a comparison of the proposed mechanisms for the
catalysis of Reactions 1 and 2 a striking similarity was observed. In each mechanism there is
a hydration of a carbon-nitrogen double bond, an inversion of the attacking water adduct
oxygen from below the plane of the pteridine ring to above the pteridine ring, and elimination
of NlO. This similarity of mechanisms helps support the conclusion that the SHMT catalysis
of the reaction shown in Equ. 2 occurs by the same mechanism proposed for the
nonenzymatic reaction as shown in Figure 2.

"'h,~
N
H
N
I
//c,
0 H
10-CHO-H4 PteGiu 0

(11S) 5,10-CHOH-H 4PteGiu0

(11 R) 5,1 O-CHOH-H 4PteGiu0 5-CHO-H 4 pteGiu.,

(anhydroleucovonn B)

Figure 2. The proposed mechanism for the interconversion of formylfolates. Reactions A, B, and C are
the accepted pathway for the interconversion of 5-CHO-lf4PteGlun lO-CHO-H4PteGlun and 5,10-CH+-
H4PteGlun. Reactions A, D, and E are the proposed pathway for both'the nonenzymatic and SHMT catalyzed
formation of 5-CHO-lf4PteGlun from 5, 10-CH+-lf4PteGiun

Table 1. Kinetic constants for conversion of 5,10-CH+-H4PteGlu0 and 5,10-CHOH-

H4PteGlun to 5-CHO-H4PteGlu0

n Km (I!M) kcat (S- 1)

1 140 ± 30 0.0001 8.0 ± 1 0.50

4 0.055 ± 0.01 0.055 ± 0.01 <0.5 0.058

683
 A study of the role of the polyglutamate forms of 5,10-CH+-H4PteGlun suggests that the
high Km value for the mono glutamate form of the substrate and its low concentration in the
cell make it a poor candidate for an important physiological role. However, the Km for the
tetraglutamate of 5,10-CHOH-fl4PteGlun is below 0.5J.1M, suggesting that the polyglutamyl
forms could be the true substrates for SHMT. Although the nonenzymatic mechanism is
suggested to be the origin of the putative (llR) 5,10-CHOH-f4PteGlun, it can not be ruled
out that there is an enzyme which catalyzes its formation in vivo from 5,10-CH+-f4PteGlun.
Even if you assume that the true substrate is the hydroxymethylene derivative of
tetrahydrofolate, it appears that the reaction catalyzed by SHMT may be too slow to account
for the 5-CHO-I4PteGlun found in the cell (Table 1). However, there are two observations
which may negate the apparent slow rate of catalysis of the reaction shown in Equ. 2. First,
in liver cells the concentration of SHMT is greater then the pool of 5-CHO-fl4PteGlun. This
suggests that only a single turnover of SHMT could account for all of the cellular 5-CHO-
fl4PteGlun. Second, the tight binding properties of the product suggests that in the cell the 5-
C H 0- H 4PteG 1un is bound to SHMT and is not available as a substrate for
methenyltetrahydrofolate synthetase.

~
H4PteG1un H2PteG1un

ser B JLThymidylate
gly gly
ser

V
ser H4PteGiun
Purines

10-CH O-H 4 PteG1lln

Figure 3. Metabolic cycles of one-carbon metabolism in procaryotes and the cytosol of eucaryotes. Cycle
A is methionine biosynthesis and the origin of S-adenosylmethionine used for many methylation reactions.
Cycle B is thymidylate biosynthesis. Cycle C is the biosynthesis of the purine ring. Cycle D is a putative
futile cycle involved in regulating one-carbon metabolism by controlling the in vivo level of 5-CHO-
H4PteGlun

POSSIBLE ROLES FOR THE FUTILE CYCLE

Figure 3 shows the major metabolic cycles that occur in the cytoplasm of eucaryotes and
procaryotes. In addition to the three biosynthetic pathways we add the futile cycle D
involving SHMT and methenyltetrahydrofolate synthetase. There are several reasons to
believe that futile cycle D may regulate the cellular level of S-CHO-H4PteGlun, and that the
level of this form of folate may in turn regulate the other three metabolic cycles. Recently,
Bertrand and Jolivet16 showed that 5-CHO-tetrahydrohomofolate was a good inhibitor of
methenyltetrahydrofolate synthetase. When MCF-7 cells were incubated in the presence of
the homofolate, both the level of 5-CHO-I4PteGlun increased two-fold in the cell, and there
was an 80% decrease in the growth rate. The decrease in growth rate was traced to the 5-
CHO-H4PteGlun inhibition of aminoimidazole-carboxamide (AICAR) formyltransferase
which is required for purine biosynthesis.

684
 In addition to AICAR formyltransferase other enzymes have been found to be inhibited by
5-CHO-H4PteGlun. These include sarcosine dehydrogenase, dimethylglycine
dehydrogenase, methionyl-tRNA formyltransferase, 5,10-methenyltetrahydrofolate
cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, and dihydrofolate reductase
(see references in 12). Of particular importance is the observation that methionyl-tRNA
formyltransferase is inhibited by 5-CHO-H4PteGlun. This enzyme is important in the
regulation and expression of proteins, and it has been suggested that the level of folates in the
cell may regulate this enzyme activity.17 There has not been a systematic study of the effect
of 5-CHO-H4PteGlun polyglutamates on other folate enzymes. We propose that this is an
area that needs further study.

NEUROSPORA CRASSA AS A MODEL TO STUDY THE FUTILE CYCLE

The existence of futile cycleD (Fig. 3) and its role in regulating one-carbon metabolism
is our current research focus. Previous studies have shown that in seeds and spores the
principle folate is 5-CHO-H4PteGlun. This would be consistent with the stability of this
compound to high temperature, alkaline pH, and oxygen. However, because it is not a
donor in any one-carbon biosynthetic reactions, this compound must first be converted back
to 5,10-CH+-H4PteGlun by methenyltetrahydrofolate synthetase. An organism that has to go
from a vegetative state to a dormant state, such as a spore, may use futile cycle D shown in
Figure 3 as the mechanism for storing its folate and one-carbon groups.
An ideal organism to test the putative role of CycleD (Fig. 3) is Neurospora crassa. This
organism has a well-defined life cycle which includes a spore stage. It grows rapidly on
simple media and can be manipulated genetically. We have shown that 5-CHO-R4PteGlun is
the major folate in the spore. We have also purified cytosolic SHMT from this organism and
shown that it exhibits all the properties previously found for both the rabbit liver isoenzymes
and the E. coli enzyme. The most important of these properties is the ability to catalyze the
hydrolysis of 5,10-CH+-H4PteGlun to 5-CHO-H4PteGlun (Equation 2) and that 5-CHO-
R4PteGlun polyglutamates are slow tight binding inhibitors. In addition, we have found that
the N. crassa enzyme is stabilized against high temperature denaturation by forming a
complex with glycine and 5-CHO-R4PteGlun polyglutamates. Future studies will determine
the role of cycle D during spore formation and regeneration to the mycelia state.

ACKNOWLEDGMENTS

This work was supported by Grant GM 28143 from the National Institutes of Health.

REFERENCES

1. S. Hopkins and V. Schirch, 1. Biol. Chern., 259:5618 (1984)

2. C. E. Grimshaw, G. B. Henderson, G. G., Soppe, G. Hansen, E. J. Mathur, and
F. M. Huennekens, 1. Biol. Chern., 259:2728 (1984)
3. E. A. Cossins, in Folates and Pterins, R. L. Blakely and S. J. Benkovic, Eds., 1:1
(1984)
4. D. W. Horne, D. Patterson, and R. J. Cook, Arch. Biochem. Biophys., 270:729
(1989)
5. S. J. Benkovic, Ann. Rev. Biochem., 49:227 (1980)
6. R. L. Kisliuk and W. Sakami, 1. Amer. Chern. Soc., 76:1456 (1954)
7. W. B. Strong and V. Schirch, Biochemistry, 28:9430 (1989)
8. P. Stover and V. Schirch, 1. Biol. Chern., 265:14227 (1990)
9. L. Schirch, Adv. Enzymol. Relat. Areas Mol. Biol., 53:83 (1982)
10. D. R. Appling, FASEB 1., 5:2645 (1991)
11. L. Schirch and M. Ropp, Biochemistry, 6:253 (1967)
12. P. Stover and V. Schirch, Biochemistry, 31:2155 (1992)
13. P. Stover and V. Schirch, 1. Biol. Chern., 266:1543 (1991)
14. D. B. Cosulich, B. Roth, J. M. Smith, M. E. Hultquist, and R. P. Parker, 1. Amer.
Chern. Soc., 74:3252 (1952)
15. P. Stover and V. Schirch, Biochemistry, 31:2148 (1992)
16. R. Bertrand and J. Jolivet, 1. Biol. Chern., 264:8843 (1989)
17. L. Gold, Ann. Rev. Biochem., 57:199 (1988)

685
COBALAMIN-DEPENDENT AND COBALAMIN-INDEPENDENT

METHIONINE SYNTHASES IN Escherichia coli: TWO SOLUTIONS TO

THE SAME CHEMICAL PROBLEM

James T. Drummond and Rowena G. Matthews

Biophysics Research Division and Department of Biological Chemistry

The University of Michigan
Ann Arbor, MI 48109

INTRODUCTION

Two genes encoding proteins with methionine synthase activity are found in Escherichia
coli. Both enzymes use methyltetrahydrofolate as a methyl donor to catalyze the conversion
of homocysteine to methionine, as shown below:

CH3-l4PteGlu 0 +Homocysteine (RSH) --------> IL(PteGlun +Methionine (RSCH3)

The metH gene encodes a cobalamin-dependent methionine synthase (MetH). This protein is
monomeric, with a deduced molecular weight of 136,055.1 It can use CH3-l4PteGlu1 as a
substrate, and requires a reducing system and AdoMet for activation.2,3 The metE gene
encodes a cobalamin-independent methionine synthase that requires methyltetrahydrofolate
substrates with at least two glutamyl residues and is completely inactive with
CH3-H4PteGlu1.4 This enzyme shows no requirement for reductive activation, but does
require phosphate ion and magnesium for optimal rates of catalysis.5 It has a deduced
molecular weight of 84,654,6 in good agreement with the value of 84,000 obtained by
ultracentrifugation of the native enzyme.5 Both the metH 1,7,8 and metE 6,9genes have been
sequenced, and their coding sequences show no detectable homologies. Thus these two
proteins appear to have evolved independently to perform highly similar methyl transfers
from N5 of methyltetrahydrofolate to the sulfur of homocysteine.

COBALAMIN-DEPENDENT METHIONINE SYNTHASE (MetH)

Assignment of catalytic functions to regions of the primary structure

The catalytic cycle of MetH is diagrammed on the next page. During turnover, enzyme-
bound cobalamin shuttles between methylcobalamin and cob(I)alamin forms, alternately
accepting a methyl group from methyltetrahydrofolate and donating it to homocysteine.
Occasionally, the cob(I)alamin prosthetic group is oxidized to form cob(II)alamin, and this
form of the enzyme is inactive. Return of the enzyme to the catalytic cycle requires a

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 687
reductive methylation, in which AdoMet rather than CH3-l4folate serves as the methyl
donor. 2,3 Huennekens and his coworkers have shown that NADPH, an NADPH-flavodoxin
oxidoreductase (the R protein) and flavodoxin (the F protein) can supply the electrons
necessary for reductive activation of the enzyme.lO

Primary Turnover Cycle Catalytic

Reactivation

o ,
..........................................
'
''
-1 e·
US--<:o(l) ~
CH 3 -H4 Folat
MS-co(ll)
0

+Ado Met
'L ................................... ..

Figure L The catalytic cycle of cobalamin-dependent methionine synthase.

Our studies have shown that the native 136 kDa MetH enzyme can be cleaved by trypsin
into two fragments, a 98 kDa N-terminal fragment with the cobalamin prosthetic group
noncovalently bound to it that extends from amino acid residues 1-896, and a 37 kDa C-
terminal domain extending from residues 900 to 1227.11 The linker region between these
two fragments contains multiple basic residues and is hypersensitive to cleavage by trypsin,
which cleaves at two closely spaced sites in this region. The location of these domains in the
primary amino acid sequence of the monomeric MetH protein is shown below. If the
prosthetic group is methylated prior to cleavage with trypsin, the isolated 98 kDa fragment is

1 649 896 900 1227

70.4kDa 37.2kDa

Figure 2. Location of functional domains within the primary amino acid sequence of cobalamin-dependent
methionine synthase.

able to catalyze the methyl transfer from CH3-l4PteGlu1 to homocysteine under anaerobic
conditions. However, enzyme slowly accumulates in the cob(II)alamin form, and can no
longer be activated in the presence of AdoMet and a reducing system. The cobalamin
prosthetic group is bound to a 28 kDa domain that forms the C-terrninal portion of the 98 kDa
fragment. This domain is shaded in the diagram above. The 28 kDa cobalamin binding
domain can be isolated in good yield by more drastic treatment of the native enzyme with
trypsin. This domain is catalytically inactive, but retains noncovalently bound cobalamin. 1
Determination of the x-ray structure of this domain is now in progress.12 The C-terminal
domain is radioactively labeled on irradiation with ultraviolet light in the presence of
[3H-methyl]AdoMet,l and appears to contain the determinants necessary for AdoMet

688
binding. This domain has recently been crystallized by Melinda Dixon in Martha Ludwig's
laboratory. Our results suggest a division of labor between theN- and C-terminal portions of
the protein, with the C-terminal domain required for catalysis of the reductive methyl transfer
from AdoMet to cobalamin, and the N-terminal region required for methyl transfer to
cobalamin and from methylcobalamin to homocysteine.
Each of the methyl transfers catalyzed by MetH is thought to proceed by a bimolecular
nucleophilic displacement (SN2), and thus places severe geometric constraints on the
positioning of the methyl donating or accepting substrate with respect to the cobalamin. In
fact, at different times in the catalytic cycle, methionine, methyltetrahydrofolate and AdoMet
must each place their methyl group in exactly the same position in space, positioned
immediately above the cobalt of cobalamin. We postulate that the unusually large size of this
polypeptide may reflect this necessity to bring each substrate in turn into position for methyl
transfer to or from the cobalamin.

Reductive activation of MetH

Met H shares the requirement for a reductive activation that involves cleavage of Ado Met
with two other E. coli proteins, pyruvate formate-lyase and the anaerobic ribonucleotide
reductase. In each case, electrons are supplied by NADPH and the electron transfer
associated with activation is mediated by flavodoxin )F protein) and an NADPH-flavodoxin
oxidoreductase (R protein)_l3,14 Both the F protein 5 and the R proteinl6 have now been
cloned and mapped in E. coli. The MetH enzyme activation by AdoMet and the RIF/NADPH
reducing system shows interesting similarities and differences when compared with the
activation of pyruvate formate-lyase and the anaerobic ribonucleotide reductase. In the latter
two cases activation is thought to involve the reductive cleavage of AdoMet to form
5'-deoxyadenosine and methionine, with concomitant formation of a glycyl radical on the
activated protein.l7 ,18,19 While AdoMet serves the function of a radical generator in

+ AdoMet + 1e· <.._(Nh-

H 0
+ 5'-dAdo + Met
H

pyruvate formate-lyase and anaerobic ribonucleotide reductase, where the 5'-deoxyadenosyl

radical formed by reductive cleavage of AdoMet is thought to abstract a hydrogen atom from
a glycine residue near the C-terminus of each of these proteins, AdoMet is postulated to play
a rather different role in methionine synthase. In MetH, reductive activation is associated
with methyl transfer from AdoMet to cobalamin,20,21 although the stoichiometry of reaction
has not been measured, and the AdoHcy product expected has not been identified. Formally,
the reaction is thought to involve coupling of the exergonic methyl transfer from AdoMet to
cobalamin with the very endergonic reduction of cob(II)alamin to cob(I)alarnin. 22 Thus in
MetH, methyl transfer from AdoMet to cobalamin serves to provide the thermodynamic
driving force to propel an endergonic reduction. Exactly how the two reactions are coupled
remains for future investigations to elucidate.

Inactivation of MetH by nitrous oxide

Our recent investigations of the mode of inactivation of MetH by nitrous oxide have
provided evidence for a close contact between residue 1177 near the C-terminus of the 37
kDa domain and the cobalamin prosthetic group (Drummond and Matthews, manuscript in
preparation). Our evidence is consistent with the postulate23 that nitrous oxide (N20) is
reduced by enzyme in the cob(I)alamin form, with concomitant generation of N2 and
hydroxyl radical, according to the formula shown below:

E-cob(I)alamin + N20 + H+ --------> E-cob(II)alamin + N2 + OH•

Our evidence suggests that hydroxyl radical, generated at the active site of the enzyme, reacts
by a regiospecific hydrogen atom abstraction from Val1177 in the C-terminal domain,

689
although we have not yet characterized the structure of the valyl adduct. These results suggest
that, at some stage in the catalytic cycle of MetH, Val1177 lies very close in space to the
cobalamin prosthetic group in the 28 kDa domain This valyl residue is embedded in a
sequence, VSGWYxxxxPD, with resemblance to the sequences surrounding the glycyl
radicals generated by activation of pyruvate formate-lyase and the anaerobic ribonucleotide
reductase. Our future efforts will be focused on characterizing the chemistry of the reductive
activation of MetH, and exploring the differences and similarities with the reductive
activations of pyruvate formate-lyase and anaerobic ribonucleotide reductase.

COBALAMIN-INDEPENDENT METHIONINE SYNTHASE (MetE)

The native MetE enzyme can be cleaved by trypsin into two fragments of approximately
equal size. This cleavage is associated with an approximately two-fold decrease in activity.
Examination of the deduced amino acid sequence of the protein provides clear indication of a
two-domain structure in which the two domains are clearly related in sequence and may well
have evolved through a gene duplication event. The two domains do not appear to function
equivalently in catalysis, however, since alkylation of Cys726, in the C-terminal domain,
results in loss of all detectable enzyme activity.6
Met E was initially purified to homogeneity by a procedure that involved crystallization as
the final step. 5 Although it was not determined whether these crystals diffracted, there is the
reasonable possibility that the availability of an efficient expression system for recombinant
MetE9 may permit the determination of the x-ray structure of this protein, and eventually a
comparison of the structure with that of domains of the MetH protein.
If the chemistry of the MetH reaction remains obscure, the chemistry of the MetE reaction
is downright mystifying. While the stereochemistry of the methyl transfer from CH3-
H4PteGlu 1 to homocysteine in the MetH reaction is known to proceed with retention at the
transferred methyl group,24 the stereochemistry of the methyl transfer catalyzed by MetE has
not yet been determined. This determination will be particularly important in defining the
mechanism of catalysis, and is currently in progress in our laboratory. Observation of
inversion at the transferred methyl group would suggest a direct nucleophilic displacement by
the sulfur of homocysteine on the methyl group of CH3-R4PteGlu3, while retention would
suggest the participation of a methylated enzyme intermediate. One way in which the
catalytic mechanisms of the cobalamin-dependent and cobalamin-independent methionine
synthases could be related is if the MetE enzyme provides a strong nucleophile to function
like cob(I)alamin in the MetH protein. Our studies have identified a highly reactive cysteinyl
residue in MetE, Cys726, and shown that alkylation of this residue by chloromethyl ketones
results in apparently complete loss of activity.6 While we are unable to deduce the role of
Cys726 in catalysis at this time, the identification of this reactive residue suggests the
possibility that this thiol may function as an intermediate methyl acceptor in catalysis,
analogous to the role of cobalamin in the reaction catalyzed by MetH.
The turnover number associated with catalysis by MetE of the methyl transfer from
CH3-H4PteGlu1 to homocysteine is 14 min-1, nearly 100 times slower than the turnover
number for the MetH enzyme. Whether MetE catalyzes the displacement of the methyl group
of methyltetrahydrofolate using homocysteine or using a thiolate residue on the protein, one
might expect the enzyme to be relatively inefficient as compared to the MetH enzyme.
Studies of the reactivity of thiolate as compared to cob(I)alamin indicate that thiolate is less
reactive by a factor of 30,000.25 Given this large difference in reactivity, perhaps we should
not be asking why MetE has such a low turnover number, but rather how it has achieved
such a high turnover.
Both MetE and MetH enzymes face the problem of activating the methyl group of a
tertiary amine towards nucleophilic attack, and in both cases the mechanism of such
activation remains the central unsolved mechanistic question associated with the catalytic
reactions.

ACKNOWLEDGEMENTS

Work from our laboratory has been funded in part by NIH Grant R37 GM29408. JTD is a

690
trainee in the Pharmacological Sciences Training Program supported by National Research
Services Award T32 GM07767 and has been an NSF Graduate Fellow.

REFERENCES
1. J.T. Drummond, R. Loo, and R.G. Matthews, 1993, Electrospray mass spectrometric analysis of the
domains of a large enzyme: Observation of the occupied cobalamin-binding domain and redefinition of
the carboxyl terminus of methionine synthase, Biochemistry: submitted for publication.

2. J.H. Mangum and K.G. Scrimgeour, 1962, Cofactor requirements and intermediates in methionine
biosynthesis, Fed. Proc. 21:242.

3. M.A. Foster, M.J. Dilworth, and D.D. Woods, 1964, Cobalamin and the synthesis of methionine by
Escherichia coli, Nature 201:39.

4. E. Burton, J. Selhub, and W. Sakami, 1969, The substrate specificity of 5-methyltetrahydropteroyl-

triglutamate-homocysteine methyltransferase, Biochem. J. 111:793.

5. C.D. Whitfield, E.J. Steers, Jr., and H. Weissbach, 1970, Purification and properties of 5-methyltetra-
hydropteroyltriglutamate-homocysteine transmethylase, J. Bioi. Chern. 245:390.

6. J.C. Gonzalez, R.V. Banerjee, S. Huang, J.S. Sumner, and R.G. Matthews, 1992, Comparison of
cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: Two
solutions to the same chemical problem, Biochemistry 31:6045.

7. R.V. Banerjee, N.L. Johnston, J.K. Sobeski, P. Datta, and R.G. Matthews, 1989, Cloning and sequence
analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and
isolation of a tryptic fragment containing the cobalamin-binding domain, J. Bioi. Chern. 264:13888.

8. I. G. Old, D. Margarita, R.E. Glass, and I. Saint Girons, 1990, Nucleotide sequence of the metH gene of
Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2, Gene 87:15.

9. E.M. Maxon, B. Redfield, X.-Y. Cai, R. Shoeman, K. Fujita, W. Fisher, G. Stauffer, H. Weissbach, and
N. Brat, 1989, Regulation of methionine biosynthesis in Escherichia coli: effect of the MetR protein in
metE and metH expression, Proc. Nat/. Acad. Sci. U. S. A. 86:85.

10. K. Fujii, and F.M. Huennekens, 1974, Activation of methionine synthetase by a reduced
triphophopyridine nucleotide-dependent flavoprotein system, J. Bioi. Chern. 249:6745.

11. J.T. Drummond, S. Huang, R.M. Blumenthal, and R.G. Matthews, 1993, Assignment of enzymatic
function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli,
Biochemistry: submitted for publication.

12. C.L. Luschinsky, J.T. Drummond, R.G. Matthews, and M.L. Ludwig, 1992, Crystallization and
preliminary x-ray diffraction studies of the cobalamin-binding domain of methionine synthase from
Escherichia coli, J. Mol. Bioi. 225:557.

13. H. Vetter, Jr., and J. Knappe, 1971, Flavodoxin and ferredoxin of Escherichia coli, Hoppe-Seyler's Z.
Physiol. Chern. 352:443.

14. J. Harder, R. Eliasson, E. Pontis, M.D. Ballinger, and P. Reichard, 1992, Activation of the anaerobic
ribonucleotide reductase from Escherichia coli by S-adenosylmethionine, J. Bioi. Chern. 267:25548.

15. C. Osborne, L.-M. Chen, and R.G. Matthews, 1991, Isolation, cloning, mapping and nucleotide
sequencing of the gene encoding flavodoxin in Escherichia coli, J. Bacterial. 173:1729.

691
16. V. Bianchi, P. Reichard, R. Eliasson, E. Pontis, M. Krook, H. Jomvall, and E. Hi'tggard-Ljungquist,
1993, E. coli Ferredoxin NADp+ reductase: Activation of E. coli anaerobic ribonucleotide reduction,
cloning of the gene lfpr) and overexpression of the protein. J. Bacterial. 175: in press.

17. J. Knappe, F.A. Neugebauer, H.P. Blaschkowski, and M. Ganzler, 1984, Posttranslational activation
introduces a free radical into pyruvate formate-lyase, Proc. Natl. Acad. Sci. USA 81:1332.

18. A.F.V. Wagner, M. Frey, F.A. Neugebauer, W. Schafer, and J. Knappe, 1992, The free radical in
pyruvate formate-lyase is located on glycine-734, Proc. Nat!. Acad. Sci. USA 89:996.

19. X. Sun, J. Harder, M. Krook, H. Jomvall, B.-M. Sj o berg, andP. Reichard, 1993, A possible glycine
radical in anaerobic ribonucleotide reductase from Escherichia coli: nucleotide sequence of the cloned nrdD
gene, Proc. Natl. Acad. Sci. 90:577.

20. R.T. Taylor and H. Weissbach, 1969, Escherichia coli B N5-methyltetrahydtofolate-homocysteine

methyl transferase: sequential formation of bound methylcobalamin with S-adenosyl-L-methionine and
N5-methyltetrahydrofolate, Arch. Biochem. Biophys. 129:728.

21. K. Fujii, and P.M. Huennekens, 1979, Methionine synthase: characterization of protein components and
mechanisms for activation and catalysis, in "Biochemical Aspects of Nutrition," K. Yagi, Ed., p. 173,
University Park Press, Baltimore.

22. R.V. Banerjee, S.R. Harder, S.W. Ragsdale, andR.G. Matthews, 1990, Mechanism of reductive
activation of cobalamin-dependent methionine synthase: an electron paramagnetic resonance
spectroelectrochemical study, Biochemistry 29:1129.

23. V. Frasca, B.S. Riazzi, and R.G. Matthews, 1986, In vitro inactivation of methionine synthase by
nitrous oxide, J. Bioi. Chern. 261:15823.

24. T.M. Zydowsky, L.F. Courtney, V. Frasca, K. Kobayashi, H. Shimizu, L.-D. Yuen, R.G. Matthews,
S.J. Benkovic, and H. G. Floss, 1986, Stereochemical analysis of the methyl transfer catalyzed by
cobalamin-dependent methionine synthase from Escherichia coli B, J. Am. Chern. Soc.108:3152.

25. K.L. Brown, 1982, Synthesis of organocobalt complexes, in "B12, Volume 1: Chemistry," D. Dolphin,
Ed., p. 245, Wiley Interscience, New York.

692
FOLATE METABOLITES AS MODULATORS OF ANTITUMOR DRUG
ACTIVITY

David G. Priest, John C. Schmitz, and Marlene A Bunni

Department of Biochemistry and Molecular Biology

Medical University of South Carolina
Charleston, SC 29425-2211

INTRODUCTION

[R,S]-5-formyltetrahydrofolate (6-CHOFH4) has shown significant

potentiation of fluorouracil (FU) activity in colorectal, breast, and head and
neck cancer .1-7 The biochemical basis for this enhanced activity is
metabolic conversion of 6-CHOFH4 to 5,10-methylenetetrahydrofolate
(CH2FH4) which in tum stabilizes the inhibitory complex formed between the
active metabolite of fluorouracil, 5-fluoro-2-deox:yuridine-5'-monophosphate
(FdUMP), and thymidylate synthase. This inhibition results in depletion of
thymidylate required for DNA synthesis and repair. 8-11
While the metabolic conversion of 5-CHOFH4 to CH2FH4 is clearly
necessary for antitumor activity, the manner and location of this
transformation in humans is not fully understood. In previous studies we
have demonstrated that administration of 6-CHOFH4 both intravenously and
orally results in plasma elevation of CH2FH4 and FH4 in addition to the
normal circulating folate 6-CH3FH4. 12 10-CHOFH4 was also detected but at
relatively low levels. We now report in vitro studies to support the concept
that the metabolites FH4 and 6-CH3FH4 are effective agents for the elevation
of intracellular reduced folates. In addition, we suggest that because these
metabolites are the predominate circulating folates following therapeutically
efficacious oral 6-CHOFH4 administration, they are themselves active
antitumor agents. Further, we demonstrate that folic acid is effective in
elevating reduced folate metabolites in human plasma and, hence, is a more
readily available alternative to 6-CHOFH4 that could have equivalent
therapeutic activity for FU modulation.

METHODS

Cell Culture

Ovarian tumor cells (OV 2008) were maintained as monolayers in

RPMI 1640 media supplemented with 10o/o fetal bovine serum in a humidified
5o/o C02 incubator at 37°C. To study the effects of exposing ovarian tumor
cells to reduced folates, logarithmically growing cells were plated at a density
of 25 x 104 cells/ml and allowed to grow for 48 hours. The reduced folates.
6-CHOFH4, 6-CH3FH4 and FH4, each at 10 11M concentration, were added to

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 693
 media that contained 50 mM ascorbate. Each folate was stable under these
conditions and the presence of ascorbate did not alter cell growth. After 4
hours incubation, cells were trypsinized, washed twice with cold PBS, and
suspended at a density of 10 x 106 cells/ml in an extraction buffer which
contained 50 mM Tris-HCl (pH 7.4), 50 mM sodium ascorbate and 1 mM
EDTA. Cells were boiled for 3 minutes to achieve lysis and centrifuged at
10,000 x g for 10 minutes at 4°C. The resultant supernatant was used to
assay for intracellular reduced folates as described below. Protein
concentrations were determined by the Bradford dye-binding assay.13

Folate Administration and Sample Preparation

5-CHOFH4 was administered orally to five healthy volunteers at a 125

mg/m2 dose.12 Folic acid was administered orally to another group of five
healthy volunteers at the same dose. All volunteers signed an informed
consent agreement before participating in the study. Before administration
and during the following 24 hour period, blood samples were withdrawn into
vacutainers containing EDTA. Plasma was separated immediately by
centrifugation at 4°C, diluted into an equal volume of 50 mM Tris-HCl buffer
(pH 7.4) that contained 100 mM sodium ascorbate and 1 mM EDTA, and
stored at -20°C until analysis. After thawing, protein was precipitated by
placing samples in a boiling water bath for 5 minutes. Following
centrifugation at 14,500 x g for 5 minutes at 4°C, the resultant supernatant
was used for estimation of folates.
Estimation of Folates

A series of radioenzymatic assays which have been previously

described, were used to measure CH2FH4, FH4, 5-CH3FH4. l O-CHOFH4, 5-
CHOFH4. and folic acid.14-18 These methods are based upon enzymatic
cycling of other reduced folates to CH2FH4 followed by entrapment into a
stable ternary complex with excess thymidylate synthase and [3H]FdUMP.19
Reaction mixtures for analysis of CH2FH4 typically contained 20 mU
thymidylate synthase and 125 nM [3H]FdUMP (20 Ci/mmole) in 200 j.tl of a
solution that contained 50 rnM Tris-HCl (pH 7.4), 50 mM sodium ascorbate
and l mM EDTA. Other folates were estimated in the same system but with
the addition of enzymes and cofactors necessary for their conversion to
CH2FH4. Following incubation for 30 minutes at 25°C, reactions were
stopped by adjustment to l% SDS and boiled for 10 minutes. Bound
[3H]FdUMP, and hence tissue CH2FH4, was determined using centrifugally
eluted mini-columns of Sephadex G-25.14

RESULTS AND DISCUSSION

Studies with human volunteers have shown that following intravenous

administration, 5-CHOFH4 is metabolized to CH2FH4 and FH4 in addition to
5-CH3FH4.12 To address the question of whether these metabolites can be
taken up and elevate intracellular reduced folate pools, a cultured human
ovarian tumor cell system was exposed to 5-CH3FH4. FH4 and the parent
compound 5-CHOFH4. CH2FH4 was too unstable with regard to dissociation
of the methylene functional group to evaluate in this system. It can be seen
in Table 1 that total intracellular folate was elevated approximately two-fold
in each case, indicating that these metabolites are effective precursors, at
least under these in vitro conditions. While each folate caused elevation of
l0-CHOFH4, the only other pool elevated was the specific media folate
added. Hence, with the exception of conversion to l0-CHOFH4. there

694
Table 1. Accumulation of Reduced Folates in Cultured Ovarian Tumor Cells
Following Exposure to 6-CHOFH4, FH4, and 6-CH3FH4. 1

Reduced Folates (pmol/mg)

Folate Total Folate
Added (pmol/mg) 5-CHOFH4 CH2FH4+FH4 5-CH3FH4 10-CHOFHi

Control 104± 6 <1 7±1 32±4 65±6

5-CHOFH4 193 ± 18 14±5 10 ±3 32±4 137 ± 15

FH4 219 ± 16 <1 30±4 31 ± 4 158 ± 11

6-CH3FH4 211 ± 16 <1 9±1 71 ± 7 131 ± 11

1ovarian tumor cells were maintained in RPMI 1640 media supplemented with 10%
fetal bovine serum for 48 hours before exposure to each reduced folate at 10 !J.M
concentration for 4 hours. Cells were trypsinized, washed twice with cold PBS and
suspended in an extraction buffer which contained 50 mM Tris-HCl (pH 7.4), 50 mM
sodium ascorbate and 1 mM EDTA. Intracellular reduced folates were measured by
the ternary complex-based assay. Total folate is the sum of the four reduced folate
pools. Data represent the mean of five determinations ± SEM.

appears to be relatively little metabolism of the individual folates subsequent

to their uptake. It is unknown whether similar behavior would occur in in
vivo systems, but if so the observed extensive accumulation of CH2FH4 and
FH4 in human plasma following N administration of 5-CHOFH412 may play
an important role in elevation of intratumor CH2FH4 and, hence, therapeutic
modulation of FU activity.
The folate metabolite pool that becomes elevated most extensively
following intravenous administration of 6-CHOFH4 is 6-CH3FH4, 12,20,21
and this derivative has been shown to potentiate FU cytotoxicity in cultured
cell systems.l1,22 Although Houghton et al. reported that 6-CH3FH4 was
unable to elevate CH2FH4 + FH4 in human tumor xenographs implanted in
nude mice as effectively as did 6-CHOFH4,23 a clinical trial24 in which 5-
CH3FH4 was used in combination with FU resulted in responses that were
equivalent to those with intravenously administered 5-CHOFH425,26 (see
Table 2). This direct therapeutic evidence that 6-CH3FH4 potentiates FU
activity strongly suggests that this folate derivative is an effective source of
intratumor CH2FH4 in humans, even though it was somewhat less effective
than 5-CHOFH4 in the mouse model system. 23
A second argument that metabolites contribute significantly to the
antitumor activity of FU is based on pharmacokinetic studies of orally
administered 6-CHOFH4. Oral administration of 5-CHOFH4 results in very
little accumulation of the parent compound but extensive elevation of
metabolites, particularly 6-CH3FH4.12.20 Yet, it can be seen in Table 2 that
orally administered 5-CHOFH427,28 was as effective as intravenously
administered 5-CHOFH425,26 in two clinical trials. Thus, since metabolites
represent the preponderance of circulating folate following oral
administration of 6-CHOFH4, and since clinical trials demonstrate that oral
5-CHOFH4 is as active as intravenously administered 6-CHOFH4, these
metabolites must be effective agents themselves in the enhancement of FU
antitumor activity.

695
 Because 5-CHOFH4 metabolites are active, an alternative potential
metabolite source, folic acid, was investigated. When this vitamin was
administered orally to volunteers at 125 mgjm2, the same metabolites,
5-CH3FH4 and CH2FH4 + FH4, were observed as following 5-CHOFH4
administration. Further, it can be seen in Figure 1 that when total
accumulation of metabolites (AUC) is compared, elevation is the same
following folic acid as after 5-CHOFH4 administration at 125 mg/m2. While

Table 2. Modulation of FU Activity by Folates in Colorectal Cancer

Clinical Trials.

Dose FU Response
(mg/m2) (mg/m2) (%) N1 Ref.

6-CH3FH4
IV 100 500 36 45 24

5-CHOFH4
lY.. 0 370 7 61 25
200 370 33 64

0 500 10 39 26
200 370 35 81

Oral 1002 375 35 18 27

1253 600 45 31 28

1N represents the number of patients participating in the clinical trial.

2100 mg of 5-CHOFH4 was administered orally every hour for 4 hours.
35-CHOFH4 was administered orally every hour for 4 hours at a dose of 125 mg/m2.

_......
.... • 5-CH3FH4
..c
~
E:
u
;:J 1:3 CH2FH4
< +FH4

Folic acid 5-CHOFH4

Figure 1. Area under the curve (AUC) or total accumulation of 5-CHsFH4 and
CH2FH4+FH4 after oral administration of 125 mg/m2 folic acid or 5-CHOFH4. 5-
CHsFH4 and CH2FH4+FH4 were estimated in plasma by the ternary complex-based
assay. AUC values were calculated by the linear trapezoidal method. Values
represent the mean from five volunteers and error bars the SEM.

696
only [S]5-CHOFH4 is active ([R,S]5-CHOFH4 was administered) and thus the
effective dose is 62.5 mg/m2, nevertheless, folic acid is clearly an excellent
source of plasma reduced folate metabolites. Hence, it is predicted to be an
attractive alternative to 5-CHOFH4 therapeutically.
Interestingly. not only is folic acid able to elevate the total reduced
folate metabolite pool, it is particularly effective in elevation of the CH2FH4 +
FH4 pool (Figure 1). If elevation of this pool leads to more extensive
intratumor elevation of CH2FH4 in vivo as suggested by the in vitro study
(Table 1), folic acid could be even more attractive as an agent to potentiate
FU therapeutically.

Acknowledgments
This work was supported by Grants from the USPHS, CA-22754 and
American Cancer Society CH-461.

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reduced folates after short-term infusion of d, 1-folinic acid, Cancer
Chemother. Pharmacal. 25:440 (1990).
22. E. Mini, T. Mazzei, M. Coronnello, L. Criscuoli, M. Gualtieri, P. Periti, and J.R.
Bertino, Effects of 5-methyltetrahydrofolate on the activity of
fluoropyrimidines against human leukemia (CCRF-CEM) cells, Biochem.
Pharmac. 36:2905 (1987).
23. J.A. Houghton, L.G. Williams, S.S.N. DeGraff, P.J. Cheshice, I.W. Waina, P.
Jadand, and P.J. Houghton, Comparision of the conversion of 5-
formyltetrahydrofolate and 5-methyltetrahydrofolate to 5,10-
methylenetetrahydrofolate and tetrahydrofolate in human colon tumors,
Cancer Corum. 1:167 (1989).
24. P. La Ciura, G. La Grotta, and E. Nigra, A. Comandone, G. Grecchi, G. Leria,
and A. Calciati, 5-Methyltetrahydrofolic acid (MFH4): An effective folate
for the treatment of advanced colorectal cancer with 5-FU, Recent
Results in Cancer Res. 110: 219 (1988).
25. C. Erlichman, S. Fine, A. Wong, and T. Elhakim, A randomized trial of
fluorouracil and folinic acid in patients with metastatic colorectal
carcinoma, J. Clin. Oncol. 6:469 (1988).
26. M.A. Poon, M.J. O'Connell, H.S. Wieand, J.E. Krook, J.B. Gerstner, L.K.
Tschetter, R. Levitt, C.G. Kardinal, and J.A. Mailliard, Biochemical
modulation of fluorouracil with leucovorin: Confirmatory evidence of
improved therapeutic efficacy in advanced colorectal cancer, J. Clin.
Oncol. 9:1967 (1991).
27. L.R. Laufman, W.D. Brenckman, Jr., and K.A. Stydnicki, Clinical experience
with leucovorin and 5-fluorouracil, Cancer 63:103 (1989).
28. J.D. Hines, D.J. Adelstein, J.L. Spiess, P. Girowski, and S.G. Carter, Efficacy of
high-dose oral leucovorin and 5-fluorouracil in advanced colorectal
carcinoma, Cancer 63:1022 (1989).

698
MTX DOES NOT AFFECT ENHANCED BIOSYNTHESIS
AND METABOUSM OF S-ADENOSYLMETHIONINE IN
TESTOSTERONE-INDUCED HYPERTROPHIC MOUSE
KIDNEY

Wanda Chmurzynska, Malgorzata Manteuffel-Cymborowska,

Malgorzata Szl4zak, and Barbara Grzelakowska-Sztabert

Nencki Institute of Experimental Biology

3, Pasteur st., 02-093 Warsaw, Poland

INTRODUCTION

In the testosterone-induced hypertrophic female mouse kidney model we recently found

that one of the anabolic effects of testosterone, apart from its profound induction of
ornithine decarboxylase, which results in the stimulation of putrescine biosynthesis, is the
enhancement of synthesis and levels of S-adenosylmethionine (AdoMet), and an increase
in cellular methylation intensity!. Synthesis of renal AdoMet may be considered to be a
folate-dependent process, because of the involvement of 5-methyltetrahydrofolate in the
methylation of homocysteine to methionine. Blocking by MTX of tetrahydrofolate
regeneration from dihydrofolate causes cellular depletion of reduced folates2 , among them
5-methyltetrahydrofolate3 A. In addition, MTX also decreases levels of AdoMef· 6 , choline
and betaine6 in various rat tissues, and causes an increase in urinary wasting of endogenous
folates in the rat kidney7 •
The aim of this study was to find out whether impairment of tetrahydrofolate
production and the depletion of metabolically linked pools of folate cofactors, caused by
the treatment of mice with MTX, could influence the metabolism of AdoMet under
conditions of testosterone-induced increased demand for this compound.

MATERIALS AND METHODS

Female Swiss mice, receiving ampicillin and streptomycin in the drinking water to
eliminate metabolism of MTX by bacteria present in the gut, were given daily i.p.
injections of MTX (0.35 or 3.5 mg/kg); after 10 days a single s.c. injection of testosterone
(125 mg/kg) was given. Antibiotics and MTX treatments were continued for additional 5

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 699
days. Mice were then sacrificed, the kidneys (and in some experiments also the liver)
dissected and processed for the estimation of MTX concentration by dihydrofolate reductase
inhibition assay 8 and for estimations of activities of dihydrofolate reductase9 , the enzymes
metabolizing homocysteine and involved in the synthesis of AdoMet and polyamines1•
Methionine uptake by the kidneys and liver was estimated 1h after i. p. injection of [methyl-
14C] methionine (10 mg/kg; 5.45 ~-tCi/30g) 1 • Mice receiving injections of vehicles (PBS and
soybean oil) served as controls.

RESULTS AND DISCUSSION

Retention of MTX and inhibition of dihydrofolate reductase activity

In control experiments we established that prolonged treatment of mice with MTX,

at doses of either 0.35 mg/kg (data not shown) or 3.5 mg/kg, resulted in the massive
accumulation of the drug in their kidneys and liver (Table 1). Although retention of MTX
in the kidneys was about 4 times lower than in liver, the inhibition of dihydrofolate
reductase activity in both organs was nearly complete. Treatment of mice with testosterone
had no effect on the amount of retained MTX (data not shown).

Table 1. MTX levels and dihydrofolate reductase activity in the kidneys and liver of MTX-
treated mice (3.5 mg/kg; 13 days).

MTX DHFR
organ treatment pmoles/g w.w.
nmoles/h/mg protein % inhibition
kidney control 0 404.1 0
MTX 317.2 (2) 25.1 93.8
liver control 0 226.3 0
MTX 1400.8 ± 463.6 (6) 20.6 88.6

MTX effects on testosterone-induced metabolic changes in mouse kidneys

Kidney enlargement and dramatic ornithine decarboxylase stimulation, characteristic

for testosterone-induced renal hypertrophy, were not affected by MTX (Table 2). Among
the other metabolic markers of testosterone-induced hypertrophy of the kidney, MTX
treatment caused a slight decrease in AdoMet synthetase activity, but did not affect the
activity of cystathionine synthase, which irreversibly commits homocysteine to the
transsulfuration pathway (Table 2).
MTX did not influence methionine synthase, the main renal enzyme involved in the
methylation of homocysteine to methionine. Betaine methyltransferase activity was
decreased in the kidneys of MTX-treated animals; however, a wide variation in the levels
of the enzyme activity in individual mice was observed. One can speculate that this
decrease may have resulted from the depletion by MTX of choline and betaine in the liver
of experimental mice, similar to that observed by Pomfret et al. 6 in rats. However, in
mouse kidneys most methionine is synthesized by methyltetrahydrofolate and vitamin B12-
dependent methionine synthase10 , thus even a pronounced decrease in betaine
methyltransferase activity would not affect the renal methionine pool.

700
 MTX influenced only to a small extent testosterone-induced renal uptake of
methionine. As shown in Fig. 1, MTX treatment resulted in a slight increase in methionine
accumulation in the kidneys and liver of both control and testosterone-treated mice. These
results point to some differences in this amino acid transport system between mouse
kidneys and mitogen-stimulated human lymphocytes, in which MTX inhibits methionine
uptake11 •

Table 2. MTX effect on testosterone-induced metabolic changes in mouse kidneys.

testosterone nmoles/h/mg protein

Enzyme -induced
stimulation· testosterone testosterone + MTX
POLY AMINE BIOSYNTHESIS

ornithine
decarboxylase 700 163.06 ± 85.91 (4) 163.96 ± 23.52 (5)
AdoMet
decarboxylase 0.22 ± 0.04 (4) 0.23 ± 0.03 (6)
MARKERS OF TESTOSTERONE-INDUCED HYPERTROPHY

cystathionine
synthase 3.4 83.26 ± 13.58 (4) 79.00 ± 7.62 (5)
AdoMet
synthetase 1.2 2.36 ± 0.23 (4) 1.79 ± 0.39 (6)
METHIONINE BIOSYNTHESIS

methionine
synthase 5.22 ± 0.76 (4) 4.67 ± 1.06 (6)
betaine
methyl transferase 1.51 ± 0.76 (4) 0.43 ± 0.96 (6)

% of body weight
relative kidney
weight 1.3 1.50 ± 0.11 (13) 1.52 ± 0.09 (15)

measured in the absence of MTX and compared with controls which received vehicle only. Mice were
treated with MTX for 15 days at a dose 3.5 mglkg. Data represent means ± SD and number of mice
is given in parenthesis.

The results presented herein support our previous results showing that some anabolic
effects of testosterone on female mouse kidneys, such as increased AdoMet levels and
augmented methylation intensity, are fulfilled by enhanced methionine uptake rather than
by its increased biosynthesis. Thus MTX-evoked disturbances in the pool of folate cofactors
had only marginal effects on testosterone-induced renal hypertrophy.

701
 dpmx10- 3/100 mg organ weight

200
- control
~ MTX
c=J testosterone
150 W4! MTX • teetoeterone

100

50

0 ...ll:::===
KIDNEY LIVER

Figure 1. Effect of MTX (0.35 mg/kg) and testosterone on radioactivity in the kidneys and liver ot mJce
pulse-labelled with [methyl -14C]methionine (1h).

ACKNOWLEDGMENT

This research was supported by grants statutable to the Nencki Institute and from the
State Committee for Scientific Research No 6 6276 9203.

REFERENCES
1. M. Manteuffel-Cymborowska, W. Chmurzynska, and B. Grzelakowska-Sztabert, Biochim.Biophys.Acta
1116:166 (1992).
2. R.C. Jackson, Pharmacol. Ther. 25:61 (1984).
3. C.J. Allegra, R.L. Fine, J.C. Drake, and B.A. Chabner, J.Biol.Chem. 261:6478 (1986).
4. V. Kesavan, P. Sur, M.T. Doig, K.J. Scanlon, and D.G. Priest, Cancer Lett. 30:55 (1986).
5. A.M. Svardal, P.M. Ueland, R.K. Berge, A. Aarsland, N. Aarsaether, P.E. Lonning, and H. Refsum,
Cancer Chemother.Pharmacol. 21:313 (1988).
6. E.A. Pomfret, K.A. da Costa, and S.H. Zeisel, J.Nutr.Biochem. 1:533 (1990).
7. J.C. Deutsch, and J.F. Kolhouse, Cancer Res. 49:5858 (1989).
8. J.R. Bertino, and G.A. Fisher, Methods Med.Res. 10:297 (1964).
9. K.G. Scrimgeour, and F.M. Huennekens, in: "Handbuch der Physiologisch und Pathologisch Chemischen
Analyse" Springer Verlag, Berlin VIB (1966) p.181.
10. B. Grzelakowska-Sztabert, W. Chmurzyiiska, M. Manteuffel-Cymborowska, and E. Sikora, Cancer Lett.
32:207 (1986).
11. K.J. Scanlon, K. Berkowitz, M.E. Pallai, and S. Waxman, Cancer Treat.Rep. 67:631 (1983).

702
THERMOlABILITY OF RESIDUAL METHYLENE-
TETRAHYDROFOIATE REDUCfASE (MR) ACfiVITY,
METHIONINE SYNTHASE ACfiVITY AND METHYL-
COBALAMIN LEVELS 1N CULTURED FIBROBlASTS
FROM PATIENfS WITH MR DEFICIENCY

D.S. Rosenblatt, H. Lue-Shing, N. Matiaszuk, L. Low-Nang, A.

Arzoumanian, and B.A. Cooper

MRC Genetics Group, Centre for Human Genetics, The Hess B. and
Diane Finestone Laboratory, Departments of Medicine, Biology, and
Pediatrics, McGill University, Montreal, Quebec, Canada

lNTRODUCfiON

The clinical findings in patients with MR deficiency, the most common inborn
error of folate metabolism, range from death in infancy to asymptomatic homocystinuria
in adulthood (1-8). Patients may have developmental delay, psychiatric manifestations,
and gait abnormalities. The biochemical manifestations include moderate homocystinuria
and homocystinemia. Plasma methionine levels are usually low or normal. In contrast
to patients with functional deficiencies of methionine synthase (cblE, cblG), megaloblastic
anemia is not found. There is a correlation between the proportion of
methyltetrahydrofolate in extracts of cultured fibroblasts, the residual MR activity, and
the clinical severity of the disease (9-11). Genetic heterogeneity of MR deficiency was
suggested when we observed varying patterns of thermostability at 55° in fibroblast
extracts (9).

MATERIALS AND METHODS

Human skin fibroblasts were grown to confluence in Eagle Minimum Essential

Medium as previously described (9,12). Lines were obtained from 15 patients referred
for the diagnosis of MR deficiency. There were 8 female and 7 male patients. For the
MR assay, cells were trypsinized and resuspended at a density of 108 cells/ml, the cell
suspension was sonicated on ice in 0.25M sucrose and was centrifuged at 5° at 50,000

Chemistry and Biology of Pteridines and Palates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 703
rpm for 45 min. For the methionine synthase assay, K phosphate, PH 7.4 was added to
the final extract that had been previously sonicated in 0.25M sucrose, for a final
concentration of 0.1M K Phosphate. Assays of both MR (9) and methionine synthase
e
(13) were performed as previously described. 4CJmethyltetrahydrofolate was purchased
from Amersham Corp. (Oakville Ont.) and purified before use as previously described
(13). Residual MR activity was determined following pre-incubation of cell extracts at
55° for 20 min. prior to the addition of labelled substrate, and expressed as per cent of
the original specific activity. Cells were grown in 25 pg/ml [57Co]cyanocobalamin
(Amersham, Corp.) for 4 days, following which the labelled cobalamins were extracted
in complete darkness in absolute ethanol at 85° and separated by high performance
liquid chromatography as previously described (13).

RESULTS

All 15 lines had low methylenetetrahydrofolate reductase activities, ranging from

undetectable to 1.76 n mols CHO/mg protein/h. It was not possible to measure
thermostability in those lines with extremely low or undetectable activities. There was
good agreement in the results seen in the two sets of sibling pairs. Lines from two sisters
who were diagnosed as teenagers showed thermostability of residual reductase activity
(47.4% and 50.4%) and relatively low levels of MeCbl (16.4% and 25.4%). Lines from
two brothers who were diagnosed as adults showed thermolabile reductase activity (0.9%
and 0%) and relatively higher levels of MeCbl (35.5% and 51.8% ). With the exception
of one cell line, all patients who presented in infancy had low levels of MeCbl. Most of
the patients with later onset disease had levels of MeCbl above 20%. The one exception
was from one of the sisters mentioned above whose level was 16.4%. There was a large
variation seen in the specific activities of methionine synthase in the cell lines. All but
two patients had values within two standard deviations of previously described mean
values for control fibroblasts (14). Although several patients with low methionine
synthase specific activities in cell extracts also had low MeCbllevels, others did not. In
those patients in whom it was possible to measure thermostability, it appeared that the
presence of a thermolabile reductase was usually associated with a later clinical
presentation.

DISCUSSION

Many of the patients with MR deficiency, particularly those with early-onset

disease, had low levels of MeCbl following incubation in [57 Co ]CN-Cbl. In addition, one
of these patients had specific activities of methionine synthase below the control range.
These findings have implications in the differential diagnosis of patients with
homocystinuria and hypomethioninemia. Patients with both cblE and cblG disease will
have low MeCbl levels in their fibroblasts, and cblG patients will also have low
methionine synthase levels in the standard assay (15). If these two tests are used to
diagnose cblE or cblG disease without looking at the incorporation of labelled [14C] from
methyltetrahydrofolate, or without performing complementation analysis, an incorrect
diagnosis may be made. Although megaloblastic anemia is usually seen in both cblE and
cblG disease, and is virtually absent in MR deficiency, misdiagnosis is still possible.
Although most of the patients with onset of disease after infancy had thermolabile
MR, this was not always the case. The thermolability described here in the severe form
of MR deficiency should be distinguished from the variant form of MR described by

704
Kang in which specific activity is less severely deficient but in which the reductase has
abnormal thermolability at 46° in lymphocyte extracts (16). The variant described by
Kang has been postulated as an independent risk factor for coronary disease (17,18), but
is not associated with the other clinical findings of severe MR deficiency. Both brothers,
one of which being completely asymptomatic as an adult (19), had thermolabile residual
activity. In contrast, the two sisters who (20) were diagnosed in adolescence both have
a thermostable enzyme. There were both thermostable and thermolabile enzyme seen
in patients with onset of disease in infancy.

ACKNOWLEDGEMENTS

The authors want to thank the clinicians who supplied cultured fibroblasts and
clinical summaries on their patients. The results have been summarized from our
previous publication with permission (21).

REFERENCES

1. D.S. Rosenblatt, Inherited disorders of folate transport and metabolism, in: "The
Metabolic Basis of Inherited Disease", C.R. Scriver, AL. Beaudet, W.S. Sly, D.
Valle, eds. McGraw Hill, New York (1989).
2. K. Narisawa, Y. Wada, T. Saito, K. Suzuki, M. Kudo, T.S. Arakawa, N.A
Katsushima, R. Tsuboi, Infantile type of homocystinuria with N5•10-methylene-
tetrahydrofolate reductase defect. Tohoku J Exp Med 121:185 (1977).
3. S.H. Mudd, B.W. Uhlendorf, J.M. Freeman, J.D. Finkelstein, V.E. Shih,
Homocystinuria associated with decreased methylenetetrahydrofolate reductase
activity, Biochem Biophys Res Commun 46:905 (1972).
4. J.M. Freeman, J.D. Finkelstein, S.H. Mudd, Folate-responsive homocystinuria and
"schizophrenia". A defect in methylation due to deficient 5,10-methylenetetra-
hydrofolate reductase activity. N Eng! J Med 292:491 (1975).
5. P.W.K. Wong, P. Justice, M. Hruby, E.B. Weiss, E. Diamond, Folic acid
non-responsive homocystinuria due to methylenetetrahydrofolate reductase
deficiency. Pediatrics 59:749 (1977).
6. E.R. Baumgartner, K. Schweizer, H. Wick, Different congenital forms of defective
remethylation in homocystinuria. Clinical, biochemical, and morphological studies,
Pediatr Res 11:1015 (1977).
7. Y.S. Kanwar, J.R. Manaligod, P.W.K. Wong, Morphologic studies in a patient with
homocystinuria due to 5,10-methylenetetrahydrofolate reductase deficiency,
Pediatr Res 10:598 (1976).
8. J.M. Visy, P. LeCoz, B. Chadefaux, C. Fressinaud, F. Woimant, J. Marquet, J.
Zittoun, J. Visy, J.M. Vallat, M. Haguenau, Homocystinuria due to
5,10-methylenetetrahydro-folate reductase deficiency revealed by stroke in adult
siblings. Neurology 41:1313 (1991).
9. D.S. Rosenblatt, R.W. Erbe, Methylenetetrahydrofolate reductase in cultured human
cells. II. Studies of methylenetetrahydrofolate reductase deficiency, Pediatr Res
11:1141 (1977).
10. D.S. Rosenblatt, B.A Cooper, S. Lue-Shing, P.W.K. Wong, S. Berlow, K. Narisawa,
R. Baumgartner, Folate Distribution in Cultured Human Cells: Studies on
5,10-CH2-H4PteGlu Reductase Deficiency, J Clin Invest 63:1019 (1979).

705
11. E.R. Baumgartner, E.L.R. Stokstad, H. Wick, J.E. Watson, G. Kusano, Comparison
of folic acid coenzyme distribution patterns in patients with methylenetetra-
hydrofolate reductase and methionine synthetase deficiencies, Pediatr Res 19:1288
(1985).
12. B.A. Cooper, D.S. Rosenblatt, Folate coenzyme forms in fibroblasts from patients
deficient in 5,10-methylenetetrahydrofolate reductase. Biochem Soc Trans 4:921
(1976).
13. D.S. Rosenblatt, B.A. Cooper, A. Pottier, H. Lue-Shing, N. Matiaszuk, K. Grauer.
Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic
anemia and homocystinuria due to a new defect in methionine biosynthesis. J Clin
Invest 74:2149 (1984).
14. D. Watkins, D.S. Rosenblatt, Genetic heterogeneity among patients with
methylcobalamin deficiency: definition of two complementation groups, cblE and
cblG. J Clin Invest 81:1690 (1988).
15. D. Watkins, D.S. Rosenblatt, Functional methionine synthase deficiency (cblE and
cblG): Clinical and biochemical heterogeneity. Am J Med Genet 34:427 (1989).
16. S.S. Kang, J. Zhou, P.W.K. Wong, J. Kowalisyn, G. Strokosch, Intermediate
Homocysteinemia: A Thermolabile Variant of Methylenetetrahydrofolate
Reductase, Am J Hum Genet 43:414 (1988).
17. S.S. Kang, P.W.K. Wong, J. Zhou, J. Sora, N. Ruggie, G. Grcevich, Thermolabile
methylenetetrahydrofolate reductase in patients with coronary artery disease,
Metabolism 37:611 (1988).
18. S.S. Kang, P.W.K. Wong, A. Susmano, J. Sora, M. Norusis, N. Ruggie, Thermolabile
methylenetetrahydrofolate reductase: An inherited risk factor for coronary artery
disease. Am J Hum Genet 48:536 (1991).
19. J.C. Haworth, L.A. Dilling, R.A.H. Surtees, L.E. Seargeant, H. Lue-Shing, B.A.
Cooper, D.S. Rosenblatt, Symptomatic and asymptomatic
methylenetetrahydrofolate reductase deficiency in 2 adult brothers. Am J Med
Genet 45:572 (1993).
20. J.M. Freeman, J.D. Finkelstein, S.H. Mudd, B.W. Uhlendorf, Homocystinuria
presenting as reversible "schizophrenia": a new defect in methionine metabolism
with reduced 5,10-methylenetetrahydrofolate reductase activity. Pediatr Res 6:423
(1972).
21. D.S. Rosenblatt, H. Lue-Shing, A. Arzoumanian, L. Low-Nang and N. Matiaszuk,
Methylenetetrahydrofolate reductase (MR) deficinecy: Thermolability of residual
MR activity, methionine synthase activity, and methylcobalamin levels in cultured
fibroblasts, Biochem Med Met Bioi 47:221 (1992).

706
ENZYMES FOR SYNTHESIS OF 10-FORMYLTETRAHYDROFOLATE
IN PLANTS. CHARACTERIZATION OF A MONOFUNCTIONAL
10-FORMYLTETRAHYDROFOLATE SYNTHETASE AND COPURIFICATION
OF 5,10-METHYLENETETRAHYDROFOLATE DEHYDROGENASE AND
5,10-METHENYLTETRAHYDROFOLATE CYCLOHYDROLASE ACTIVITIES.

Edwin A. Cossins 1, Carole D. Kirk 1, Helena C. Imeson 2

and Li-li Zheng3

1Department of Botany, University of Alberta,

Edmonton, T6G 2E9, Canada
2Department of Biology, University of Lethbridge,
Lethbridge, T1K 3M4, Canada
3Department of Horticulture, Xiongyue Agricultural College,
Xiongyue, The People's Republic of China

INTRODUCTION

Plants require folates for the biosynthesis of purines, serine, methionine,

formylmethionyl-tRNA and thymidylate (1). In leaves, mitochondrial folates mediate
glycine cleavage and serine formation during photorespiration (2). Plants also have
enzymes (1 ,3) that interconvert methylene- and formyltetrahydrofolates. Alternatively,
these species generate lO-HCO-H4PteGlu from formate (1). Recent work from
Rabinowitz's laboratory (4,5) on the latter reaction in spinach leaves, identified a
monofunctionallO-HCO-H4PteGlu synthetase protein whose primary structure is like the
synthetase domain of the mammalian and yeast trifunctional C1-THF synthase. Partial
purification of the related dehydrogenase and cyclohydrolase activities suggested they
may occur as a bifunctional complex in spinach leaves (4). However, it is not clear
whether this structural organization is typical of higher plants in general or may reflect a
possible chloroplastic origin.
The present study has characterized these activities from a non-photosynthetic
plant tissue. The affinity of plant lO-HCO-H4PteGlu synthetase protein for H4PteGlun
substrates has also been assessed.

METHODS

Seeds of pea (Pisum sativum L. cv. Homesteader) were germinated and cotyledon
extracts used as an enzyme source. PteGlun (n = 2-5) were from Dr. B. Schircks
Laboratories, Matrex Green A was from Amicon; all other chromatographic media were

Chemistry and Biology of Pteridines and Palates, Edited by

J .E. Ayling et al., Plenum Press, New York, 1993 707
from Sigma. Lactobacillus casei DHFR was from Biopure (Boston, MA).

Enzyme Assays

10-Formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydro-

folate cyclohydrolase (EC 3.5.4.9) and 5, 10-methylenetetrahydrofolate dehydrogenase
(EC 1.5.1.5) were assayed spectrophotometrically (6, 7). Units (umoles product
formed/min) are based on an extinction coeffficient of24.9 uM-1 cm-1 (7).

Tissue Extraction and Protein Fractionation

Cotyledons (500 g) of 1 day seedlings were ground in 800 ml of 50 mM Tris-HCl

(pH 7.5) containing 25% v/v glycerol, 10 mM 2-mercaptoethanol and 1 mM PMSF.
The homogenate was filtered and centrifuged (10,000g for 15 min) to give Step 1 protein
followed by fractionation with streptomycin sulphate (Step 2 protein) and (NH4)2S04 to
give Step 3 protein. After dissolving in 8-10 ml of 5 mM KH2P04 (pH 7.5) containing
glycerol, 2-mercaptoethanol and PMSF (as above), protein was chromatographed as
summarized in Tables 1 & 2. SDS-PAGE was according to Laemmli (9) with silver
staining. Cross reactivity of synthetase and dehydrogenase/cyclohydrolase protein with an
antibody to the pure spinach lO-HCO-H4PteGlu synthetase was examined in Western
blot and ELISA assays. Good reaction was obtained with 10 ng synthetase and a 1/200
dilution of spinach antibody. Alkaline phosphatase conjugated to a secondary goat anti-
rabbit antibody was used. No cross-reactivity was apparent with different dilutions of
dehydrogenase/ cyclohydrolase protein.

Synthesis of (6S)-H4PteGlu Polyglutamates

PteGlun (n = 2 - 5) were converted to H2PteGlun by dithionite reduction and

purified on Bio-Gel P-2 in the presence of argon (10). After lyophilisation, (6S)-
H4PteGlun were generated using L. casei DHFR. (6S)-H4PteGlun concentrations were
measured by reaction with rabbit liver cSHMT (11), using a molar exctinction coefficient
(A492 nm) of 40,000 (12).

RESULTS

When cotyledon extracts were fractionated through Steps 1 to 4 of the purification

protocols (Tables 1 & 2) synthetase, dehydrogenase and cyclohydrolase activities
appeared to copurify. However, under the conditions employed for Matrex Green A
chromatography, synthetase protein was not retained. In contrast, dehydrogenase and
cyclohydrolase activities were retained by Matrex Green A and only released when the
cr concentration of the eluting buffer was raised. Subsequent hydroxylapatite
chromatography of synthetase protein (Table 1), followed by SDS-PAGE, gave a major
silver-staining band of protein (approx Mr 56,000). This Step 7 synthetase protein
lacked NADP-dependent and NAD-dependent dehydrogenase activity as well as
cyclohydrolase activity. The native molecular weight of this synthetase protein, as
judged by gel filtration on Sephacryl S-300, was approximately 110,000. We conclude
that this plant synthetase is a homodimer of similar size to that reported from spinach
leaves (4). Similarities between the synthetases from these different higher plant tissues
was also indicated by strong cross-reaction, in ELISA and Western blot analyses, of
purified pea cotyledon synthetase with antibodies raised to the purified spinach leaf
enzyme (data not shown).
Co-purification of dehydrogenase and cyclohydrolase activities occurred during
the fractionation steps shown in Table 2. These activities also co-eluted from

708
 Table 1. Purification of plant 10-Formyltetrahydrofolate synthetase activity.

Fractionation Protein Activity Purification Yield

Step (mg) Units Units/mg (fold) (%)

1. Crude homogenate 3506.3 52.8 0.0150 1 100

2. Streptomycin S04 1489.4 42.4 0.0285 2 so
3. 55-70% (NH4)2so4 240.4 40.9 0.1701 8 70
4. Sephacryl S-300 53.8 21.8 0.4052 27 41
5. DEAE-Cellulose 7.4 16.8 2.2703 151 32
6. Matrex Green A 3.6 13.0 3.6111 241 25
7. Hydroxylapatite 0.3 3.2 10.5490 703 6

heparin agarose. SDS-PAGE of this protein showed a single, silver-staining band

(approx. Mr 57,000) and Sephadex G-75 chromatography gave Mr values of
approximately 60,000.

Table 2. Co-Purification of plant 5, 10-Methylenetetrahydrofolate dehydrogenase

and 5,10-Methenyltetrahydrofolate cyclohydrolase activities.

FractiOnation Dehydrogenase Cyclohydrolase Ratio

Step Units Units/mg Units Units/mg (Dehyr/Cyclo)

1. Cmde homogenate 17.7 0.0048 6.1 0.0017 2.82

2. Streptomycin S04 20.3 0.0117 4.1 0.0023 n.d 1
3. 55-70% (NH 4)2so 4 5.9 0.0296 2.5 0.0125 2.37
4. Sephacryl S-300 6.7 0.0630 2.7 0.0258 2.44
5. Matrex Green A 4.1 2.1632 2.6 1.3556 n.d 1
6. Hepann agarose 1.3 32.5000 0.5 12.5000 2.30

1n.d = not determmed

This Step 6 protein (Table 2) lacked lO-formyl-H4PteGlu synthetase activity and

failed to cross-react with antibodies raise against this protein from spinach leaves (Table
3). Based on these data we conclude that the dehydrogenase/cyclohydrolase activities are
probably associated with a monomeric, bifunctional protein. Complexes of this type, but
of larger molecular size and containing distinct subunits, occur in some bacteria (3).
Apart from recent work with pea leaf mitochondria (13) there is very little
information on the folylpolyglutamate specificites of plant folate-dependent enzymes. In
the present work we found that purified pea cotyledon synthetase protein showed no
activity with PteGlu, H2PteGlu or their pentaglutamates (Table 3) but these folates did
not inhibit activity when H4PteGlu or H4PteGlu5 were provided. Furthermore, the
synthetase protein displayed an increasing affinity for folate polyglutamates as the chain
length was increased from mono- to pentaglutamate (Table 3). The affinities shown for
ATP and formate were also enhanced when H4PteGlu5 was provided (Table 3). These
data are in close agreement with studies of the mammalian trifunctional enzyme (14).

709
 Table 3. Major Properties of Plant Synthetase and Dehydrogenase/Cyclohydrolase.

Synthetase Dehydrogenase/Cyclohydrolase
Property examined (Step 7 Protein) (Step 6 Protein)

Mr (gel filtration): ca.llO,OOO ca.60,000

Mr based on SDS-PAGE: ca.56,000 ca.57,000
pH optimum: 7.5 6.5
Cross-reactivity (see text) Yes No
Reaction requirements: Mg2 + ,ATP,HCOO- NADP(not NAD),H4PteG!u,
Apparent Km values:
H 4PteGlu 40uM 7.7uM
H4PteGlu5 3uM n.d
ATP (with H4PteG!u) 94uM
ATP (with H4PteGlu5) 12uM
Hcoo· (with H4PteGlu) 7.6mM
HCOO- (with H4PteG!u5) 35uM
NADP 10.7uM

In other kinetic studies with purified dehydrogenase/cyclohydrolase protein (Table

3), we determined the apparent Km values for the dehydrogenase reaction. The values
for H4PteGlu (7.7 uM) and NADP (10.7 uM) are similar to those reported for the
mammalian liver enzyme (3). Further characterization of this plant dehydrogenase-
/cyclohydrolase complex including its intracellular compartmentation is in progress.

ACKNOWLEDGEMENTS

This work was supported by grants to E.A.C. from the Natural Sciences and
Engineering Research Council of Canada. Professor Jesse Rabinowitz is thanked for the
antibodies to spinach 10-formyltetrahydrofolate synthetase and for valuable discussions.
We also acknowledge Professor Verne Schirch for kindly providing mammalian serine
hydroxymeth yItransferase.

REFERENCES

1. E.A.Cossins. in: The Biochemistry of Plants, Vol2, D.D. Davies, ed., 365. Academic Press, New
York (1980).
2. E.A.Cossins. in: The Biochemistry of Plants, Volll, D.D.Davies, ed., 316. Academic Press, New
York (1987).
3. R.E.MacKenzie. in: Folates and Pterins, Vol1, R.L.Blakley and S.J.Benkovic, eds., 255. Wiley, New
York (1984).
4. J.M.Nour and J.C.Rabinowitz. J.Biol.Chem.266:18363 (1991).
5. J.M.Nour and J.C.Rabinowitz. J.Biol.Chem.267:16292 (1992).
6. D,R.Appling and J.C.Rabinowitz. J.Biol.Chem.260:1248 (1985)
7. N.P.Curthoys, J.M.Scott and J.C.Rabinowitz. J.Biol.Chem.247:1959 (1972).
8. Z.S.de Mata and J.C.Rabinowitz. J.Biol.Chem.255:2569 (1980).
9. U.K.Laemmli. Nature 227:680 (1970).
lO.P.Stover and V.Schirch. Anal.Biochem. 202:82 (1992).
ll.P .Stover and V.Schirch. J.Biol.Chem. 266:1543 (1991).
12.W.B.Strong and V.Schirch. Biochemistry 28:9430 (1989).
13.V.Besson,F.Rebeille,M.Neuburger,R.Douce,and E.A.Cossins. Biochem. J.in press: (1993).
14. V.Schirch and W.B.Strong. Arch.Biochem.Biophys.269:371 (1989).

710
CLONING OF THE GENES ENCODING THE SERINE
HYDROXYMETHYLTRANSFERASES FROM SACCHAROMYCES CEREVISIAE

Brian V. Taylor\ J. Bryan McNeil\ Evan M. Mclntosh2,

Fang-rang Zhang1 and Andrew L. Bognar1

1Department of Microbiology
University of Toronto
Toronto, Ontario, Canada MSS 1A8
2Biology Department,

York University,
Toronto, Ontario, Canada

INTRODUCTION

Serine hydroxymethyltransferase genes have only recently been cloned from

eukaryotic sources. These include the cytosolic gene from Neurospora crassa (1) and
both the human mitochondrial and cytosolic genes (2). The complete amino acid
sequences of the rabbit SHMT enzymes have been determined (3,4). In this report,
we describe the isolation of DNA fragments specific for both the mitochondrial and
cytoplasmic SHMT genes (SHM1 and SHM2) from the yeast Saccharomyces cerevisiae.
DNA fragments were amplified by the polymerase chain reaction using degenerate
oligonucleotide primers based on conserved amino acid sequences present in SHMT
enzymes of eukaryotic origin. The fragments were then cloned into plasmid vectors
and used as probes to screen a yeast genomic library for the corresponding intact
genes.

MATERIALS AND METHODS

The polymerase chain reaction was done in 30 cycles using 2 min at 45° C for
annealing, 2 min at no C for reaction and 1 min at 94° C for denaturation. The yeast
SHM genes were isolated from a genomic library constructed in the shuttle vector
Yep13 (5). DNA manipulations and colony blots were done as described in Sambrook
et al. (6) and all DNA modifying enzymes were utilized following the manufacturer's
instructions. DNA sequencing was done using the Sequenase kit from USB.

RESULTS

Six different oligonucleotide primers were synthesized corresponding to the

blocks of identical amino acids among the Neurospora crassa and the rabbit SHMT

Chemzstry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 711
enzymes, shown in Figure 1. Two separate primers :were made corresponding to the
sequence NKYSEG, differing only in the degenerate codons used for serine, one
primer using TCN and the second using AGY. All other primers were fully degenerate
for all amino acids. Each primer also contained the sequence CCGGTTGAATTC, 5'
to the homologous sequences, containing an EcaRI restriction site. A DNA fragment
of 700 bp was amplified by PCR when the primers corresponding to the sequences
NKYSEG (with TCN for serine) and PEFKEY and were utilized. This combination
gave the best yield of amplified fragment and was used for cloning experiments,
although all the primers produced amplified bands in some combination.
The amplified fragments were digested with EcoRI and ligated into £caRl-digested
pUC19. Plasmids containing inserts were isolated and the insert DNAs were
sequenced from both ends to verify that they contained coding sequences homologous
to SHM genes. Two separate sequences were found, both containing open reading

1. MSTYSLSETHKAMLEHSL VESOPQVAE IMKKEVQRQRES I ILIASEHVTSRAVFDALGSPMS

2. MATAVNGAPRDAALWSSHEQMLAQPLKDSOAEVYDIIKKESNRQRVGLELIASENFASRAVLEALGSCLN
3. KAAQTQTGEASRGWTGQESLSDTOPEMWELLQREKDRQCRGLELIASENFCSRAALEALGSCLN
NKVSEGLPGARYYGGNQHIOEIEVLCQNRALEAFHLDPKQWGVNVQCLSGSPANLQVYQAIMPVHGRLMGLDLPHGGHLS
NKYSEGYPGQRVYGGTEHIDELETLCQKRALQAYGLOPQCWGVNVQPYSGSPANFAV'tTALVEPHGRIMGLOLPDGGHLT
NKVSEGYPGKRYYGGAEVVOEIELLCQRRALEAFDLOPAQWGVNVQPYSGSPANLAAYTALLQPHDRIMGLOLPDGGHLT
HGYQTPQRKISAVSTYFETMPVRVNIDTGLIOYDTLEKNAQLFRPKVLVAGTSAYCRLIOYERMRKIAOSVGAYLVVDMA
HGFMTDKKKISATSIFFESMAYKVNPDTGYlOYDRLEENARLFHPKLIIAGTSCVSRNLOVGRLRKIAOENGAYLMADMA
HGYMSDVKRVSATSIFFESMPYKLNPQTGL!OYEQLALTARLFRPRLIIAGTSAVARLIOVARMREVCOEVKAHLLADMA
HI SGl lAS EV IPSPF LYADVVTTTTHKSLRG PRGAMI FF- RGVRSVDAKTGKETLYDLEDKINFSVFPGHQGGPHNHT1T
HISGL VVAGVVPSPFEHCHVVTTTTHKSLRGCRAGM!FYRRGVRSVDPKTGKE ILYNLESL!NSAVFPGLQGGPHNHA!A
HISGlVAAKVIPSPFKHADVVTTTTHKSLRGARSGLIFYRKGVRTVDPKTGQEIPYTFEDRINFAVFPSLQGGPHNHAIA
ALAVALKQAASPEFKEYQQKVVANAKALEKKLKELGYKLVSDGIDSHMVLVDLRPIGVDGARYEFLLEQINlTCNKNAVP
GVAVALKQAMTPEFKEYQRQVVANCRALSAALVELGYKIVTGGSONHLILVOLRSKGTDGGRAEKVLEACSIACNKNTCP
AVAVALKQACTPMFREYSLQVLKNARAMADALLERGVSLVSGGTONHLVLVDLRPKGLOGARAEKVLELVSITANKNTCP
GOKSALTPGGLRIGTPAMTSRGFGEAOFEKVAVFVDEAVKLCKEIQASLPKEANKQKDFKAKIA-TSDIPR-INELKQEI
GOKSALRPSGLRLGTPALTSRGLLEKOFQKVAHFIHRGIELTVQIQDDTGPRAT-LKEFKEKLAGDEKHQRAVRALRQEV
GORSAITPGGLRLGAPALTSRQFREOOFRRVVDFIDEGVNIGLEVKRKTA KLQDFKSFLLKDPETSQRLADLRRRV
AAWSNTFPLPVEGWRYDAGL
ESFAALFPLPGLPGF
QQFARAFPMPGFPEH
Figure 1. PCR primers used for amplification of SHM gene-specific DNA fragments.

8 8g X E Rv E Rv Rv8/S
pSH36

8/S H HSpSp 8cH EEHSa Sa Sm8/S

I I II I I I
I X' I I I pSH3

8/SH EEHSa Sa Sm H 8/S

pSH2

1 kb
Figure 2. Restriction maps of S. cerevisiae DNA in plasmids containing the SHM
genes. pSH36 contains the intact SHMl gene, while pSH3 and pSH2 contain the
SHM2 gene. The open reading frames of the intact genes are indicated by the solid
boxes. Restriction enzyme sites are: B, Bam HI; Be, Bel I; Bg, Bgl II; B/S, Bam HI site
in the vector ligated to a Sau 3AI site in the genomic DNA; E, Eco RI; H, Hind III;
Rv, Eco RV; Sa, Sal I; Sm, Sma I; Sp, Sph I.

712
frames with predicted amino acid sequences homologous to those of known SHMT
enzymes and the corresponding genes were designated SHM1 and SHM2. Probes were
prepared from these cloned fragments and used to screen a yeast genomic library. One
clone, pS36 was found containing the intact SHM1 gene on a 5.6 kbp BamHI-Hindiii
fragment, which was subcloned into pUC19 (see Figure 2). Two clones were found,
pS2 and pS3, containing the SHM2 gene. Clone pS2 contained a large insert of yeast
genomic DNA (> 10 kb). The PCR-amplified DNA probe hybridized to a 1 kbp
Hindiii fragment and a 1.2 kb Sali-EcoRI fragment and these fragments were
subcloned into pUC19 and sequenced. Translation of the sequence at the 5' end of the
Sal I- Eco RI fragment showed the insertion site in the vector followed by sequences
with homology to the amino terminal sequence of SHMT enzymes but lacking the
initiator methionine, indicating that the gene was incomplete. Rescreening of the yeast
genomic library led to the isolation of pS3, which contains the intact SHM2 gene.
Interestingly, when the sequences of the genomic clones were determined in the
regions of the PCR primer, and compared to the sequences of the amplified DNA
fragments, which contain the actual primers, at least one mismatch was found in each
primer and one primer had three mismatches.
Both the SHM1 and SHM2 genes were subcloned into pUC19. The entire pSH36
insert was subcloned into pUC19 in both orientations. These plasmids were unable to
transform the E. coli glyA strain to glycine prototrophy. The pSH3 insert cloned
downstream of the lac was unable to complement the glyA strain but a subclone, in
which the DNA 5' to the Sph I site was deleted, did complement the glyA strain,
indicating that the cloned SHM2 was a functional gene.

J.
1. MFPRASALAKCMATVHRRGLLTSGAQSLVSKPVSEGOPEMFD!LQQERHRQKHS!TL!
2. MPYTLSDAHHKLITSHLVDTDPEVDS!IKDEIERQKHS!DL!

Figure 3. Alignment of the predicted amino acid sequences of the SHM1 (1) and
SHM2(2) gene products. The arrow indicates the predicted cleavage site in the leader
sequence.

DNA sequence determination of the regions encoding the amino terminal regions
of the SHM genes suggested that SHM1 encodes the mitochondrial SHMT and SHM2
encodes the cytosolic enzyme. The amino terminal region of the SHMl product
extends 16 amino acids farther than the SHM2 product when the two sequences are
aligned and the leader sequence has the characteristics of a mitochondrial leader
sequence (Figure 3). The putative leader sequence lacks acidic amino acids or
extensive hydrophobic stretches. In addition, There is a sequence in the leader:
VHRRGL which corresponds well to a consensus recognition sequence proposed for
the protease that cleaves mitochondrial leader peptides (7).

DISCUSSION

The putative mitochondrial gene, SHM1, did not complement the E. coli glyA
strain and does not express SHMT activity in E. coli. It is unlikely that this represents
an inactive or pseudogene in yeast, since repeated PCR amplifications did not yield
sequences homologous to a third gene, representing the active mitochondrial gene but
did consistently amplify the SHM1 gene. The plasmid used for the expression studies
contains 900 bp of yeast DNA between the lac promoter in the vector and the
initiation codon of the SHM1 open reading frame. Yeast non-coding sequences are AT
rich and contain long stretches of consecutive T residues, which may act as termination

713
signals in E. coli. We are deleting some of this intervening sequence to try to obtain
high expression of the enzyme. An alternate explanation for the lack of activity may
be that the mitochondrial leader sequence, which would not be cleaved in E. coli,
interferes with enzyme activity. Expression would then require deletion of the leader
sequence codons and insertion of an ATG codon at the predicted site of cleavage
using in vitro mutagenesis.
We have recently become aware of a report of the sequencing of the gene
corresponding to SHM1 (8). These authors identify the gene as encoding the cytosolic
SHMT because the leader sequence does not correspond to the mitochondrial
consensus. Their sequence differs from ours (unpuplished) by an A residue 81 bp
upstream of our predicted initiation codon, resulting in a 5' extension of the open
reading frame into nonsense sequences.

REFERENCES

1. C.R. Mclung, C. Davis, K. Page and S.A. Denome, Mol. Cell. Biol. 12:1412-1421
(1992).
2. B. Shane, personal communication, J. Biol. Chern, in press (1993).
3. F.B. Martini, B. Maras, P. Tanci, S. Angelaccio, S. Pascarella, D. Barra, F. Bossa
and V. Schirch, J. Biol. Chern. 264: 8509-8519 (1989).
4. F.B. Martini, S. Angelaccio, S. Pascarella, D. Barra, F. Bossa and V. Schirch, J.
Biol. Chern. 262: 5499-5509.
5. J.R. Broach, J.N. S,trathern and J.B. Hicks, Gene 8:121-133 (1979).
6. J. Sambrook, E.F. Fritsch abd T. Maniatis, "Molecular Cloning a Laboratory
Manual" , 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor NY (1989).
7. B.Schmidt, E. Wachter, W. Sebald and W. Neupert, European J. Biochem. 144:581-
588 (1984)
8. F. Doignon, N. Biteau, M. Crouzet and M. Aigle, Yeast 9:189-199 (1993).

714
SERINE HYDROXYMETHYLTRANSFERASE: ROLE OF THE ACTIVE

SITE LYSINE IN THE MECHANISM OF THE ENZYME

Douglas Schirch, Sonia Delle Pratte, Sandra Iurescia, Sebastiana

Angelaccio, Roberto Contestabile, Francesco Bossa and Verne Schirch

Department of Biochemistry and Molecular Biophysics

Box 614, MCV Station
Virginia Commonwealth University
Richmond, VA 23298

Dipartimento di Scienze Biochimiche and Centro di Biologia Molecolare del

Consigilio Nazionale delle Ricerche
Unviersita La Sapienza
00185 Rome, Italy

INTRODUCTION

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate requiring enzyme

which catalyzes the reaction shown in Equation 1. This enzyme uses both the
monoglutamate and polyglutamate forms of tetrahydrofolate (H4PteGlun) and 5,10-
methylenetetrahydrofolate (5,10-CH2-H4PteGlun). In eucaryotic organisms there are both
cytosolic and mitochondrial isoenzymes. The role of the cytosolic isoenzyme is to provide
5,10-CH2-R4PteGlun for the biosynthesis of thymidylate, purines, and methionine.!

f4PteGlun + L-Serine .. 5,10-CH2-R4PteGlun + Glycine (1)

Pyridoxal phosphate is covalently attached to SHMT as an internal aldimine bond between

the C4' aldehyde of pyridoxal-P and the ~::-amino group of Lys-229 in E. coli SHMT. 2-7 All
pyridoxal-P enzymes have been found to have the coenzyme bound as an internal aldimine to
a Lys residue. For several of these enzymes, especially aspartate aminotransferase, the active
site lysine has been changed to another amino acid by site-directed mutagenesis.8 Both
kinetic studies and 3-d structural determinations suggest that in aspartate aminotransferase the
active site lysyl residue has two functions.9 First, it is required to break the bond between
pyridoxal-P and the amino acid product. Without this step the enzyme can not effectively
release the amino acid product, which remains bound as an external aldimine. Second, the
active site lysyl group serves as the base in the transfer of the a-proton of the amino acid to
the 4'-carbon of pyridoxal-F.
We have investigated the role of the active site Lys-229 in E. coli SHMT by changing this
residue to a Gin, His, or an Arg (K229Q, K229H, and K229R SHMTs). Each enzyme was
purified to homogeneity and its catalytic and spectral properties examined. For each enzyme
kcat was close if not equal to zero. However, the K229Q enzyme catalyzed a single turnover
of either serine to glycine in the presence of R4PteGlu, or glycine to serine in the presence of
5,10-CH2-H4PteGlu3. Since these mutant enzymes exhibited no steady state catalytic
activity the purification procedure had to follow other criteria for the presence of the enzyme

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 715
in different fractions. This manuscript describes the detailed purification procedure, and lists
the major catalytic and spectral properties of the mutant enzymes.

PURIFICATION PROCEDURE
Growth of E. coli.
The mutant enzymes are expressed from a plasmid containing the glyA gene in the E. coli
strain GS245, which has a deletion of the wild type glyA gene. One liter of 1.5X LB broth
containing 50 Jlg per L ampicillin was inoculated with a single colony of GS245 E. coli
grown overnight at 37 °C on LB agar plates supplemented with 50 Jlg of ampicillin. This
liter of overnight culture was used to inoculate 20 liters 1.5 X LB broth supplemented with
0.5 % glucose and 100 mM potassium phosphate, pH 7 .0. The cells were grown with
aeration at 37 °C until the O.D.6Qo reached 6 to 7 (normally this took 6 hours). The cells
were harvested by centrifugation at 6,000g in a Dupont GSA rotor. Normally 200 to 250 g
of cells were obtained.

Cell lysis and homogenate

The cells were suspended in 1.5 L of lysis buffer (6 g of Tris base and 9.6 g of disodium
EDTA, pH 7.8). To the resuspended cells 200 mg of lysozyme, dissolved in the lysis
buffer, were added. After 15 min at 23 °C the cells were divided into 6 centrifuge bottles
(500 ml) and frozen at -20 °C overnight. The next morning the cell lysate was thawed and
each centrifuge bottle was filled to the top with 20 mM potassium phosphate containing 5
mM 2-mercaptoethanol and 0.2 mM pyridoxal-F. The cells were centrifuged at 12,000g for
30 min at 4 °C. The supernatant was decanted and saved for the first purification step. The
total volume of homogenate was usually about 1.5 L.

Ammonium sulfate fractionation

Solid ammonium sulfate was added to 50% of saturation (313 g/L) to the supernatant
of the cell lysate. After stirring for 10 min the solution was centrifuged for 30 min at
12,000g. To the supernatant ammonium sulfate was added to 75% of saturation (176g/L)
and stirred for 10 min. The solution was centrifuged as above and the supernatant discarded.
The pale yellow precipitate was dissolved in 200 ml of 20 mM potassium phosphate, pH 7.2,
containing 5 mM mercaptoethanol. This solution was either dialyzed against two changes of
the dissolving buffer, or passed through a Pellicon concentrator. Using either method the
salt was removed until the conductivity had fallen to 4,000 jlMHOS. A pale yellow solution
remained. Throughout the remainder of the purification all buffers contained 5 mM 2-
mercaptoethano1.

DEAE Sephadex column

A 7 em x 15 em DEAE Sephadex column was equilibrated with 20 mM potassium
phosphate, pH 7.2 at 23 °C. As the desalted enzyme solution was added, the column, a
yellow band adhered to the top. After all the enzyme solution had been added the column
was washed with another 200 ml of 20 mM potassium phosphate buffer. The enzyme was
then eluted with a linear NaCl gradient. The gradient was made with one liter of A buffer (20
mM potassium phosphate, pH 7.2) and one liter ofB buffer (100 mM potassium phosphate
and 400 mM NaCl, pH 6.4). After one-half of the gradient a yellow band of protein moves
down the column and is completely eluted by the end of the gradient. The yellow band is
followed by a brown protein. Normally complete resolution between the yellow and brown
bands is not complete. The tubes containing the yellow protein and the first part of the
brown protein were pooled, yielding about 600 ml. The proteins were precipitated by the
addition of solid ammonium sulfate to 75% of saturation (516 g/L). The solution was
centrifuged as before and the precipitate, which is now distinctly yellow, dissolved in about
50 ml of 20 mM potassium phosphate, pH 7.2. This was dialyzed overnight against 2
changes of the same buffer at 4 °C.

716
First hydroxylapetite column

A 6 em x 8 em hydroxylapatite column was equilibrated with 30 mM potassium

phosphate, pH 7 .2. The dialyzed enzyme solution was added to the column. A reddish-
brown band formed at the top of the column, but a yellow solution passed directly through
and was collected. The column was washed with the 30 mM potassium phosphate buffer
until all the yellow solution had eluted. To this solution was added solid ammonium sulfate
to 40% of saturation ( 243 giL) and pyridoxal-P to 0.2 mM.

Phenyi-Sepharose column
The yellow protein solution with 40% saturated ammonium sulfate was added to a 4 em
x 12 em column of phenyl Sepharose equilibrated with a 40% saturated ammonium sulfate
solution in 20 mM potassium phosphate and 0.2 mM pyridoxal-P, pH 7.2. A yellow band
formed at the top of the column. The proteins were eluted with a linear gradient (A buffer
was 400 ml of the 40% ammonium sulfate equilibration solution, and the B buffer was 400
ml of 20 mM potassium phosphate, pH 7.2). A yellow band moved down the column and
was eluted at the end of the gradient. A colorless protein was eluted immediately after the
yellow band of protein. Normally, base line resolution was not obtained between these two
proteins. The yellow protein fractions were collected until the absorbance at 278 nm stopped
decreasing. The fractions containing the yellow solutions were pooled and concentrated with
75% ammonium sulfate as described above. The dissolved precipitate was dissolved in a
small volume and dialyzed overnight against 20 volumes of 20 mM potassium phosphate, pH
7.2.

Second hydroxylapatite column

A 6 em x 8 em hydroxylapatite column was equilibrated with 20 mM potassium BES, pH
7 .2. The enzyme solution was also dialyzed for 4 h against this equilibrating buffer and
added to the column. A yellow band of protein formed at the top. The enzyme was eluted
with a linear gradient of 200 ml equilibration buffer and 200 ml of 50 mM potassium
phosphate, pH, 7 .2. A yellow band of protein moved down the column at the beginning of
the gradient. The absorbance at 278 nm was monitored and the first peak of yellow protein
was pooled and concentrated with ammonium sulfate. After dialysis against 20 mM
potassium phosphate the enzyme was stored in 1.5 ml vials at -70 °C. SDS-PAGE gels
showed the SHMT to be better than 95% pure. Normally about 300 mg of enzyme was
obtained for each mutant protein.

SPECTRAL AND CATALYTIC PROPERTIES OF THE MUTANT ENZYMES

The K229Q SHMT
In addition to the 278 nm absorption maximum of the protein this enzyme was yellow
and exhibited a 424 nm peak. This is characteristic of wild type enzyme with a protonated
internal aldimine. Since this mutant enzyme can not form an internal aldimine, it was shown
that acid precipitation released 0.6 equivalents of serine and 0.4 equivalents of glycine.
Therefore, the K229Q SHMT is purified with tightly bound amino acid substrates as external
aldimines. This was supported by the observation that the addition of NaCNBH3 did not
significantly alter the spectrum of the enzyme over a 15 min period. Addition of NaCNBH3
to the wild type enzyme results in a rapid reduction of the internal aldimine to a secondary
amine with a concomitant shift of the absorption maximum from 422 nm to 325 nm.
However, saturation of the wild-type enzyme with either serine or glycine to form the
external aldimine blocks reduction of the external aldimine by NaCNBH3.
The addition of D-alanine resulted in a slow decrease in the absorbance at 422 nm with a
concomitant increase in absorption at 325 nm. Dialysis left a protein with no absorption
maxima above 300 nm. This absorbance change in the presence of D-alanine is characteristic
of the wild type enzyme and is the result of the enzyme undergoing a half-transamination
reaction to yield pyruvate and pyridoxamine phosphate, which does not stick to the enzyme,
leaving apoSHMT. To the apoenzyme was added pyridoxal phosphate and either serine or
glycine. In each case the absorbance at 422 nm returned, and amino acid analysis after acid

717
precipitation confirmed that the enzyme contained one equivalent of either serine or glycine.
Addition of tetrahydrofolate to the K229Q•SHMT•Ser complex followed by acid precipitation
showed that the one equivalent of serine had been converted to glycine. Likewise, when
CH2-H4PteGlu3 was added to the K229Q•SHMT•Gly complex followed by acid
precipitation, 80% of the glycine had been converted to serine.

K229R SHMT

This mutant enzyme exhibited pyridoxal-P absorption maxima at 332 nm and 390 nm with
the 332 nm band being 1.5-fold higher than the 390 nm band. These two absorption maxima
are close to those of the pyridoxal phosphate aldehyde (388 nm) and its hydrated adduct (330
nm). Saturation of K229R SHMT with serine resulted in a decrease of the 332 nm band and
disappearance of the 390 nm band and the appearance of a new absorption band at 420 nm.
However, rapid removal of the serine by filtration at 4 °C resulted in a return to the original
spectrum within 2 min. These results suggest that this mutant enzyme is purified without
bound amino acid substrates and that the pyridoxal-P is bound as a free aldehyde (390 nm
band) and possibly a hydrated pyridoxal-P (332 nm band). The shift to 420 nm upon the
addition of serine shows that this enzyme can form an external aldimine, but it must be
unstable since removal of serine results in a rapid loss of the bound serine.
K229H SHMT

This mutant enzyme exhibits only a single pyridoxal-P absorption band at 334 nm.
Addition of serine or glycine did not alter the spectrum of the enzyme. Also, changes in pH
or the addition of NaCNBH3 did not alter the spectrum. We conclude that this mutant
enzyme has pyridoxal-P bound in a nonreactive form. No further studies were done with
this mutant enzyme.
CONCLUSIONS

The results with K229Q SHMT indicate that the active site lysine is required to release the
amino acid product, either serine or glycine, as found in other pyridoxal-P enzymes.
However, unlike aspartate aminotransferase, the active site lysine in SHMT does not appear
to be the base which removes the pro2S proton of glycine (alpha proton) to form the
quinonoid complex. This step is essential in the conversion of glycine to serine. The active
site base which accepts this proton is still not known.
ACKNOWLEDGMENT

This research was supported by Grant GM 28143 from the National Institutes of Health.
REFERENCES

1. L. Schirch, Adv. Enzymol. Relat. Areas Mol. Biol., 53:83 (1982)

2. V. Schirch, S. Hopkins, E. Villar, and S. Angelaccio, J. Bact., 163:1 (1985)
3. L. L. Ilag and D. Jahn, Biochemistry 31:7143 (1992)
4. T. Yoshimura, M. B. Bhatia, J. M. Manning, D. Ringe, and K. Soda,
Biochemistry, 31:11748
5. B. Grimm, M. A Smith, and D. vonWettstein, Eur. J. Biochem., 201:579 (1992)
6. M.D. Toney and J. F. Kirsch, J. Biol. Chern., 266:23900 (1991)
7. D. L. Smith, S.C. Almo, M.D. Toney, and D. Ringe, Biochemistry 28:8161 (1989)
8. M.D. Toney and J. F. Kirsch, Protein Science 1:107 (1992)
9. A Arnone, P. Christen, J. N. Jansonius, and D. E. Metzler, in Transaminases
(P. Christen and D. E. Metzler, Eds.) pp326-357, Wiley, New York (1985)

718
PURIFICATION OF NEUROSPORA CRASSA CYTOSOLIC SERINE

HYDROXYMETHYLTRANSFERASE

Heidi Kruschwitz, Darin McDonald, Edwin Cossins, and Verne Schirch

Department of Biochemistry and Molecular Biophysics

Box 614 MCV Station
Virginia Commonwealth University
Richmond, VA 23298

Department of Botany
University of Alberta
Edmonton, Alberta, T6G 2E9, Canada

INTRODUCTION

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of

serine to glycine with tetrahydrofolate serving as a one-carbon acceptor (Reaction 1). This
reaction is the major source of activated one-carbon groups required for the biosynthesis of
purines, thymidylate, methionine, and choline. SHMT has been purified and characterized
from many sources, including mammalian liver, prokaryotes, and several plant
species.1,2,3,4,5 The mammalian and prokaryotic enzymes require pyridoxal phosphate as a
cofactor, which exhibits specific absorption bands corresponding to different reaction
intermediates. These absorption bands have been utilized in studies to determine both
reaction specificity and the mechanism of the enzyme. The enzymes from rabbit liver and E.
coli show very broad reaction specificity, catalyzing the cleavage of various 3-hydroxy
amino acids, the transamination of D and L-alanine, and the decarboxylation of
aminomalonate.1,2 SHMT is present in both the cytosol and mitochondria of eukaryotes,
however the isozymes have been separately purified only from rat and rabbit liver. The
isozymes from these two sources appear to exhibit the same spectral and catalytic properties. I
SHMT has also been purified from several plant species. These enzymes also have a
pyridoxal phosphate bound to the active site of the purified enzyme. However, it has been
reported that the enzyme from mung bean is not a pyridoxal phosphate dependent enzyme. 5

L-Serine + I4PteGlun .. Glycine+ S,lO-CH2-H4PteGlun (1)

We report in this study the purification and characterization of cytosolic SHMT from
Neurospora crassa mycelia and compare its properties to the enzymes purified from other
sources.

PURIFICATION PROCEDURE

The enzyme does not appear to be present in high concentrations in wild type N. crass a,
which makes its purification from this strain difficult. However, studies have shown that

Chemistry and Biology of Pteridines and Folates, Edited by

I.E. Ayling et al., Plenum Press, New York, 1993 719
the met-6 mutant of N. crassa shows increased SHMT specific activity under certain
conditions. There are two folylpolyglutamate synthetase activities in the cytosol of N.
crassa. One of these activities is responsible for the synthesis of I4PteGluz derivatives from
H4PteGlu derivatives, while the other is required for the synthesis of H4PteGluz+n
derivatives. The met-6 mutant lacks the latter of these two activities, resulting in a
methionine dependency.6 Previous studies have shown that decreasing the L-methionine
concentration in the media from the optimum 0.5 mM to 0.1 mM results in a 3-fold increase
in SHMT specific activity compared to wild type N. crassa.7 Furthermore, growing wild
type N. crassa in the presence of 10 mM glycine results in a 2-fold increase in SHMT
specific activity.8 To purify SHMT, the met-6 mutant was grown in media supplemented
with 10 mM glycine and only 25 ~ D, L-methionine.

Growth of Neurospora crassa

The Neurospora crassa methionine auxotroph, met-6 (FGSC no. 1330) was maintained
on agar slants of Vogel's media containing 2% (w/v) sucrose, 2% (w/v) agar, and 1 mM
D, L-methionine. For the purification of SHMT, fresh agar slant cultures of the met-6
mutant were used to inoculate two 1 L sidearm flasks, each containing 1 L of Vogel's media
supplemented with 2% sucrose and 1 mM D,L-methionine. A sterile loop was used to
transfer an inoculum from the agar slants to the 1 L flasks. The 1 L cultures were incubated
at 30 oc with aeration for approximately 24 hours. These cultures were then used to
inoculate a 20 L carboy containing 20 L of Vogel's media supplemented with 2% sucrose, 50
J.LM D,L-methionine, 10 mM glycine, and 0.5 mM pyridoxine. Approximately 50 J.Lg/ml of
ampicillin was also included to prevent bacterial contamination. Each of the 1 L cultures was
poured into the 20 L carboy, which was then incubated at 30 °C with aeration for about 36
hours. Mycelia were harvested by pouring the 20 L culture through a colander. The mycelia
were rinsed thoroughly with distilled water and stored at -70 °C.

Cell Lysis and Heat Step

To purify the enzyme, about 450 g (wet weight) of mycelia were resuspended in 800 ml
of 40 mM dibasic potassium phosphate containing 100 mM D,L-serine, 5 mM 2-
mercaptoethanol, 0.5 mM EDT A, and 0.1 mM pyridoxal phosphate, followed by
homogenization in a Waring blender. The mycelia were lysed by passing the suspension
through a SLM Aminco French Press cell once at a pressure of approximately 20,000 psi.
After the suspension had been pressed it was heated to 65 oc and immediately cooled to 4
oc. This was followed by centrifugation at 13,000g for 30 minutes at 4 OC. The pellet was
then discarded. All remaining buffers contained 5 mM 2-mercaptoethanol and 0.5 mM
EDTA.

DEAE-Sepbadex Column

The pH and conductivity of the supernatant were adjusted to 7.2 and 3000 J.LMHOS,
respectively, and the solution was immediately applied to a DEAE-Sephadex column (7 x 13
em) equilibrated in 20 mM potassium phosphate, pH 7 .2. The column was washed with
equilibration buffer until the absorbance at 280 nm was less than 0.1. The enzyme was
eluted with a linear gradient of 1 L of equilibration buffer and 1 L of 100 mM potassium
phosphate, pH 6.5, containing 400 mM sodium chloride. The eluant was collected in 20 ml
fractions that were assayed for SHMT activity using a coupled enzymatic assay. The SHMT-
catalyzed aldol cleavage of serine with I4PteGlun to glycine and 5,10-CHz-H4PteGlu was
measured at 340 nm by coupling with the 5,10-CHz-tetrahydrofolate dehydrogenase activity
of C1-tetrahydrofolate synthase, which converts NADP+ to NADPH. Fractions containing
activity were pooled, and the protein was precipitated by addition of solid ammonium sulfate
to 75% of saturation (516 g/L). This suspension was centrifuged at 13,000g for 20
minutes, and the pellet was resuspended in equilibration buffer. The solution was then
dialyzed overnight against equilibration buffer.

720
Blue-Sepharose Column
The next day, the dialysate was applied to a Blue-Sepharose column (7 x 4 em)
equilibrated with 20 mM potassium phosphate, pH 7.2. The column was washed with
equilibration buffer until the absorbance at 280 nm was less than 0.1. The enzyme was then
eluted with 20 mM potassium phosphate, pH 7.2 containing 1 M sodium chloride. The
eluant was collected in 20 ml fractions that were assayed for enzyme activity using the
coupled enzymatic assay. Fractions containing SHMT were pooled and ammonium sulfate
was added to 40% of saturation (243 giL). This solution was then centrifuged at 13,000g for
20 minutes at 4 oc and the pellet was discarded.
Phenyi-Sepharose Column
The supernatant from the 40% ammonium sulfate fractionation was immediately applied
to a Phenyl-Sepharose column (3 x 12 em) equilibrated in 50 mM potassium phosphate, pH
7.2, containing 40% ammonium sulfate and 0.1 mM pyridoxal phosphate. The enzyme was
eluted with a linear gradient of 250 ml of equilibration buffer and 250 ml of 20 mM
potassium phosphate, pH 7.2. The enzyme elutes near the end of the gradient. The eluant
was collected in 10 ml fractions that were assayed for activity using the coupled enzymatic
assay. Fractions containing activity were pooled, and the protein was precipitated by the
addition of ammonium sulfate to 75% of saturation. The precipitate was then dissolved in 20
mM N, N-bis-(Hydroxyethyl)-2-aminoethanesulfonic acid (BES), pH 7.2, and exhaustively
dialyzed against this buffer.
Hydroxylapatite Column
The dialyzed enzyme was applied to a hydroxylapatite column (3 x 3 em) equilibrated
with 20 mM BES, pH 7.2. After thoroughly washing the column with equilibration buffer,
the adsorbed SHMT was eluted with a linear gradient of 200 ml of equilibration buffer and
200 ml of 50 mM potassium phosphate, pH 7.2. Fractions containing activity were pooled,
and the enzyme was precipitated by the addition of ammonium sulfate to 75% of saturation.
The precipitate was resuspended and dialyzed against an appropriate buffer.
The enzyme obtained from the hydroxylapatite column is greater than 95% pure based
on SDS-PAGE analysis. The overall purification is 746-fold with a 30% yield (Table I).
Approximately 50 mg of enzyme was obtained from 450 g (wet weight) of mycelia.

Table I. Purification of N. crassa serine hydroxymethyltransferase

Purification Step Vol (ml) mg/ml Total Units Specific Activity Fold %Yield
(Jlmol/min) (Jlmol/min/mg) Purification

French Press 1930 75.0 9650 0.07 1 100

Heat Step 1760 11.8 8448 0.41 6 87
DEAE-Sephadex 61 11.4 4117 5.9 84 43
Blue-Sepharose 196 0.62 2450* 21 300 25
Phenyl Sepharose 96 0.94 3360 37 552 35
Hydroxylapatite 55 0.96 2772 50 746 29

*The low activity after this step could be due to high concentrations of sodium chloride.

721
DISCUSSION

The most important question we wished to address was how N. crassa SHMT
compares to the mammalian SHMTs purified and characterized from other organisms. The
overproduction of SHMT by the met-6 mutant of N. crassa allowed for a simple and rapid
purification of the enzyme. Partial sequencing of the pure enzyme was carried out by a group
of collaborators at the University of Rome. However, during this process the sequence for
the cloned N. crassa cytosolic SHMT gene was published.9 Since the partial sequence we
obtained was identical to the published sequence, we concluded that we had purified the
cytosolic form of the enzyme. This sequence was also used to obtain a calculated extinction
coefficient of 37,500 M-lcm-1_10
Like the mammalian enzymes, N. crassa SHMT is a tetramer of identical 54 kDa
subunits. This was determined using SDS-PAGE analysis and agrees with the predicted
amino acid sequence obtained from the cloned cytosolic SHMT gene, which would result in a
protein with a molecular weight of 53 kDa.9 Molecular sieve chromatography experiments
using a TSK G4000 HPLC column result in a native molecular weight value of 230 kDa,
indicating a tetramer of identical subunits. These results are similar to those obtained for the
rabbit isoenzymes which are also tetramers of identical 54 kDa subunits.ll
The bound pyridoxal phosphate of the N. crassa enzyme displays the same spectral
properties as the enzyme from other sources. Furthermore, the enzyme shows the same
reaction specificity, catalyzing the cleavage of serine, allothreonine, and 3-phenylserine, as
well as the transamination of D-alanine. The enzyme also displays absorption maxima at
either 492 nm or 502 nm due to the SHMT•Gly•R4PteGlun and SHMT•Gly•5-CHO-
H4PteGlun ternary quinonoid complexes, respectively. As with the rabbit liver cytosolic
enzyme, N. crass a SHMT is stabilized to heat denaturation by the polyglutamate forms of 5-
CHO-H4PteGlun. Although the dissociation constants for folylpolyglutamate derivatives
have not been determined, these studies indicate that the N. crassa enzyme probably has a
polyglutamate binding site, and thus a much higher affinity for polyglutamate folate
derivatives. Like the rabbit liver enzyme, N. crassa SHMT also catalyzes the synthesis of 5-
CHO-H4PteGlun from 5,10-CH+-H4PteGlun in the presence of glycine. Both enzymes
show biphasic kinetics consisting of a rapid burst phase followed by a much slower rate.
Stopped-flow kinetic studies have shown that 5-CHO-IilPteGlun polyglutamates are slow,
tight-binding inhibitors of the N. crassa enzyme. Therefore, like the rabbit liver enzyme, the
N. crassa enzyme can synthesize its own inhibitor in the presence of glycine.

ACKNOWLEDGMENTS

This work was supported by Grant GM 28143 from the National Institutes of health.

REFERENCES
1. L. Schirch, in "Folates and Pterins: Chemistry and Biochemistry of Folates" (R. L.
Blakley and S. J. Benkovic, eds) Vol. 1, p. 399-432. Wiley-Interscience, New York
(1984).
2. V. Schirch, S. Hopkins, E. Villar and S. Angelaccio, J. Bacterial. 163:1-7 (1985).
3. D. Henricson and I. Ericson, Phys. Plantarum 74:602-606 (1988).
4. M. Sakamoto, T. Masuda, Y. Yanagimoto, Y. Nakano and S. Kitaoka, Agric. Bioi.
Chern. 55:2243-1149 (1991).
5. N. Sukanya, M. Vijaya, H. S. Savithri, A. N. Radhakrishnan and N. A. Rao, Plant
Physiol. 95:351-357 (1991).
6. E. A. Cossins and P. Y. Chan, Phytochemistry 23:965-971 (1984).
7. E. G. Burton and R. L. Metzenberg, Arch. Biochem. Biophys. 168:219-229 (1975).
8. E. A. Cossins, P. Y. Chan and G. Combepine, FEBS Letters 54:286-290 (1975).
9. C. R. McClung, C. R. Davis, K. M. Page and S. A. Denome, Mol. Cell. Bioi. 12:1412-
1421 (1992).
10. H. Edelhoch, Biochemistry 6:1948-1954 (1967).
11. L. Schirch,Adv. Enzymol. Relat. Areas Mol. Biol. 53:83-112 (1982).

722
PURIFICATION AND PROPERTIES OF RABBIT LIVER

5,10-METHENYLTETRAHYDROFOLATE SYNTHETASE

Patrick Stover, Teng Huang, Verne Schirch, Bruno Maras, Sofia Valiante and
Donatella Barra

Department of Biochemistry and Molecular Biophysics

Box 614, MCV Station
Virginia Commonwealth University
Richmond, VA 23298

Dipartimento di Scienze Biochimiche and Centro di Biologia Molecolare del

Consigilio Nazionale delle Ricerche
Unviersita La Sapienza
00185 Rome, Italy

INTRODUCTION

The only known enzyme to use 5-formyltetrahydrofolate and its polyg1utamate forms (5-
CHO-H4PteGlun) is 5,10-methenyltetrahydrofolate (5,10-CH+-H4PteGlun) synthetase.1,2
This enzyme catalyzes the ATP dependent reaction shown in Equation 1. All attempts to
demonstrate the reverse reaction have failed, suggesting that this reaction is functionally
irreversible. Since 5-CHO-H4PteGlun and 5,10-CH+-H4PteGlun can be reversibly
interconverted nonenzymatically, the role of ATP in this reaction is apparently to shift the
equilibrium far to the right. It has been proposed that methenyl-tetrahydrofolate synthetase is
a salvage enzyme which converts the nonenzymatically formed and nonmetabolically active
5-CHO-R4PteGlun back into the one-carbon donor folate pool.l However, it is also possible
that methenyltetrahydrofolate synthetase is part of a regulation system in the cell in which its
substrate 5-CHO-H4PteGlun plays a role as a regulator of one-carbon metabolism by
inhibiting other folate requiring enzymes.

5-CHO-R4PteGlun + Mg-ATP .. 5,10-CH+-R4PteGlun + Mg-ADP +Pi (1)

This enzyme has been purified from both eucaryotic and procaryotic sources, and in
each case is a monomeric protein about 28 kDa in size.l-3 The most extensive studies have
been done with the rabbit liver enzyme. I The properties of this enzyme relate to its putative
function in the cell. Although the enzyme is present in cells at low concentrations, a rapid
purification procedure has been developed which permits obtaining 15 to 20 mg of
homogeneous enzyme from 15 rabbit livers in 3 days. This purification procedure is
different than the published procedure and will be given in detail in the following paragraphs.
A key to the new purification procedure was the observation that the enzyme is stabilized by
non-ionic detergents like Tween-20. The presence of this detergent at concentrations as low
as 0.1% permits long term storage at -70 OC without loss of activity.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 723
PURIFICATION PROCEDURE

Homogenate and polyethylene glycol (PEG) precipitation

Fifteen frozen rabbit livers were broken into small pieces by placing them in a plastic
bag and fracturing with a hammer. After partial thawing the liver pieces were placed in a
blender and homogenized with 1500 ml of homogenization buffer (20 mM dipotassium
phosphate, 20 mM 2-mercaptoethanol, 12% (v/w) polyethylene glycol 3350 (PEG)).
Homogenization was performed for 45 seconds and the resulting slurry centrifuged at
10,000g for 25 min. The pellet was discarded and the volume of the supernatant recorded.
The supernatant was brought from 12% to 40% PEG by the addition of solid PEG. After
stirring for a few min the slurry was again centrifuged for 25 min at 1O,OOOg. The pellet
contains the trifunctional enzyme CHetrahydrofolate synthase, which can be purified to
homogeneity by a previously published procedure. However, the methenyltetrahydrofolate
synthetase activity was in the supernatant. The volume of the supernatant was between 1 and
1.5 L. If lipid debris was floating on the supernatant, it was passed through glass wool.

DE-52 cellulose column

DE-52 cellulose was added to a 10 em x 40 em column containing a course glass frit

until the bed volume of the cellulose was 7.5 em high. The column was equilibrated with 50
mM potassium phosphate, pH 7.6, containing 20 mM 2-mercaptoethanol. The 40% PEG
supernatant was gently added and allowed to slowly drip through the column overnight.
This is best performed at room temperature. The methenyltetrahydrofolate synthetase is
stable in the 40% PEG, and the PEG keeps the column from cracking when it runs dry so
that one can let the column finish overnight. The methenyltetrahydrofolate synthetase activity
sticks to the column.
The following morning methenyltetrahydrofolate synthetase activity was eluted. Two
liters of 10 mM potassium phosphate, pH 7.2, containing 20 mM 2-mercaptoethanol and
0.5% Tween-20 were gently added in 50 ml aliquots to the top of the column and 50 ml
fractions were collected. The synthetase activity began to elute after about 200 ml of buffer
had passed through the column. Once activity began to elute, 100 ml fractions were collected
and each fraction assayed for methenyltetrahydrofolate synthetase activity. All fractions
containing activity were pooled, resulting in about 1 to 1.5 liters of solution.

S-CHO-H4PteGiu affinity column

The pooled fractions from the DE-52 column were applied directly to a 2 em x 8 em
column of 5-CHO-f4PteGlu linked to Sepharose and equilibrated with the elution buffer
used for the DE-52 column. This column was kept at 4 °C and the DE-52 supernatant was
allowed to pass through the column. This normally took 10 to 12 hours. Again, the column
does not crack if all of the supernatant passes through the column, permitting the column to
be run overnight.
After all the DE-52 supernatant had passed through the affinity column, it was washed
with 300 ml of 10 mM potassium phosphate, pH 7.2, containing 20 mM 2-mercaptoethanol,
0.5% Tween-20, and 500 mM NaCl. This was followed by washing with another 300 ml of
this buffer, but lacking the 500 mM NaCl. Methenyltetrahydrofolate synthetase was eluted
by adding the washing buffer containing 100 JlM (6RS) 5-CHO-H4PteGlu. Twenty ml
fractions were collected and aliquots assayed for synthetase activity. The active fractions
were pooled, resulting in a volume of about 350 ml.

Hydroxylapetite column

Active fractions from the 5-CHO-~PteGlu affinity column were applied to a 2 em x 10

em column containing hydroxylapetite equilibrated with 5 mM potassium N,N-bis-
(hydroxyethyl)-2-aminoethanesulfonate (KBES), pH 7.2. The methenyltetrahydrofolate
synthetase passed through the column and was collected in 50 ml fractions. After all the
enzyme solution had passed through the column, it was washed with 5 mM KBES, pH 7.2,
until all activity had eluted. The final volume was usually about 500 ml.

724
 This synthetase solution was very dilute and contained both excess 5-CHO-f4PteGlu and
Tween-20. Each of these molecules stabilize the synthetase. Concentration of the dilute
solution can be accomplished by several methods. One method used was to lyophilize the
solution and redissolve the dried paste in a small volume of KBES buffer. The excess 5-
CHO-H4PteGlu and Tween-20 were removed by passing the enzyme solution through a
Sephadex G-25 column (3 em x 20 em) equilibrated with 20 mM potassium phosphate, pH
7 .2, 5 mM 2-mercaptoethanol and 0.1% Tween-20. An alternative method used to
concentrate and remove the excess 5-CHO-H4PteGlu and Tween was to dialyze the dilute
synthetase solution against 20 mM potassium phosphate, pH 7 .2, under a vacuum until the
volume was reduced to less than 15 mi. The enzyme was then further dialyzed to remove
additional5-CHO-R4PteGlu and Tween. However, this final dialysis buffer contained 0.1%
Tween-20. The final enzyme preparation was judged to be pure by SDS-PAGE
electrophoresis. Two closely spaced bands were sometimes found on the stained gel, but
both were due to methenyltetrahydrofolate synthetase.

DETERMINATION OF THE AMINO ACID SEQUENCE

The two bands that were sometimes found on the SDS-PAGE gels were sequenced and
shown to be the same enzyme differing only by the presence of an Ala residue at the
aminoterminus of the lower running band. Using a variety of cleavage methods, including
trypsin, chymotrypsin, and cyanogen bromide, sets of overlapping peptides were sequenced
by the Edman method. The sequencing results suggest that most of the purified enzyme is
blocked at the aminoterminus. However, our longest protein sequence is probably missing
only 2 or 3 residues at the aminoterminus. There is also some ambiguity in the sequence of a
few residues at the carboxylterminus. The complete amino acid sequence has been
determined except for these few residues at the carboxyterminus and the blocked
aminoterminus.
Analysis of the predicted secondary structure by the methods of Gamier et al., and Chou
and Fasman, suggests that methenyltetrahydrofolate synthetase is about 40% alpha helix,
23% extended conformation, 22% turns, and 15% coiJ.4,5 Analysis of its hydrophobicity by
the method of Kyte and Doolittle, shows that the enzyme is mostly hydrophilic.6 The
hydrophilic character of the enzyme does not explain the observation that Tween-20 helps
solubilize and stabilize the enzyme.

PROPERTIES OF METHENYLTETRAHYDROFOLATE SYNTHETASE

Molecular sieve chromatography shows that the synthetase is a monomer protein with
a molecular weight of about 28 kDa. It has a broad substrate specificity with respect to the
trinucleotide, with ATP and CTP exhibiting the same activity. Mg(II) is required in
equivalent concentration with ATP, suggesting that its only role in this reaction is to form a
complex with the substrate. Other divalent metal ions, such as Co(ll), Mn(II), and Ca(II),
also give full activity in place of Mg(II). The enzyme activity is largely located in the cytosol,
although small amounts of activity (less than 10%) could be located in the mitochondria.
The enzyme catalyzes a random sequential mechanism. The affinity for substrates
show that the Krn for MgATP and 5-CHO-H4PteGlu are 0.3 mM and 0.5 11M, respectively.
The kcat for this enzyme is 300 min-I at 30 oc and pH 6.0.

CONCLUSIONS
Even though cell homogenates of rabbit liver contain only small amounts of
methenyltetrahydrofolate synthetase, the activity of this enzyme is high and can be readily
measured. We first proposed that this enzyme probably served as a salvage pathway for
converting 5-CHO-H4PteGlu back to a metabolically active form of one-carbon folate
compounds. I This is based on the observation that no enzyme has yet been found which
uses 5-CHO-R4PteGlu as a one-carbon donor. The origin of cellular 5-CHO-f4PteGlu was
thought to be the nonenzymatic hydrolysis of 5,10-CH+-H4PteGlu to 5-CHO-H4PteGlu.
However, this is a slow reaction, and with the high activity of the synthetase there should be
no accumulation of 5-CHO-H4PteGlu in the cell. Recently we have shown that serine
hydroxymethyltransferase catalyzes the hydrolysis of S,10-CH+-H4PteGlu to 5-CHO-

725
I4PteGlu in an irreversible reaction.? Together, the methenyltetrahydrofolate synthetase and
serine hydroxymethyltransferase reactions result in a futile cycle in the interconversion of
5,10-CH+-H4PteGlu and 5-CHO-H4PteGlu. Futile cycles are often part of a regulation
mechanism for controlling the cellular level of an important metabolite. Others have shown
that cellular concentrations of 5-CHO-f4PteGlu in mammalian cells represent about 10% of
the total folate pool, and in at least one case, raising this level two-fold results in an 80%
decrease in cell growth.8,9 These observations suggest that 5-CHO-H4PteGlu may be a
regulator of one-carbon metabolism and that methenyltetrahydrofolate synthetase is part of
the regulation system for maintaining its cellular concentration. We have also observed that
5-CHO-H4PteGlun polyglutamates are slow tight binding inhibitors of SHMT and that in
liver the concentration of SHMT is greater than the concentration of 5-CHO-f4PteGlun.10
This suggests that in vivo most of the 5-CHO-f4PteGlun is bound and not readily available
to methenyltetrahydrofolate synthetase as a substrate. This could account for why there is a
pool of 5-CHO-f4PteGlun in most cells.
ACKNOWLEDGMENT

This research was supported by Grant GM 28143 from the National Institutes of Health.
REFERENCES

1. S. Hopkins and V. Schirch, V. J. Bioi. Chern. 259:5618 (1984).

2. C. E. Grimshaw, G. B. Henderson, G. G. Soppe, G. Hansen, E. Mathur, and
F. M. Huennekens, J. Bioi. Chern. 259:2728 (1984)
3. R. Bertrand, R.E. MacKenzie, and J. Jolivet, J., Biochern. Biophys. Acta 911:154
(1987)
4. J. Garnier, D. J. Osguthorpe, and B. Robson, J. Mol. Bioi. 120:97 (1978)
5. P. W. Chou and G. D. Fasman, Annu. Rev. Biochem. 47:251(1978)
6. J. Kyte and R. F. Doolittle, J. Mol. Bioi. 157:105
7. P. Stover and V. Schirch, J. Bioi. Chern. 265:14227 (1990)
8. D. W. Home, D. Patterson, and R. J. Cook, Arch. Biochem. Biophys. 270:729
(1989)
9. R. Bertrand and J. Jolivet, J. Bioi. Chern. 258:8843 (1989)
10. P. Stover and V. Schirch, J. Biol. Chern. 266:1543 (1991)

726
 FOLATE METABOLISM IN PREGNANCY

J. M. Scott, J. McPartlin, A. Molloy, H. McNulty, A. Halligan,

M. Darling and D.G. Weir

Departments of Biochemistry and Clinical Medicine

Trinity College Dublin
Rotunda Hospital Dublin

INTRODUCTION
Folate plays a crucial and indispensible role in cell division so it is not
surprising that it is important in pregnancy. This importance was first
highlighted by the historical observation made by Lucy Wills in India 1 that the
latter stages of pregnancy are frequently associated with a megaloblastic anaemia
that is folate responsive, i.e. in pregnancy not only is extra folate needed but it is
frequently lacking. Subsequent studies over the years have found that folate
deficiency in the second and particularly the third trimester is common in some
countries and quite rare in others. The determining feature was found to be
adequate folate nutrition for the mother not only during pregnancy but before the
pregnancy began2. The most often suggested mechanism for folate deficiency in
pregnancy is increased requirement for the rapidly growing fetus and placenta.
However the mechanism is unclear and certainly cannot be accounted for by
transfer of the vitamin to the fetus. One possible explanation is that the events of
fetal/placental growth cause an increase in the rate of catabolism of the vitamin.
Early studies in our laboratory demonstrated that the mechanism of folate
catabolism in the rat was cleavage of the C9-N10 bond with excretion of a mixture
of pteridines and p-aminobenzoylglutamate (pABGlu) with the latter being largely
acetylated to acetamidobenzoylglutamate (apABGlu)3. Subsequent studies by us in
the rat4 and by Krumdieck 5 in man confirmed this mechanism. These studies
used radioactive tracers. They had the disadvantage that they assumed that the
exogenous tracer would equilibrate with the endogenous pool given sufficient
time. In addition while they could be used to compare rates of catabolism in
specific circumstances between animals, say treated with convulsant drugs and
controls 6 , they did not measure true endogenous rates of catabolism.
Furthermore, since they used radioactive tracers they were unsuitable for routine
studies on humans.

Recently we have developed methods to determine the rate of catabolism of

endogenous folate by HPLC both in the rat 7 and in mans. Using these methods we
have determined the rate of catabolism in normal and pregnant rats. We have
also carried out a more limited study on pregnancy in women.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et at., Plenum Press, New York, 1993 727
 The above considerations of folate deficiency in late pregnancy should not be
confused with the newly demonstrated role of folic acid and folate in the aetiology
of neural tube defects (NTDs) such as Spina Bifida and Anencephaly. The
protective effect of folic acid in this context is specific to the period of the closure of
the neural tube between 24 and 28 days after conception. At this point the fetus is
small and the folate related event would be unlikely to be due to increased
catabolism producing an increased requirement. Rather the effectiveness of such
folic acid would appear to be due to (i) treating a state of folate deficiency in some
women, (ii) supplying extra folate which some women need to carry out a folate
related event involved in the closure of the neural tube (iii) a combination of(i) and
(ii).

MATERIALS

The materials used were as described in the two papers recently published
on the method for analysis of folate catabolism in rat urine 7 and in human
urine 8.

METHODS
Exact details of the HPLC procedures used to quantitate the folate catabolites
in rat urine 7 and human urine8 were as previously described.

Three groups of six rats were compared.

(1) Non pregnant rats : (2) Pregnant rats pair fed against these non-pregnant
controls : (3) Pregnant i·ats freely fed. The rate of catabolism was followed for a
total of 30 days to include a base line value before pregnancy and the period up to
post partum. Student's t-test for unpaired data was used to compare the mean
apABGlu excretion and weight data with each of the non-pregnant groups. P<
0.05 was considered significant. Results were expressed as mean ± SEM.

In the human studies six normal parous volunteers were followed for their
catabolite excretion from their first booking clinic until post partum. They
consumed a complete liquid enteral feed (Fortisip by Cow and Gate) for an 18 hour
wash out period, followed by a 24 hour collection period. Six non pregnant healthy
women of the same age consuming the same diet were used as controls.

RESULTS
As can be seen from Figure 1, both groups of pregnant rats showed
increases in weight reaching significance just before parturition at 21 days. The
excretion of the folate catabolite apABGlu showed elevation in both pregnant
groups compared to the non pregnant control group (Fig. 2). This was significant
by day 6 in the Pregnant Free-Fed group and by day 12 in the Pregnant Pair-Fed
Group. In both groups the values peaked at day 18 and most importantly fell
before parturition with day 18 being significantly greater than day 20 in both
groups. This drop in the rate of catabolism in late pregnancy (Fig. 2), where
weight continued to increase (Fig. 1), made weight gain alone an improbable
explanation of these results.

To further explore this relationship the weights of these rats were monitored
throughout pregnancy (Fig. 1). Non pregnant rats of equal weight were examined
at these different times to see what level of catabolite they were excreting. These
rats were of course older to achieve a similar weight. This comparison showed a
marginal increase in apABGlu excretion with increasing weight (0. 7 nmol per
lOg increase in weight) in non pregnant animals (Fig. 3).

728
 350
PREGNANCY
0 Non·pregnant Fr1HHed
• Pregnam Pa1r-fcd
• Pregnant Free~ led
•• P<0.01
300

§
:c01 250
~

200

150~------~----~----~------~----~
-7 0 7 14 21 28
Days

Figure 1. The increase in weight over the 30 day period of the study in the three groups indicated

60 PRE G NANCY
0 Non-pregnant Free--fed

• Preg.nant Paw·fed
50 • Pregnant Ftee·fed
p <0.05

'0 .. p <0.01
p <0.001
"§
40
'6
E
c:
::J
<.? 30
co
~
a.
ttl

20

10 6 22
-2 2 10 14 18 26 30

Days

Figure 2. The excretion of the folate catabolite apABGlu over the 30 day period of the study in the
three groups indicated.

729
 50
-- Pregnant Pa~r·fed
-.- Pregnant Free-led
- - D-- Non-pregnant we1ght matched

-- -o-- (To - - - and ----- )

40

30

20

10
200 220 240 260 280 300

Weight (g)

Figure 3. The excretion of the folate catabolite apABGiu over the 30 day period of study in the final
group indicated.

In the study on human pregnancy there was a significant increase in excretion of

catabolite in the pregnant women during their 2nd and 3rd trimester when
compared to values for the same women in their 1st trimester or with their post
partum values or with the six non-pregnant controls (Fig. 4). Again as in the rat
study the rate of catabolism fell before the end of pregnancy and was reflected in a
significantly greater excretion in the 2nd trimester when compared to the 3rd
trimester.

300
• Tot:tl
liD apABGiu
pABGiu

0 folate
200

DAILY FOLATE
BREAKDOWN
(~g fol ate equi\'alents)

100

NON-
I'REG ANT
1st 2nd 3rd I POST
J>ARTUM
TRIMESTERS

Figure 4. The excretion of folate catabolites apABGiu and pABGlu, intake folates added to give total
loss of folate in six pregnant women. Also included are their postpartum excretion rates and
excretion of a group of six normal non pregnant control women.

730
DISCUSSION
The increases shown in folate catabolism would appear to be related to
pregnancy itself and not merely be a reflection of the increase in weight associated
with the latter stages of pregnancy.

In the rat model it was clear that non pregnant rats of weights comparable
to the weights found in late pregnancy excreted far less catabolite than the
pregnant counterpart (Fig. 3). Furthermore in both the rat and the human study
there was a statistically significant reduction in catabolism prior to parturition
(Fig. 2,4). Thus in the face of continuous weight gain at the end of pregnancy the
rate of catabolism actually falls. These decreases coincide in both species with a
time when there is a shift from hyperplastic growth to hypertrophic growth9,IO.
This latter finding may give a clue as to the nature of the increased rate of
catabolism seen in pregnancy. It would appear to be caused by a pregnancy
related event that diminished before parturition. An attractive possibility to
explain the initial increase in catabolism and the drop would be the rate of DNA
biosynthesis. A further attraction of this model is that uniquely amongst all folate
dependent reactions DNA biosynthesis produces the most labile of all of the folate
cofactors, namely dihydrofolate. The increased need to generate this labile species
may have a chemical price for the body, in that it may be more susceptible to
catabolism. Provided adequate dietary folate is available to replace this loss the
animal will suffer no disadvantage. However, should initial stores in pregnancy
be low or the amount recommended for ingestion in pregnancy (RDA) be
inadequate, then damage to the health of the mother and the embryo may ensue.
In this context the levels of inescapable catabolism found in our human subjects
may not be catered for by existing recommendations for pregnancy. These values
for the US call for 400 J.Lg per day in pregnancyll and the EC is shortly to come out
with a similar recommendation (in press). Slightly higher values of 370-470J.Lg
have been recommended by the WHO/FAO (1988)12.

More recent recommendations that have emerged from the U.K. suggesting
that a total intake of 300 J.Lg per day can cater for the increased needs of
pregnancy13 seem seriously low.

Allowing for 50% bioavailability and two standard deviations of the mean
level of catabolism, in excess of 600J.Lg per day would seem more appropriate.

REFERENCES

1 Wills, L., 1931 Treatment of "pernicious anaemia of pregnancy and tropical

anaemia" with special reference to yeast extract as a curative agent, B r.
Med. J. 1 : 1059.
2 Chanarin, I., 1990, "The Megaloblastic Anaemias" Blackwell Scientific
Publications, Oxford.
3 Murphy, M., Keating, M., Boyle, P., Weir, D.G. and Scott, J.M. 1976, The
elucidation of the mechanism of folate catabolism in the rat. Biochem.
Biophys. Res. Comm. 71 : 1017
4 Scott, J.M., McPartlin, J., Geoghegan, F., Courtney, G., McNulty, H., and Weir,
D.G., 1989, Folate catabolism in rat and man : investigation of short-and
long-term components of catabolite excretion. In Chemistry and Biology of
Pteridines, Curtius H.C. ed. Walter de Gruyter and Co. Berlin.
5 Krumdieck, C.E., 1978, A long-term study of the excretion of folate and pterins
in a human subject after ingestion of14C folic acid with observations on the
effects of diphenylhydantoin administration. Ann. J. Clin. Nutr. 31: 88.

731
 6 Kelly, D., Weir, D.G., Reed, B. and Scott, J.M., 1974, The effect of anticonvulsant
drugs on the rate of folate catabolism in mice. J. Clin. I nuest. 64 : 1089.
7 McNulty, H., McPartlin, J., Weir, D., and Scott, J.M., 1993, Reversed-phase
high-performance liquid chromatographic method for the quantitation of
endogenous folate catabolites in rat urine. J. Chromat. (In Press).
8 McPartlin, J., Courtney, G., McNulty, H., and Weir, D.G., 1992, The
quantitative analysis of endogenous folate catabolites in human urine.
Anal. Biochem. 206: 256.
9 Winick, M. and Noble, A. 1965, Quantitative changes in DNA, RNA and protein
during prenatal and postnatal growth in the rat. Deul. Biol. 12:451.
10 Winick, M., Brasel, J.A., and Rosso, P., 1972, Nutrition and cell growth. In :
Winick M. ed.Nutrition and cell growth, John Wiley, New York.
11 National Research Council, 1989, Recommended dietary allowances. National
Academy Press, Washington, D.C.
12 WHO/FAO 1988, Requirements of Vitamin A, iron, folate and vitamin B12 .
Rome : Food and Agricultural Organisation of the United Nations.
13 COMA, 1991, Dietary reference values for food, energy and nutrients for the
United Kingdom. COMA Report 41 H.M. Stationery Office London.

732
INFLUENCE OF GESTATION AND LACTATION ON THE LEVELS OF
PLASMA FOLATES IN SOWS

Masahiro Natsuhori, Ei-ichi Kokue and Minoru Shimada

Department Veterinary Medicine

Faculty of Agriculture
Tokyo University of Agriculture and Technology
Fuchu, Tokyo 183, Japan

INTRODUCTION

Since the folate level decreases in plasma before its depletion, and this decrease in
plasma is paralleled by a reduction in red blood cell folate, the levels in plasma are
important clinical indicators that reflect folate status of the body of many mammalian
species. 1' 2 Pregnancy is one of the important factor that is associated with negative folate
balance, including progressively reduced serum and erythrocyte folate levels, and increased
urinary folate excretion. 1 N5-methyltetrahydrofolic acid (CH3-H4PteGlu) generally
comprises most or all of the plasma folate in many mammalian species. Recently,
however, tetrahydrofolic acid (H4PteGlu) was found to exist as the primary form of folate
in the plasma of pigs,3 suggesting interspecies differences in folate metabolism.
This study was undertaken to investigate the plasma levels of maternal folates during
gestation and lactation in sows. The levels of H4PteGlu and CH3-H4PteGlu were
determined simultaneously by using high-performance liquid chromatography with
electrochemical detection (HPLC-ECD). 4 Then the influence of gestation and lactation on
the levels of plasma folates, and specificity of folate metabolism in pigs will be discussed.

MATERIALS AND METHODS

Forty commercial pigs (Large White, body wt 120-150 kg, 2-4 yr old) which were
housed in individual gestational cages with slatted floors on a commercial farm (Miyata
Pig Farm, Asamizodai, Kanagawa, Japan) were used. Pigs were fed daily 2.5 kg of
commercial basal diet which contains folates about 0.5 mglkg feed, as described
previously. 3
Blood was taken from tail vein in EDTA-coated test tubes before and during
gestation, and during lactation. Plasma samples were obtained by centrifugation at 2000
g for 5 min. Sodium ascorbate (3 mglml plasma), an antioxidant, was added to the

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 733
samples and the mixtures were kept at -20°C until folate analysis. Plasma folates were
simultaneously determined by using HPLC-ECD, as described by Shimoda.4 Folate
standards (H4PteGlu and CH3-H4PteGlu) were from Sigma Chemicals (StLouis, MO). All
other reagents for the analysis were from Wako Pure Chemicals (Osaka, Japan).
Student's t-test for grouped data was used for the comparison of the levels of before,
during and after gestation, and during lactation.

RESULTS AND DISCUSSION

Figure 1. shows plasma levels of H4PteGlu and CH3-H4PteGlu during gestation.

Almost all animals showed decrease of plasma folates during gestation. Although levels
of plasma H4PteGlu distributed in wide range, most of the levels dropped drastically.
While the levels of CH3-H4PteGlu distributed in narrow range, and their changes were
rather little during gestation.

30 H4 PteGiu CHTH 4 PteGiu

-
E
:::::,
0
E 20
3
c..i
c
0
(.)

Cll
E 10
([)
Cll
0:::

0 30 60 90 120 0 30 60 90 120
Days of gestation

Figure 1. Individual levels of plasma folates (pmoVml) during gestation (day 1 to 117) in 16 sows. Lines
are drawn between the levels of the same animal.

Figure 2. shows plasma levels of H4PteGlu and CH3-H4PteGlu before gestation, early
gestation (day 1 to 60), late gestation (day 61 to 117), and during lactation in sows. As
pregnancy advanced, the levels of H4PteGlu decreased significantly. At the late gestation,
the mean level of H4PteGlu decreased to about 50 % of the level before gestation, which
was contrast to the little changes observed in the levels of CH3-H4PteGlu. During
lactation, plasma level of H4PteGlu was increased but not up to the level before gestation.
In the previous study, 3 most of the examined animals including humans, rats, mice,
horse, dogs and cows had no or trace (less than 1 pmol/ml) amounts of H4PteGlu in
plasma. And only CH3- H4PteGlu was detected in these species, which was quite contrast
to pigs.

734
 Decreases of plasma folates during gestation have been reported in several species
including humans,5- 7 rats, 8 guinea pigs,9 and pigs.10 Matte et al. 10 observed a biphasic
decrease of total folates level in serum during gestation in sows. This study also
demonstrated the progressive decrease of plasma H4PteGlu during gestation. However no
statistical significance was observed among the levels of CH3-H4PteGlu throughout the
reproductive cycle. This result highly suggests the specificity of homeostasis of plasma
folates in pigs, since the other species have CH3-H4PteGlu as the principal congener in
plasma and the level is generally considered to decrease during pregnancy. 14

:€0
E
s
u
c
0
(.)
ell
E
~
0::
before early late
gestation gestation gestation lactation

Figure 2. Plasma levels of H4PteGlu and CH3-H4PteGlu before gestation (n=16), early gestation (n=28),
late gestation (n=29), and during lactation (n=15) in sows. Values are presented by mean +standard error.
*:
t,:l: : Significant vs before gestation ( t ; p<O.l, :1: ; p<O.Ol). Significant vs late gestation (p<O.Ol).

A relation between maternal folate status and reproductive performance is reported

to exist in several species including humans,5- 7 rats, 8 guinea pigs,9 and pigs.n-B It has
been considered that folate requirement increases during pregnancy. 14 In humans, daily
doses of 1 mg or less of folic acid are considered to be adequate for the treatment of
severe nutritional megaloblastic anemia or pregnancy-induced anemia.6"15 In pigs, it is
reported that dietary supplementation or intramuscular injection of folic acid during
gestation and lactation improved reproductive performance by preventing the decrease in
plasma levels and increasing the number of pigs born alive per litter.u-!3 The
improvement in litter size is considered to be a result of increased embryo survival during
the first 30 days of gestation. 16 With regard to a bioavailability of folic acid, Kokue et al. 17
recently observed much better bioavailability of folic acid that reflected significant
increases in the levels of plasma H4PteGlu and CH3- H4PteGlu after intravenous and
intramuscular administrations than after oral administration of folic acid to pigs, although
in their experiment pigs used were not pregnant. According to these results, it may be
considered that plasma H4PteGlu is the clinical indicator of maternal folate status of pigs.
Besides plasma folate, pigs have another species specificity in folate binding proteins
in plasma. Mantzos et al. 18 have reported that only pigs out of several animal species (pig,
sheep, goat, cattle, horse, rabbit, dog, rat, guinea pig, and chicken) examined had
unsaturated avid specific binders of folic acid in plasma. The maximum binding capacity
for folic acid has been estimated to be 14.0 to 26.5 nglml of plasma. 18 They reported
plasma binding of H4PteGlu in pig plasma. 19 We are also confirming the property of

735
protein bindings of H4PteGlu in pig plasma.20 Therefore these results also suggest the
specificity in the homeostasis of folate in pigs. Further research is needed for studying this
protein(s) in pig plasma, related to tissue distribution of H4PteGlu. In this respect, we are
now characterizing this high-affinity binding protein(s) in pig plasma.

REFERENCES

1. L.B. Bailey, J.Nutr. 120: 1508-1511 (1990).

2. A.J. Clifford, M.K. Reid, H.G. Muller, and N.D. Bills, J.Nutr. 120: 1633-1639 (1990).
3. M. Natsuhori, M. Shimoda, E. Kokue, T. Hayama, and Y. Takahashi,
Am.J.Physiol. 261: R82-R86 (1991).
4. M. Shimoda, J. Vet.Med.Sci. 54: 249-253 (1992).
5. J. Ek and E.M. Magnus, Acta.Obst.Gynecol.Scand. 60: 247-251 (1981).
6. J.A. Pritchard, P.J. Whalley, and D.E. Scott, Am.J.Obst.Gynec. 104: 388-395 (1969).
7. I.J. Temperley, M.J.M. Meehan, and P.B.B. Gatenby, Brit.J.Haemat. 14: 13-19 (1968).
8. S.W. Thenen, Nutr.Res. 11: 105-116 (1991).
9. N. Habibzadeh, C.J. Schorah, and R.W. Smithells, Brit.J.Nutri. 55: 23-35 (1986).
10. J.J. Matte, C.L. Girard, and G.J. Brisson, J.Anim.Sci. 59: 158-163 (1984).
11. J.J. Matte, C.L. Girard, and G.J. Brisson, J.Anim.Sci. 59: 1020-1025 (1984).
12. M.D. Lindemann, and E.T. Kornegay, J.Anim.Sci. 67: 459-464 (1989).
13. R.M. Friendship and M.R. Wilson, Can. Vet.J. 32: 565-566 (1991).
14. R.S. Hillman, in: "The Pharmacological Basis of Therapeutics (8th ed.)", A.G. Gilman,
T.W. Roll, A.S. Nies and P. Taylor eds., Pergamon Press, New York (1990).
15. I.H. Rosenberg and J. Selhub, in: "Folates and Pterins Volume 3. Nutritional,
Pharmacological and Physiological Aspects" R.L. Blakely ed. Wiley-interscience,
New York (1986).
16. G.F. Tremblay, J.J. Matte, J.J. Dufour and G.J. Brisson. J.Anim.Sci. 67: 724-732
(1989).
17. E. Kokue, T. Sekiya, M. Shimoda, and M. Natsuhori. Vet.Q. in press.
18. J.D. Mantzos, V. Alevizou-Terzaki and E. Gyftaki. Acta Haematol. 51: 204-210
(1974).
19. J.D. Mantzos, E. Gyftaki, V. Alevizou-Terzaki, E.Manesis, and B. Malamos.
Jahrestag.Ges.Nuclearmed., Antwerpen (1971).
20. K. Sasaki, M. Shimoda, Y. Aoki, M. Natsuhori and E. Kokue. in "Advances in
Experimental Medicine and Biology: Chemistry and Biology of Pteridines and
Folates", J.E. Ayling, M.Gopal Nair, and C.M. Baugh eds. Plenum Publishing
Corporation, New York (1993).

736
IDENTIFICATION OF ENDOGENOUS TETRAHYDROFOLATE AND
10-FORMYLTETRAHYDROFOLATE AS MAJOR FOLATES IN RAT BILE

Ho-Chul Shin, Minoru Shimoda, Eiichi Kokue, and Yuji Takahashi 1

Department of Veterinary Medicine, Faculty of Agriculture,

Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183
1Research Institute for Animal Science in Biochemistry and Toxicology,

Sagamihara, Kanagawa 229, Japan

INTRODUCTION

The enterohepatic circulation of folates plays an important role on the homeostasis of

folates 1• The principal folate in bile has been known to be 5-methyltetrahydrofolate (5-
CH3-H4PteGluf However the other endogenous folates in bile, other than 5-CH3-
H4PteGlu, have not been well established. Recently, we have developed a sensitive
method to determine oftetrahydrofolate (HlteGlu) and 5-CH3-H4PteGlu in plasma using
high-performance liquid chromatography with electrochemical detection system (HPLC-
ECD)3. The present study was carried out to identify the other folates in rat bile using this
method.

MATERIALS AND METHODS

Female Sprague-Dawley rats (about 13 weeks old, Clea Japan Inc., Tokyo, Japan)
were used. The bile sample was collected via bile duct catheter under urethan anesthesia
(intraperitoneal injection, 0.8 - 1.0 g!kg of body weight). The bile samples were collected
into ice-cold test tubes containing 0.5 % sodium ascorbate solution and stored at -80°C
until the HPLC analyses.
Chromatographic analysis was performed using HPLC systems. Analytical detectors
included an electrochemical detector (L-ECD-6A, Shimadzu Co. Kyoto, Japan) and a
photodiode array detector (Multi-340, Jasco, Tokyo, Japan). The mobile phase was a
acetate or phosphate buffer.
For the identification of bile folates, the retention time profiles and electrochemical

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 737
properties of the bile folates and standards were examined using HPLC-ECD method. The
retention time profiles were determined for the various pH values of acetate buffer and for
the various fractions of acetonitrile (AN) in the mobile phase. The voltammograms were
obtained by responses versus applied potentials of the electrochemical detector. For further
identification, the spectral curves of UV absorbance of the bile folates and standards were
obtained using HPLC with a photodiode array detection system (HPLC-PAD).

RESULTS AND DISCUSSION

The chromatogram of rat bile obtained from HPLC-ECD analysis was shown in Fig.
1. The peaks A and B had the same retention time as those of standard 10-HCO-H4PteGlu
and H4PteGlu, respectively.

I 01 nA

standard

----bile

0 4 8 12 16 20

RETENTION TIME (min)

Figure 1. HPLC-ECD chromatogram of rat bile folates and standards.

The consistency of retention time between the substances in bile and standards was
obtained for the various pH values of acetate buffer and for the various concentrations of
AN in the mobile phase (Table 1). This observation indicates that bile substances from
peaks A and B have the same values for the negative logarithm of acid dissociation con-
stant (pKa) and the same lipid solubility with 10-HCO-HlteGlu and H4PteGlu, respec-
tively.
The voltammograms of the bile substances indicating peaks A and B were almost
identical to those of standards including 10-HCO-H4PteGlu and H4PteGlu, respectively
(Fig. 2). This findings indicate that the bile substances have same electrochemical proper-
ties or redox potentials with those of standard folates.
The UV absorbance spectral curves of the bile substances obtained from peaks A and
B were very similar to those of standard 10-HCO-HlteGlu and H4PteGlu, respectively
(Fig. 3). This observation indicates that the bile substances have the same UV absorption
characteristics with those of standard folates.

738
 The rates of bile secretion of 10-HCO-HlteGlu, HlteGlu and 5-CH3 -HlteGlu
were 314 ± 181, 321 ± 179 and 449 ± 198 ng!hr (mean± SD), respectively.
It has been suggested that 5-CH3-HlteGlu is the principal folate in rat bile 2 • Howev-
er, this study demonstrated that 10-HCO-HlteGlu and HlteGlu were also secreted as
major folates into bile. These nonmethylated folates may play an important role in the
folate homeostasis through enterohepatic circulation.

Table 1. Retention time profiles.

Condition Retention times (min)

pH AN Peak-A 10-HCO-H4PteGlu Peak-B H4 PteGlu

3.5 6% 10.63±0.03 10.73±0.13 9.22±0.05 9.19±0.08

4.0 6% 8.67±0.05 8.63±0.12 9.28±0.06 9.18±0.07
4.5 6% 6.82±0.11 6.82±0.07 7.94±0.08 7.87±0.11
5.0 6% 5.43±0.13 5.31±0.02 6.56±0.10 6.42±0.03

4.5 3% 15.01±0.01 15.03±0.68 23.63±1.02 23.73±1.40

4.5 4% 11.58±0.64 11.31±0.24 16.35±0.19 16.27±0.15
4.5 6% 6.82±0.11 6.82±0.07 7.94±0.08 7.87±0.11
4.5 8% 5.42±0.08 5.42±0.02 5.85±0.00 5.82±0.04

Values are means ± SD (number of determinations = 3).

100
w
0
z
0
0...
(/)
w
a:
~
:::!
LL
0
~ 0
.0 .1 .2 .3 .4 .0 .1 .2 .3 .4

OXIDATION POTENTIAL (Volt)

Figure 2. HPLC-ECD voltammograms of bile folates (solid line) and standards (dotted line).

739
 10 -HCO-H 4 PteGiu
w 100
0
z
~
a:
0
(/)
(])
<(
w
>
~
w
a: 0

200 250 300 350 200 250 300 350

WAVELENGHTH (nm)

Figure 3. UVabsorption spectra of the bile folates (solid line) and standards (dotted line) obtained from
HPLC-PAD.

REFERENCES

1. S.E. Steinberg, C.L. Campbell, and R.S. Hillman, J. Clin. Invest. 64:83(1979).
2. S.E. Steinberg, Am. J. Physiol. 246:G319(1984).
3. M. Natsuhori, M. Shimoda, E. Kokue, T. Hayama, and Y. Takahashi, Am. J.
Physiol. 261:R82 (1991).

740
DEVELOPMENT OF A SENSITIVE ASSAY FOR DETECTION OF URACIL IN DNA

Benjamin C. Blount and Bruce N. Ames

Division of Molecular and Cellular Biology

University of California, Berkeley, CA 94720

INTRODUCTION

Folate deficiency causes chromosomal breakage. Chromosome abnormalities can

lead to cancer and other pathologies. We are interested in determining the role that uracil
misincorporation into DNA plays in folate deficiency-induced chromosome breaks.
Previous work with cultured human cells indicates that methotrexate inhibition of
thymidylate (TMP) synthesis results in substantial increases of deoxyuridylate (dUMP) and
subsequent incorporation of uracil into DNA in the place of thymine. 1 A number of
researchers postulate that folate deficiency causes a futile cycle of uracil insertion and
removal from DNA that accounts for the observed chromosome breaks4•7 and an increased
risk of cancer 1•3 (Figure I). Our goal is to determine if uracil misincorporation into DNA is
occurring in humans due to folate deficiency or marginal folate intake. To accomplish this
we developed a sensitive gas chromatography- mass spectrometry (GCMS) method for the
quantitation of uracil in DNA
Folate Deficiency

1
Figure 1. Proposed mechanism for folate
deficiency causing an increased risk of
cancer. Low folate partially inhibits thymidylate tdUTP i ITP

/ ~
synthesis, causing an increase in dUTP levels and a
decrease in TfP. DNA polymerase then incorporates
dU into the DNA. 1 Uracil-DNA glycosylase removes
the uracil and repair enzymes excise several Attempted Repair ,.__.. dU Misincorporation
nucleotides on either side of the lesion. DNA
polymerase may insert another dU as it attempts to 1
fill this gap, resulting in repeated excision and Double Strand DNA Breaks
repair. If several uracils are on opposite strands in
the same vicinity, double strand DNA breaks occur. 5
Chromosome breaks decrease genetic stability and 1
Increased Risk of Cancer
increase cancer risk. 6

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 741
METHOD

The method for quantitation of uracil in DNA is outlined in Figure 2. Briefly, DNA is
isolated from the tissue of interest by phenoVchloroform extraction. Uracil is specifically
removed from the DNA by incubation with 1 unit uracil-DNA glycosylase (UDG). Three
hundred picograms of the internal standard (ISN2-uracil) is then spiked into the sample.
After removing residual proteins and biological polymers with an acetonitrile precipitation,
the sample is derivatized with ditrifluoromethylbenzyl bromide (DTF.MBzBr) in
triethylamine. The sample is then cleaned up by separating excess derivatizing reagent and
other unwanted materials from the compounds of interest with a C-18 solid phase extraction
cartridge. The 100% ethanol eluant from these columns is concentrated under a gentle
stream of nitrogen and injected into a gas chromatograph fitted with a 12 meter DB-5
capillary column. A temperature gradient from 80°C to 280°C at 25°C per minute results in
sufficient resolution of uracil-diDTFMBz from interfering compounds. The uracil-
diDTFMBz and 1sN2-uracil-diDTFMBz are quantified by selected ion monitoring. The
detection limit is 30 picograms of uracil in 100 J..l.gs ofDNA (1 uracil per 106 thymine).

add internal
Ura-DNA-glycosylase standard DTFMBzBr C-18 cleanup
DNA - - - - - - + - - - + Uracil Uracil-diDTFMBz Quantitation
Digestion ACN Derivitization GCMS analysis
precipitation

Figure 2. Procedure for quantitation of trace amounts of uracil in DNA

Samples are analyzed by using electron impact mass spectrometry (El-MS) after the
resolved compounds exit the gas chromatograph. Uracil-diDTFMBz and its stable isotope
internal standard each have unique fragmentation patterns that make El-MS feasible. Figure
3 shows the EI mass spectrum and structure ofuracil-diDTFMBz.

227
100 Uracil-di DTFMBz
MW=564
<!)
80
a
u
CF3 FF

'"~.0~
"0
s::
:s 60
.D 294
<
Hyo
C~J~CH 2 H
<!)
;> 40
-.;:::;
~ 96 268 564
~ 20 177 545
0 I 3f7 l
100 200 300 400 500
Mass/Charge (m/z)
Figure 3. Uracil-diDTFMBz electron impact mass spectrum and structure. Note the
prominent molecular ion at 564. This fragment is monitored later for quantitation of trace amounts
of uracil. The EI mass spectrum of ISN2-uracil-diDTFMBz is identical except 564=566, 545=547,
337=339 & 294=295 as a result of the heavier ISN(s) in the internal standard.

742
We found that the molecular ions of uracil-diDTFMBz (m/z=564) and 15N2-uracil-
diDTFMBz (m/z=566) are the best to monitor for sensitive detection of the two
compounds. Mass 294 is more abundant but it has a lower signal to noise ratio. The
sensitivity of this assay was enhanced by monitoring only mass 564 for uracil detection and
566 for the 15N2-uracil internal standard. A typical chromatogram is shown in Figure 4.

. - 15 N2-Uracil-diDTFMBz
internal standard

7.40 7.80 8.20 8.60

Retention Time (minutes)

Figure 4. Typical ion chromatogram of uracil from DNA as analyzed by our method. In
this example 75 pg of uracil was spiked with 300 pg of internal standard. The upper trace represents
mass 566; the peak at 7.79 minutes is the 15N2-uracil-diDTFMBz internal standard. The lower
trace is mass 564; the peak at 7.79 minutes is uracil-diDTFMBz.

Quantitation is accomplished by integrating the two peaks (such as those in figure 4) and
adjusting the uracil-diDTFMBz according to the amount of internal standard recovered.
The stable isotope labeled uracil has an identical recovery rate as uracil: approximately 60%.
El-MS of these compounds yields identical, linear responses from <10 pg to 5 ng. The
efficacy of the internal standard is demonstrated in figure 5.

. -!!!
2.00
....
.~
= ICIC
a.l
c...,. 1.00
....00
IC
,_,
I(')

...."'
0.00
0 200 400 600

Uracil (pg)

Figure 5. Calibration curve of uracil and 15N2-uracil-diDTFMBz. Uracil (30-600 picograms)

is spiked with 300 picograms 15N2-uracil. The ratio of ions 564:566 is linear when plotted against
the actual starting amount of uracil. The data are clearly linear (R2 = 0.997). This indicates 15N2-
uracil is an excellent internal standard under these conditions.

743
RESULTS

We are using this assay to study the effect of an organism's folate status on the uracil
content of its DNA. We induced folate deficiency in Fisher 344 rats either by feeding a
folate deficient diet or injecting methotrexate. These animals were also given a cell
proliferative stimulus (partial hepatectomy) to exacerbate misincorporation of uracil into the
DNA. Seven days after surgery the animals were killed and their entire liver removed.
Uracil content of this hepatic DNA was determined using our method. The following
statistically significant differences are of interest:
1. The methotrexate treated animals had higher levels of uracil in DNA than any other
group.
2. Folate sufficient and deficient animals had the same amounts of uracil in their DNA
before surgery.
3. Folate deficient animals maintained on this diet after surgery had a significant increase
in DNA uracil levels. Folate supplementation after surgery prevented this increase.

CONCLUSIONS

We have developed an extremely sensitive gas chromatography-mass spectrometry method

for the detection of uracil in DNA. This assay is specific for uracil and relatively fast. A
stable isotope internal standard makes adjusting for sample loss accurate and easy. We are
using this method to examine DNA from human white blood cell and sperm and folate
deficient rodents. Preliminary results from folate deficient rats indicate that folate status
does effect the amount of uracil in DNA. These experiments will help us to elucidate the
role that uracil misincorporation into DNA plays in folate deficiency-induced chromosome
breaks.

REFERENCES

1. Goulian, M., Bleile, B., & Tseng, B.Y. Proceedings of the National Academy of
Sciences (1980) 77(4): 1956-1960.
2. Reidy, J. Mutation Research (1988) 200: 215-220.
3. Krumdieck, C.L. "Nutritional Factors in the Induction and Maintainence ofMalignancy"
Bristol-Meyers Nutritional Symposia volume 2, pp 225-245, Academic Press, N.Y.
4. MacGregor, J.T., Schlegal, R., Wehr, C.M., Alperin, P. & Ames, B.N. Proceedings of
the National Academy of Sciences (1990) 87: 9962-9965.
5. Dianov, G.L., Timchenko, T.V., Sinitsina, O.I., Kuzminov, A.V., Medvedev, O.A., &
Salganik, R.I. Molecular and General Genetics (1991) 225: 448-452.
6. Croce, C.M. Cell (1987) 49: 155-156.
7. Everson, R.B., Wehr, C.M., Erexson, G.L., & Macgregor, J.T. Journal of the National
Cancer Institute (1988) 80(7): 525-529.

744
5-METHYLTETRAHYDROFOLATE URINARY EXCRETION: MODELING BY
CULTURED HUMAN KIDNEY CELLS

Kenneth E. McMartin, 1 Khandoker M. Morshed, 1 Debra

J. Hazen-Martin,2 and Donald A. Sens2

1Louisiana State University Medical Center

Shreveport, LA 71130
2Medical University of South Carolina

Charleston, SC 29425

INTRODUCTION

Urinary folate excretion appears to be controlled by the degree of reabsorption

in the renal proximal tubule. 1 We have studied the membrane binding and
intracellular transport of folate by cultured human proximal tubule (HPT) cells and
have suggested that these cells serve as good models for renal folate reabsorption. 2
However, these studies were conducted with cells grown on plastic, which exposes only
the apical side to the media. To produce a better model of the transcellular transport
of folate, HPT cells have been grown on microporous filter inserts in which the
confluent monolayers effectively separate the apical (AP) and basolateral (BL)
chambers, allowing for separate manipulation of their media contents.3 Initial
experiments on the transport of 5-methyl-tetrahydrofolate (SM) in these inserts
produced surprising results. A large amount of SM was transported to the BL
chamber and this was not suppressed by excess amounts of SM, suggesting leakage
through a paracellular pathway. Such a pathway should be minimized by the existence
of functional tight junctions, which are normally present in confluent cultures of HPT
cells. 3•4 The presence of tight junctions is typically determined by measuring the
transepithelial electrical resistance (TER) across the monolayer on porous membrane
supports.5 The present studies were designed to assess the tight junction status during
the folate transport experiments in order to overcome the "disappointing" results.

METHODS

Stock cultures of HPT cells were grown in serum-free media (DME/Ham's

F12) supplemented with hormones and growth factorsY Cells were sub-cultured using
trypsin-EDTA and seeded in a 1:1 ratio onto collagen-coated membrane inserts
(Millicell PCF, 12 mm D, 0.4 J.L pore size, 0.6 cm2 area) at passages 4-8 with the same

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 745
media in the AP (0.3 ml) and BL (1.5 ml) chambers. Cells were grown to confluency
(at 7 d2•3) as determined daily by TER. TER measurements were conducted using an
EVOM epithelial voltohmmeter (World Precision Instruments).
Paracellular permeability was assessed by measuring the AP-BL and BL-AP flux
of the membrane impermeable probe inulin after cultures became confluent (day 6).
The growth media was removed and both sides of inserts were rinsed thrice in uptake
buffer (pH 7.4) containing (in mM): NaCl, 107; KCl, 5.3; CaC12, 1.9; MgC12, 1.0;
NaHC0 3, 26.2; D-glucose, 7; HEPES, 20. Cells were then incubated in these buffers
containing [14C]-inulin (either AP or BL addition) at 25°C. At timed intervals, 10-20
,u.l aliquots were removed from AP or BL chambers (replaced volumetrically) and
analyzed for 14C-content by scintillation counting.
Substrate uptake under "open" tight junction conditions was studied by removing
the growth media and rinsing the respective sides of the inserts at 4°C in apical (AP)
and basolateral (BL) uptake buffers with similar composition except the pH was
adjusted to 5.5 for AP buffer (containing MES) and to 7.4 for BL buffer (containing
HEPES). 2 Incubations were run at 37°C for 15-120 min with either the AP or BL
uptake buffer containing 10 nM of [3H]-5M or tracer amounts of [14C]-inulin or [14C]-
paraaminohippurate (PAH). Nonspecific controls contained a 500 fold excess of
unlabeled SM. In contrast, uptake under "closed" tight junction conditions was studied
by preincubating thrice-washed inserts for 60 min at 37°C in pH 7.4 uptake buffers
(AP & BL) to allow for recovery of TER values (Fig.1). Incubations were then run
at 37°C following the addition of 10 nM [3H]-5M to either the AP or BL buffer. At
30 - 120 min, the AP and BL buffers were collected for analysis of transfer of label to
the opposite chamber, then the inserts were rinsed 3X with ice-cold uptake buffers.
The amount of AP-bound folate was determined by pH 3.0 buffer incubation followed
by pH 7.4 uptake buffer rinse. 2 Intracellularly transported substrate was determined
by solubilizing cells in 0.1% Triton X-100. All values determined by liquid scintillation
analysis. The total uptake or transfer values were measured in incubations with
labeled 5M only, nonspecific values were measured in those with excess unlabeled 5M
and specific values were calculated by subtraction.

RESULTS

Initial studies of 5M uptake and transmembrane transfer by HPT cells were

carried out under "open" conditions, i.e. 5M was added to the incubation buffer prior
to recovery of TER. Under these conditions, cultured HPT cells bound and
intracellularly transported 5M in significant amounts when applied to the AP chamber
of filter inserts (Table 1). In contrast, neither inulin nor PAH were taken up to any
extent when applied AP. However, large portions of all 3 substrates were transferred
to the BL side, presumably by leakage pathways. 5M transfer was not suppressed by
excess amounts of unlabeled substrate, indicating a nonspecific pathway. Similar
results were obtained when substrates were added initially to the BL chamber (at 15
min, AP-BL and BL-AP fluxes of 5M were 0.8 ± 0.2 and 0.5 ± 0.1 nmol/h/cm2 and
of inulin were 1.1 ± 0.2 and 0.6 ± 0.1 nmol/h/cm2, respectively).
TER values increased during the first 4 d in culture (Fig.1) and maintained a
stable value for up to 2 wk, correlating in time with microscopic evidence of the
development of confluency and the presence of tight junctions. The TER was
abolished immediately upon replacement of the growth medium with fresh growth
medium (data not shown) or with pH 7.4 uptake buffer (Fig.1). TER recovery to its
initial state occurred within 60 min, suggesting that "tight" junctions had transiently
opened and then reclosed.

746
 Table 1. Cellular uptake of 5-CH3-THF, inulin and PAH by cultured HPT cells.

Substrate Condition AP binding Intracellular transport BL transfer

(%) (%) (%)
5-CH3-THF Total 0.28 ± 0.12 0.20 ± 0.05 16.05 ± 1.93
Specific 0.22 ± 0.11 0.12 ± 0.03 0.70 ± 1.41
Inulin 0.01 ± 0.00 0.02 ± 0.00 11.02 ± 0.90
PAH 0.02 ± 0.00 0.05 ± 0.01 18.37 ± 1.32
Incubations (60 mm) were earned out tmmedtately after replacement of growth medta wtth pH 5.5 assay
buffer (open tight junction conditions). Values for binding to AP membrane, transport into HPT cell,
and transfer into BL chamber represent the percent of the amount of substrate added to AP chamber.
Values represent mean ± SD (n=4 for folate, n=2 for others).

........ 1600
N
E

l __l_l
0
*Ill A 8
E
..c 1200

Vl
0
........,
Q)
0
T o-o-o-O
0/ ~ l
T

.L
T T
1 1
c 800
0
......
Ill

0
T/ 1
Til
/1
Ill
0

/
Q)
0::

0
400
·;:
0
0
......
0
0
I
Q)
0
w 0
0 2 3 4 5 6 7 0 30 60 90 120
Days of Culture Time (min)

Figure 1. Panel A shows the development of TER by HPT cells during 7 d of culture in standard growth
medium. Panel B shows the time-dependent recovery of TER at 25°C on d 6 after replacement of
standard growth medium at time 0 with pH 7.4 buffer. Values represent mean net TER (after subtraction
of "TER" values from collagen-coated inserts containing no cells) ± SEM of 4 cell isolates.

To determine whether the abolition of TER was associated with a loss of the
permeability barrier, the AP-BL and BL-AP flux of inulin was studied. Under "open"
conditions (5 min after TER was abolished), inulin flux was substantial in both
directions (AP-BL and BL-AP flux of 5.4 ± 0.5 and 7.0 ± 0.8 nmol/h/cm2). When
studied after recovery of TER ("closed" conditions), the flux was reduced to 0.4 and
0.3 nmol/h/cm2, respectively, suggesting reestablishment of the tight junction barrier.
The transmembrane transfer of 5M was dramatically decreased (100 fold) when
uptake studies were conducted under "closed" conditions, i.e. after recovery of TER.
(Table 2). Transmembrane transfer under these conditions was partly suppressed by
excess 5M, suggesting movement through a specific cellular pathway rather than via
paracellular leakage. Cellular 5M uptake (AP membrane binding and intracellular
transport) occurred by specific processes and was similar whether 5M was applied AP
or BL.

747
 Table 2. Folate transport by HPT cells with closed tight junctions.

Time Cellular Uptake Membrane Transfer Flux

Direction (min) Sample (fmoljmg protein) (fmol/h/cm2)

AP -BL 30 Total 640 7.52

Specific 520 1.19
120 Total 1466 7.06
Specific 1376 5.65

BL - AP 30 Total 672 5.89

Specific 590 2.89
120 Total 1012 5.08
Specific 842 2.92
IncubatiOns were earned out 1 h after replacement of growth medta Wtth pH 7.4 assay buffer (closed ttgilt
junction conditions). Cellular uptake represents the sum of the amount bound to the AP membrane and
that transported into the cell. Values from one experiment.

SUMMARY AND CONCLUSIONS

Initial attempts to model urinary folate reabsorption using cultures of HPT cells
on porous filter inserts produced disappointing results in that large amounts of SM
were transported across the epithelial monolayer in a nonspecific manner. Since the
impermeable molecule inulin was also transported, there apparently existed a
significant leakage pathway in the way that the cultured cells were used for transport
studies. SM was bound to the AP membrane and taken up into the HPT cell by
specific processes, while inulin was excluded, suggesting that the HPT cells were
nevertheless operating functionally. TER values from cultured HPT cells plateaued
at a high level when cells became confluent, suggesting an epithelial layer with
functional tight junctions. However, when growth media were removed and replaced
with transport buffers, there was an immediate loss of TER that fully recovered if the
transport buffers were preincubated for 60 min. Under these conditions, transport
studies showed the expected results - no movement of inulin through the cell layer and
much reduced transfer of folate through the paracellular pathway. These results
suggest that a transient opening of tight junctions occurs when growth media are
replaced with biological buffers (or with fresh growth media), but that recovery of tight
junction function occurs with time.

ACKNOWLEDGEMENTS

Supported by NIH grant R01-AA05308.

REFERENCES

1. R.T. Muldoon, B.E. Eisenga, K.M. Morshed, and K.E. McMartin, J Nutr. 122:2415-23 (1992).
2. K.E. McMartin, K.M. Morshed, D.J. Hazen-Martin, and DA. Sens,Amer! Physiol. 263:F841-8 (1992).
3. J.G. Blackburn, D.J. Hazen-Martin, C.J. Detrisac, and DA. Sens, Kidney Int. 33:508-16 (1988).
4. C.J. Detrisac, MA. Sens, A.J. Garvin, S.S. Spicer, and DA. Sens, Kidney Int. 25:383-90 (1984).
5. B. Gumbiner, Amer J Physiol. 253:C747-58 (1987).

748
CONTRIBUTION OF PLASMA PROTEIN BINDING TO THE STABILITY
OF TETRAHYDROFOLATE IN PIG PLASMA

Kazuaki Sasaki, Minoru Shimada, Yukiko Aoki

Masahiro Natsuhori and Ei-ich Kokue

Department of Veterinary Medicine

Faculty of Agriculture
Tokyo University of Agriculture and Technology
Fuchu, Tokyo 183, Japan

INTRODUCTION

Plasma folate takes important roles in folate homeostasis in the body 1. The principal
folate in plasma has been recognized to be is 5-methyltetrahydrofolate in many species2•
Recently Natsuhori et al. 3 demonstrated that the principal folate in pig plasma is
tetrahydrofolate (H4 PteGlu). H4 PteGlu is, however, very susceptible to oxidation and
plasma contains oxidizing agents such as oxygen and peroxides. This study examined
stability of HlteGlu in pig plasma with relation to its protein binding.

MATERIALS AND METHODS

Blood was obtained from 5 adult Goettingen miniature pigs. The pooled plasma was
adjusted pH to 3.0 with 1 N HCl and kept at 4 °C for 30 min, in order to degrade endoge-
nous H4PteGlu. After adjusting pH to 7.4 with 1 N NaOH, 1 ml aliquots of the treated
plasma were stored at -80 °C until experiment.
After adding HlteGlu into the plasma sample, its stability was monitored using a
reversed phase high-performance liquid chromatography with electrochemical detection 4 •
Sodium ascorbate was added into the sample to stop degradation of H4 PteGlu.
HlteGlu solution and sodium ascorbate solution were added into theylasma sample,
and the mixture was ultrafiltrated with Amicon Micropartition SystenfBJ(Japan Grace,
Tokyo, Japan). Ultrafiltrates were analyzed by the HPLC system and unbound concentra-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 749
tion of HlteGlu was determined. The percentages of plasma protein binding was
calculated from total and unbound concentration.

RESULTS AND DISCUSSION

Figure 1 shows the stability of H4PteGlu in pig plasma at various concentrations. At

concentrations less than 35 ng!ml, HlteGlu was stable in the plasma sample where almost
all H4PteGlu bound to plasma proteins (more than 90 %). At concentrations more than 80
ng!ml, HlteGlu rapidly degraded and its concentration reached about 40 ng!ml by 30 min
after the addition of H4PteGlu. Thereafter H4PteGlu was relatively stable in the plasma
sample. Percentages of plasma protein binding of H4PteGlu were lower as the concentra-
tion was higher as is shown in Figure 1.

150 %binding

E 0 55% •58%
OJ
s D 72% •82%
0
c a 90% 4 96%
0
(.)

ell
E
({)
ell
0::::
0

0 30 60 90 120
Time after addition (min)
Figure 1. Stability of H4PteGlu in pig plasma at various concentrations.

plasma

buffer (pH 7.4)

c
0
eft

0 20 40 60
Time after addition (min)

Figut·e 2. Stability of H4PteGiu in plasma and plasma ultrafiltrate of pigs.

750
 In Figure 2 the stability of HlteGlu was compared between plasma and its ultrafil-
trate. In plasma filtrate HlteGlu rapidly degraded with a half-life of about 5 min even at
concentrations less than 35 ng/ml. This result suggests that binding proteins protect
H4PteGlu from degradation in pig plasma.
Binding kinetics of HlteGlu in pig plasma was also examined in this study. As is
shown in Figure 3, Scatchard plot showed 2 kinds of binding protein. Dissociation con-
stant and binding capacity of the high affinity binder were 1.9 x 10-9M (0.86 ng/ml) and
7.5 x 10-8M (33.3 ng/ml), respectively. The low affinity binder showed a linear binding in
the used concentration range (20 - 500 ng/ml).

12

0 100 150 200

Cb (ng/ml)
Figure 3. Scatchard plot ofHlteGlu binding h1 pig plasma.

The binding of H4PteGlu was competitively inhibited by folic acid, which indicates
that the binders for H4PteGlu in pig plasma are so called folate binding protein (FBP). As
is shown in Table 1, however, the inhibition was slight at the same concentration with
HlteGlu. Therefore, it is suggested that the high affinity binder in pig plasma has higher
affinity to H4PteGlu than folic acid, although it is generally accepted that high affinity
FBPs in plasma have higher affinity to folic acid than reduced folates such as HlteGlu5 •

Table 1. Effect on plasma protein binding of H4PteGlu

of folic acid spited at the same concentration.

Treatment 67nM llOnM

H4PteGlu 92.5±2.0 85.3±1.4

H4PteGlu 76.4±4.0 75.0±0.7
+
Folic acid

751
 Since binding kinetics of HlteGlu in this study indicates that almost all HlteGlu in
plasma can bind to the high affinity binder at basal levels of H4PteGlu, this binder may
mainly contribute to the stability of HlteGlu in pig plasma.

REFERENCES

1. S. E. Schteinberg, Am. J. Physiol. 246: G319-324 (1984).

2. N. W. Flodin, in : "Current Topics in Nutrition and Disease vol. 20, Alan R. Liss, Inc,
New York (1988).
3. M. Natsuhori, M. Shimoda, E. Kokue, T. Hayama andY. Takahashi, Am. J. Physiol.
261: R82-86 (1991).
4. M. Shimoda, J. Vet. Med. Sci. 54: 249-253 (1992).
5. J.D. Mantzos, V. Alevizou-Terzaki and E. Gyftaki, Acta Haematol. Basel 51: 204-210
(1974).

752
LIGAND INDUCED CONFORMATION CHANGE
IN FOLATE BINDING PROTEIN

Niels C. Kaarsholm,I, Anne-Marie Kolstrupl, Susan E. Danielsen\

Jan Holm,2 and Steen I. Hansen3

1Novo Research Institute

Novo Nordisk NS
Denmark-2880 Bagsvaerd
2Department of Clinical Chemistry

Central Hospital of Nyk0bing Falster

Denmark-4800
3Department of Clinical Chemistry

Central Hospital of Hiller0d

Denmark-3400

INTRODUCTION

We have employed circular dichroism (CD) and fluorescence spectroscopy to charac-

terize structural implications of folate (PGA, pteroylglutamate) binding to soluble folate
binding protein (FBP) from cow's milk, a 222 residue protein that displays high-affinity
ligand binding1•2 • These tools provide spectroscopic signatures of FBP conformation and
show that folate binding affects the structure of the FBP molecule.

METHODS

Protein Preparation. FBP was isolated from cow's whey and purified as described1 •
A concentrated stock solution (10 mg!ml) of FBP in 0.2 M acetate buffer, pH 5.0, was
stored frozen until required. Protein concentrations were determined by UV absorbance
using E280 = 6.816 x 104 M-1cm-1• This value is based on a molecular weight of 29,000
da1 •

Circular Dichroism Spectroscopy. CD spectra were recorded on a Jobin Yvon

Model IV dichrograph at 25 °C. Spectra for determination of secondary structure (180-
260 nm) were recorded with a cell pathlength of 0.01 em. The protein concentration was
typically about 1 mg!ml and the ionic strength kept sufficiently low so that the total

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 753
 3
/'

2 I \
I \
!J \
!J \
w \
<l 0 /---
/
-1 I
I
I
-2 I
~'/

-3
180 200 220 240 260

Wavelength (nm)
Figure 1. Far-UV CD spectra of FBP and FBP-PGA complexes. The spectrum of 25 t-tM unligated FBP
( ) is compared with that of the FBP-PGA complex (25 ~-tM FBP + 25 t-tM folate) in 10 mM
sodium acetate, pH 5.0 (----).Note that 25 t-tM folate per se has no measurable CD spectrum under
these conditions.

G)
C)

~ 200
C)
l:ll
G)

'g
"' 100
~

0 E:::__ __._____::::::::~~Cialao--...l

300 350 400 450 500

Wavelength (nm)
Figure 2. Tryptophan emission spectra (excitation 290 nm) of FBP and the effect of folate binding in 10
mM Tris/Cl04-, pH 7.0, at 25 °C. Fluorescence emission spectra for 0.4 t-tM FBP ( ) and the effect
of PGA additions: 0.082 nM ( - - - - ) , 0.166 (-- -), 0.248 (" ....), 0.329 (-·-), 0.410 (-··-),
0.610 ( - - - - --), and 0.807 (- - - ) t-tM.

754
optical density of protein plus solvent was less than 1.0 at 190 run. Typical protein
concentrations were 5 1-1M. All spectra were smoothed with a Fourier transform algorithm
and the appropriate background spectra subtracted. The result, ~E, is based on the molar
concentration of peptide bond. Secondary structures were predicted from 180-260 nm CD
spectra using singular value decomposition combined with variable selection3• For this
purpose, a set of 22 reference proteins with known CD spectra and X-ray structures was
used4•

Fluorescence Spectroscopy. Tryptophan emission spectra were recorded with a

Perkin Elmer luminescence spectrometer model LS-50 at 25 oc. Spectra obtained for the
appropriate solvent blanks were subtracted from all samples. In PGA titrations, correction
for a small but significant inner-filter effect was made by parallel titrations of a
tryptophan solution with the same OD280 as the protein solution in question5 • Both
excitation and emission slits were set at 5 nm.

RESULTS

PGA Induced Changes in Secondary Structure of FBP. Figure 1 shows the far-
UV CD spectrum of 25 1-1M FBP at pH 5. The spectrum shows two negative bands with
the minimum at 209 nm and a shoulder located at 220 run. A positive band shows
maximum at 192 run, and the cross-over point is located at 199 run. Figure 1 also shows
the spectrum of a mixture of 25 1-1M FBP and 25 1-1M PGA at pH 5. As a result of
complex formation, the intensity of the positive band increases and the maximum shifts
to 194-195 nm. The crossover point is red-shifted by 2 run and the relative intensities of
the negative bands at 209 and 220 nm are inverted. Note that 25 1-1M PGA per se has no
measurable CD spectrum at 0.01 em cell pathlength. A secondary structure analysis for
free and ligated FBP showed that complex formation resulted in a loss of 10% antiparallel
B-strand.

Effect of PGA Binding on FBP Tryptophan Fluorescence. The tryptophan

emission of FBP provides an additional spectroscopic signature of PGA binding. Figure
2 shows the effect of increasing amounts of PGA on the tryptophan emission spectrum
of 0.4 1-1M FBP. At pH 7 ligand binding strongly quenches the tryptophan fluorescence
with a concomitant 13 nm blue-shift of the emission maximum from 346 to 333 run. At
346 nm, the signal is quenched 4.7-fold at saturating (PGA]. For comparison, the same
additions of PGA to a 2 1-1M solution of tryptophan quenches only 6 % of the fluorescence
at 346. A similar pattern was found at pH 5.0 (data not shown). At pH 3.0 PGA had
virtually no effect on FBP tryptophan fluorescence (data not shown). Figure 3 shows that
the extent of the quench at 350 run as a function of the PGA concentration (pH 5-9)
resulted in a titration curve consisting of two straight lines with an intersection point
corresponding to the stoichiometry of PGA binding. The observed stoichiometry of about
0.6 mol PGNmol FBP is very close to that obtained from dialysis experiments using
radiolabeled PGA2 • We conclude that tryptophan is present at the PGA binding site and/or
ligand binding induces a conformation change which strongly affects tryptophan environ-
ment in FBP. Figure 3 also shows that fluorescence at 350 run is virtually unaffected by
the PGA concentration at pH 3-4. This observation suggests a drastic decrease in the
ability of FBP to bind PGA as compared to pH 5-7. The pH-dependence of PGA-
induced changes in fluorescence is thus compatible with previous studies showing a rapid
dissociation of radiolabeled PGA from FBP at pH 3.5 as compared to pH 5-1'.

755
 ,......
E 300
0
=
II)
~
200
4)
0
c4)
0
(/) 100
....
4)

0
:::l
~ 0
0 200 400 600 800

[PGA] (nM)

Figure 3. Quench of FBP tryptophan fluorescence as a function of PGA addition at various pH. The protein
concentration was 400 nM, excitation was effected at 290 nm. pH 3 (+), 4 {t.), 5 (0), 6 ( .t. ), 7 (e), 8 (D), and
9 {•).

REFERENCES
1. I. Svendsen, B. Martin, T.G. Pedersen, S.I. Hansen, J. Holm, and J. Lyngbye, Carlsberg Res. Commun.
44, 89. {1979).
2. S.I. Hansen, J. Holm, J. Lyngbye, T.G. Pedersen, and I. Svendsen, Arch. Biochem. Biophys. 226, 636.
(1983).
3. J.P. Hennessey, and W.C. Johnson, Biochemistry 20, 1085. (1981).
4. W.C. Johnson, Proteins 7, 205. (1990).
5. B. Birdsall, R.W. King, M.R. Wheeler, C.A. Lewis, S.R. Groode, R.B. Dunlap, and G.C.K. Roberts, Anal.
Biochem. 132, 353. (1983).
6. S.l. Hansen, J. Holm, and J. Lyngbye, Biochim. Biophys. Acta 535, 309. {1978).

756
THE HIGH-AFFINITY FOLATE BINDING
PROTEIN IN NORMAL AND MALIGNANT
MAMMARY GLAND TISSUE

Jan Holm\ Steen I. Hansen3, Knud S0ndergaard2 and

Mimi H0ier-Madsen4

1Department of Clinical Chemistry

2Department of Pathology
Central Hospital of Nyk0bing Falster
Denmark-4800
3Department of Clinical Chemistry

Central Hospital of Hiller0d

Denmark-3400
4Laboratory for Autoimmune Serology

State Serum Institute Copenhagen

Denmark-2300 S.

INTRODUCTION

Human milk contains a soluble 25 kDa folate binding protein (FBP) as well as a
membrane-derived FBP with a hydrophobic glycosyl-phosphatidylinositol tail; the
enzyme phosphatidylinositol specific phospholipase C reduced the apparent size of the
latter FBP from 100 to 25 kDa1• The two FBPs from human milk had identical N-
terminal amino acid sequence for 39 cycles and shared antigenic determinant!1·3 • A high
correlation between the concentrations of FBP and folate in milk suggested the secretion
of a folate mammary FBP complex into milk4• In the present study we have characterized
and compared FBP in the normal mammary gland and mammary tumors.

METHODS

Mammary gland tissue was homogenized in 5 mM Tris/HCl buffer (pH 7.4, 4°C)
containing 5 mM mannitol (5 ml of buffer/g of tissue). Homogenate solubilized (2h, 4°C)
with Triton X-100 (20 gil) was centrifuged (1000 g, 30 min) and the supernatant used for
analysis.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 757
 30 40
B/F B/F
30

20
10
10

0 0
0 1 2 3 0 1 2 3 4
B nM B nM
Figure 1. Scatchard plots showing high-affinity binding of 3H-folate to normal mammary gland tissue
homogenate (left) and mammary tumor tissue homogenate (right). Each point represents a single result.
Abscissa, bound folate (B). Ordinate, bound/free folate (B;F).

2 3

106
80

49.5

32.5

27.5

18.5

Figure 2. SDS-PAGE and immunoblotting with rabbit anti-human milk FBP

of normal mammary gland homogenate (lane 1), non-malignant (lane 2)
and malignant (lane 3) tissue from mammary tumors. Molecular markers
at the left.

758
 In some experiments with normal mammary gland tissue the upper lipid-containing
(triglyceride-rich) layer from 1000 g supernatant was mixed with Triton X-100 (30 gil),
stirred for 90 min and centrifuged (35000 g, 30 min); the pellet was used for analysis. All
tissue preparations were stripped for endogenous folate by dialysis against 0.2 M acetate
of pH 3.5. Binding of 3H-folate was determined by equilibrium dialysis (37°C, pH 7.4f

RESULTS AND DISCUSSION

The FBP concentration in homogenate, expressed as maximum 3H-folate binding,

was 0.7 nmol!g protein; FBP was localized in the pellet from the triglyceride-rich layer.
Binding of 3H-folate to homogenate from mammary gland and mammary tumor
tissue was of a high-affinity type with a concentration of free 3H-folate at half saturation
of binding (S0.5) of 0.05 nM corresponding to a binding affinity (1/S 05) of 2 x 1010 M-1•
Binding displayed apparent positive cooperativity as indicated by convex upward
Scatchard plots (Figure 1) and Hill coefficients of 1.59 (normal mammary gland) and 1.36
(mammary tumor), both significantly (P <0.001) higher than 1.0.
There was virtually no dissociation of 3H-folate from FBP at pH 7.4 Dissociation
was rapid at pH 3.5, however, with a residual acid-dialysis resistant binding of 21% in
normal mammary gland. The residual binding was only 4-5% in mammary tumor tissue.
The relative molecular mass of FBP in homogenate from mammary gland and
mammary tumor tissue was estimated by gelfiltration5• The homogenate was pre-exposed
to an excess of 3H-folate prior to Ultrogel AcA 44 gel chromatography. The elution
diagram contained a 100 kDa and a 25 kDa peak of 3H-folate-bound FBP. Immunoreac-
tive FBP determined by an enzyme-linked immunosorbent assay involving rabbit anti
human milk FBP was found in the effluenf.
Sodium dodecylsulfate polyacrylamide (12%) gel electrophoresis (SDS-PAGE) and
immunoblotting with rabbit anti-human milk FBp5 was performed on homogenate of
normal mammary gland and mammary tumor tissue. As can be seen (Figure 2) a band of
65 kDa was detectable in all preparations. Two additional bands of 25-27 and 27-32 kDa
in tumor tissue.
Immunohistochemistry on paraffin-embedded sections of formalin-fixed mammary
tumor tissue was performed using rabbit anti human milk FBP serum as the primary
antibody and biotin-conjugated goat anti rabbit immunoglobulin as the secondary
antibody. Streptavidin-alkaline phosphatase was used for visual detection. Figure 3 shows
immunostaining of malignant as well as non malignant tissue from a mammary tumor; no
staining in control experiments with non-immune rabbit serum.
The FBP concentration was 1.24 nmol maximum 3H-folate bindinglg protein in
malignant tissue and 0.43 nmol!g protein in adjacent non-malignant tissue of mammary
tumors from 11 patients (mean values); the difference was significant (p <0.005, paired
test; Wilcoxon signed ranks test). A significant (P <0.01) difference was also found
between the triglyceride concentration in malignant tissue, 0.96 nmol/l, and adjacent non-
malignant tissue, 0.50 mmol/l.
The parallel elevation of triglyceride and FBP in malignant as compared to non-
malignant tissue from mammary tumors is compatible with the localization of FBP in the
triglyceride-rich fraction of mammary gland tissue.

759
 Figure 3. Upper part, immunostaining of mammary tumor sections after exposure to
rabbit anti human milk FBP serum. Lower part, control sections exposed to pre-
-immune rabbit serum.

REFERENCES
1. S.I. Hansen, and J. Holm, Biosci. Rep. 12:87 (1992).
2. I. Svendsen, S.I. Hansen, J. Holm, and J.Lyngbye, Carlsberg Res. Commun. 47:371 (1982).
3. M. H0ier-Madsen, S.I. Hansen, and J. Holm, Biosci. Rep. 7:553 (1987).
4. J. Selhub, R. Arnold, A.M. Smith, and M.F. Picciano, Nutrition Res. 4:181 (1984).
5. J. Holm, S.I. Hansen, M. H0ier- Madsen, and L. Bostad, Biochem. J. 280:267 (1991).

760
DETERGENT-INSOLUBILITY DURING THE BIOSYNTHESIS OF
MEMBRANE FOLATE RECEPTOR-2

Simon Rijnboutt, Ger Stroul, Engelien Bijleveld, George Posthuma#,

Manohar Ratnam*, Jan Schornagel@, and Gerrit Jansen

Dept. of Oncology, Medical Faculty, Free University, Amsterdam

®Dept. of Internal Medicine, The Dutch Cancer Institute, Amsterdam
'Tiept. of Cell Biology, Medical School, University of Utrecht
The Netherlands
'Dept. of Biochemistry and Molecular Biology, Medical College of Ohio
Toledo, Ohio

INTRODUCTION

Membrane folate recepter (MFR) type 2 is one of the two isoforms originally identified
in human placenta 1.1n the rough Endoplasmic Reticulum MFR-2 is provided with N-linked
oligosaccharide chains and a glycosyl phosphatidylinositol (GPI) membrane-anchor, which
has been shown to serve as a signal for the targeting of proteins to the apical plasma membrane
domain in epithelial cells 2 It has been suggested that GPI-linked proteins are sorted in the Golgi
0

complex into microdomains enriched in glycolipids and cholesterol which leads to directional
transport to the apical domain J,4,so Recently, it has been shown that newly synthesized GPI-
linked proteins have less lateral mobility when they arrive at the plasma membrane as compared
to GPI-linked proteins that were allready present at the cell surface, again suggesting clustering
of GPI-linked proteins at the plasma membrane 6 0

At the plasma membrane, MFR-2 has been implicated in the receptor-mediated uptake of
naturally occurring folates and folate-based chemotherapeutic drugs 7 Specialized structures
0

at the plasma membrane called caveolae have been suggested to be involved in the internalization
ofMFRs 8 and it has been observed that GPI-linked proteins and integral membrane proteins
are internalized via distinct pathways 9 However, the internalization-mechanism has not yet
0

been entirely elucidated and therefore, we are currently studying the biosynthesis, internalization
and intracellular distribution of MFR-2 in KB cells, a human nasopharyngeal cell line
expressing high amounts ofMFR-2 10 0

Chemistry and Biology of Pteridines and Folates, Edited by

JOEO Ayling et alo, Plenum Press, New York, 1993 761
MATERIALS AND METHODS

KB cells were cultured in a 5% C02 atmosphere in RPMI 1640 without folic acid (Gibco),
supplemented with 10% dialyzed fetal calf serum and 1 nM of folinic acid. For metabolic
labeling cells were depleted from methionine and pulse-labeled at 37°C with [3 5S)methionine
for 15 minutes. Cell surface iodination was performed at 0°C using lactoperoxidase and 1251in
the presence of glucose-oxidase and Hp2• Next, the cells were chased at 37°C for the indicated
periods of time. After the chase period cells were occasionally incubated with lOOmU/ml

chase 0 hrs. chase 4 hrs.

~e
chase (hours) 0.2% saponin
lysis lcmp. ('C)
lysis rcmp. ('C)
46·
46-

30-

30-

Figure 1. MFR-2 becomes partly insoluble in Triton X -100 during passage ofthe Golgi complex.
KB cells were metabolically labeled with [35S]methionine and chased for the indicated periods of time.
Molecular mass markers (kDa) are indicated on the left.

neuraminidase at 0°C and lysed in PBS containing 1% Triton X-100 and 1 mM PMSF at 0°C.
Next aliquots of the lysates were incubated at either 37°C or ooc in the presence of 0.2%
saponin. Control samples were kept at 0°C. Detergent-insoluble material was subsequently
removed by centrifugation at 10,000 x gat 4°C. Finally MFR-2 was immunoprecipitated and
analyzed on SDS-PAGE or isoelectric focussing gels with a linear pH-range from 4.4 to 8.2.
Iodination of Pte-ASA-LYS was performed using Iodobeads (Pierce) after which Pte-[1251]-
ASA-LYS was purified by thin layer chromatografy.

RESULTS AND DISCUSSION

After pulse-chase labeling MFR-2 was initially detected as a 34 kDa band, which could
be completely solubilized in 1% Triton X-100 at ooc (Figure lA). After 4 hours of chase
(Figure lB) all ofthe protein was converted to the 36-38 kDamature protein, due to conversion
of the high mannose-type N-linked oligosaccharides to the complex-type in the Golgi complex.
Scanning of these bands in a laser densitometer revealed that only 35% of the mature species
could be solubilized at these conditions. Complete solubilization of mature MFR-2 was
observed after incubation of the lysate at 37°C or at 0°C in the presence of 0.2% of the
cholesterol-complexing agent saponin.
This result shows the existence of at least two differently organized pools ofmature MFR-
2 in post-Golgi membranes. Furthermore, cholesterol is probably involved in maintainance of
MFR-2 in Triton X-1 00-insolubl.) clusters. Figure 1C shows the time course ofbiosynthesis of
MFR-2 with respect to the acquisition of Triton X-100 insolubility. Conversion of34 kDa
MFR-2 into the 36-38 kDamatureproteinoccurs just before TritonX-1 00 insolubility ofmature
MFR-2 is initiated (Figure 2; compare chase= 60 and chase= 90 minutes). This indicates that
Triton X-100 insolubility is a late- or post-Golgi phenomenon as was also indicated by
treatment of these samples with endoglycosidase H (not shown). Similar results were obtained

762
 A
B 0.2% saponin CEJ
lysis tcmp.(0C) ~

Pte- ['"1)- ASA - Lys

46-

c 10

~ 8 30-
-.:;
tJ
0 6
0 4
E
p.,
2
0
control 0.2 % saponin

- 37 °C

Ftgure 2 Only the pool ofMFR-2 soluble m Tnton X-100 at 0°C IS mtemahzed K.B cells were
cell surface wdmated and chased for 2 or 60 mmutes Next they were mcubated With or Without
neuramtmdase at 0°C and lysed m the absence or presence of 0 2% sapomn MFR-2 was
Immunoprectpttated and analyzed on IEF-gels

for another GPI-lmked protem, placental alkalme phosphatase, m BeWo chonon carcmoma
cells 5 11
Smce MFR-2 IS Implicated to partlctpate m the receptor-medtated uptake of naturally
occurnng folates and folate-based chemotherapeutic drugs the mtemahzatton ofMFR-2 was

0.2% saponin - +
neuraminidase - + - +

chase (min) 0 0 I 60 0 0 I 60

Ftgure3 BothMFR-2poo\satthecell5urfacearecapableofbmdmghgand K.Bcellsweredepleted

fromhgandwtthNa-acetatebufferpH4 5at0°C Next,thecellsweremcubatedwtthPte-[1251]-ASA-Lys
(A), mad1ated with UV-hght and lysed as m F1gure 1 Ahquots of the Iysates were analyzed by SDS-
PAGE(B)andbygamma-countmg(C) Molecularmassmarkers(kDa)aremdtcatedontheleft(B)

763
studied. KB cells were cell-surface iodinated at 0°C and chased for 1 hour at 37°C. Next, the
cells were incubated at ooc with 100 mU/ml neuraminidase to desialylate all MFR-2 at the cell
surface and lysed in Triton X-100 at 0°C with or without 0.2% saponin. The lysates were
immunoprecipitated for MFR-2 and the immunoprecipitates were analyzed on lD-IEF-gels.
Neuraminidase completely desialylated extracellular MFR-2, thereby increasing its iso-
electric point. During the chase period at 37°C part of the MFR-2 was internalized by the cells,
as is monitored by MFR-2 molecules which were protected from the neuraminidase treatment.
A control experiment showed that all MFR-2 molecules at the cell surface were accessible for
extracellularly added neuraminidase (data not shown). All of the internalized MFR-2 could be
solubilized in Triton X-100 at 0°C. Incubation of the lysate with 0.2% saponin only increased
the solubilized amount ofextracellular MFR-2. This result suggests that only MFR-2 molecules
outside Triton X-100 insoluble clusters are internalized at the plasma membrane.
Finally we determined if both pools ofMFR-2 present at the cell surface were capable of
binding ligand. Therefore, KB cells were washed at 0°C with Na-acetate buffer pH 4.5 to
deplete all extracellular MFR-2 from ligand. Next the cells were incubated with Pte-[l 25 I]-ASA-
Lys (Figure 3A), a photoaffinity analog offolic acid. Next, the cells were washed to remove
excess ofligand, irradiated with UV-light and lysed as in Figure 1. Aliquots of the lysates were
analyzed by SDS-PAGE (Figure 3B) and by gamma-counting (Figure 3C). Pte-[1 251]-ASA-Lys
labeling of MFR-2 was very specific, since only one band corresponding to MFR-2 was
detected after SDS-PAGE (Figure 3B). In Figures 3B and 3C is shown that MFR-2labeled by
Pte-[l 25I]-ASA-Lys has identical solubilization characteristics as in Figures 2 and 3, indicating
both MFR-2 pools present at the plasma membrane exhibit ligand binding activity.

In conclusion the present results show that MFR-2 is organized in at least two different
pools at the plasma membrane, one of which can be solubilized in Triton X-100 at 0°C. The
second pool can only be solubilized at 37°C orin the presence ofcholesterol-complexing agents
such as saponin. This indicates that MFR-2 is not homogeneously distributed at the plasma
membrane and suggests that Triton X-100 insoluble MFR-2 is present in microdomains. As has
been observed in another study 12 , maintainance ofthese microdomains depends on the presence
of cholesterol as was suggested by the facilitated solubilization at 0°C in the presence of
saponin. The insolubility ofMFR-2 in Triton X-100 at 0°C is acquired after its synthesis in the
rough endoplasmic reticulum during passage of the Golgi Complex, probably the trans-Golgi
reticulum. At the plasma membrane internalization ofMFR-2 molecules was only observed for
those which were not localized in Triton X-100 insoluble clusters. Both cell surface pools of
MFR-2 have ligand binding capacity.

Acknowledgements

We thank dr JB Hynes (Univ. ofS. Carolina, Charleston, SC, USA) for providing us with
Pteroyl-N'-(4-azido-salicylyc)-L-Lysine (Pte-ASA-Lys). We thank Tom van Rijn and Rene
Scriwanek for excellent darkroom services. This study was supported by the Dutch Cancer
Society (Grant NKI 92-43).

REFERENCES

1. Ratnam, M., Marquardt, H., Duhring, JL., andFreisheim, JH. Biochemistry 28:8249-8254
(1989).
2. Lisanti, MP., and Rodriquez-Boulan, E. Trends Biochem. Sci. 15:113-118 (1990).
3. Van Meer, G., Stelzer, EH., Wijnaendts-van-Resandt, RW., and Simons, K. J. Cell
Biol. 105:1623-1635 (1987).

764
4. Simons, K., and van Meer, G. Biochemistry 27:6197-6202 (1988).
5. Brown, DA., and Rose, JK. Cell68:533-544 (1992).
6. Haanan, LA., Lisanti, MP., Rodriquez-Boulan, E., and Edidin, M. J. Cell Biol. 120:353-
358 (1993).
7. Jansen, G., Schomage1, JH., Westerhof, GR., Newell, DR., and Jackman, AL. Cancer Res.
50:7544-7548 (1990).
8. Rothberg, KG., Ying, YS.,Kolhouse, JF., Kamen, BA., andAnderson,RGW. J. CellBiol.
110:637-649 (1990a).
9. Bamezai, A., Go1dmacher, VS., and Rock, KL. Eur. J. Immunol. 22:15-21 (1992).
10. Anthony, AC. Blood 79: 2807-2820 (1992).
11. Cemeus, DP, Ueffing, E., Posthuma, G., Strous, GJ., and van der Ende, A. J. Bioi.
Chern. 268: 3150-3155 (1993).
12. Rothberg, KG., Ying, YS., Kamen, BA., and Anderson, RGW. J. Cell Biol. 111:2931-
2938 (1990b).

765
THE REDUCED FOLATE/METHOTREXATE CARRIER AND A MEMBRANE-

ASSOCIATED FOLATE BINDING PROTEIN AS TRANSPORT ROUTES FOR NOVEL

ANTIFOLATES: STRUCTURE-ACTM1Y RELATIONSHIPS.

Gerrit Jansen, 1 G. Robbin Westerhof,1 Jan H. Schornagel,2 Ann L. Jackman, 3 and

F. Thomas Boyle4
1Department of Oncology 2Department of Internal Medicine
Free University Hospital Netherlands Cancer Institute
Amsterdam, The Netherlands Amsterdam, The Netherlands

3Drug Development Section Zeneca-Pharmaceuticals (ICI)

4

Institute of Cancer Research Macclesfield, Cheshire

Sutton, Surrey, United Kingdom United Kingdom

INTRODUCTION

The role of membrane transport is an important parameter in the biological evaluation

of novel antifolates. 1-3 In this respect the reduced folate/methotrexate carrier (RFC) is
considered to be the major transport route for these compounds in neoplastic cells. 4
However, there is accumulating evidence that other transport routes for (anti)folates may
be relevant as well. In particular, a membrane-associated folate binding protein (mFBP)
has been identified as a potential transport route in a variety of normal cells (e.g.
haematopoietic progenitor cells) 5•6 and some tumor types like ovarian carcinoma.6•7
In this study we have used, as a model system, human CCRF-CEM and murine L1210
leukemia cell lines expressing different levels of RFC and/or mFBP 8-10 to establish the
role of these membrane transport proteins in the cytotoxic effects of a series of
quinazoline-based antifolate inhibitors of thymidylate synthase (TS). 1•11 -13 Furthermore,
some structure-activity relationships demonstrated in this study may be further exploited
in antifolate drug design for selective transport via carrier- and/or receptor-mediated
transport routes.

RESULTS AND DISCUSSION

The chemical structures of the 13 quinazolinone-based antifolate inhibitors of TS,

included in this study, are shown in Figure 1. 2-Desamino-2-methyl-N10-propargyl-5,8-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 767
dideazafolic acid (ICI-198583, 1) and its N10-methyl thiophene analogue (ICI-D1694, 2)
were selected as the basic structures from which modifications were introduced in the
quinazoline ring (compounds 3-9), heterocyclic benzoyl ring (compounds 10,11) and the
glutamate side chain (compounds 12,13). The substrate affinity for RFC and mFBP, the
growth inhibitory effects (against leukemia cells expressing either RFC or mFBP), and
some other characteristics of the TS inhibitors are shown in Table 1. These results show
that RFC exhibited an efficient substrate affinity for all analogues, except (.2). The fact
that the RFC also has a low affinity for folic acid and N10-propargyl-5,8-dideazafolic acid
(CB3717) suggests that 2NHz14-oxo oxidized structures have a poor affinity for RFC. The
highest binding affinity of mFBP (relative to folic acid) was noted for (.2) (1.2).
Modifications at the N-3 position (.2.) or replacement of the 4-oxo group for 4-H (2)
resulted in a poor binding affinity of mFBP for these compounds (0.0009 and 0.019
relative to folic acid, respectively). Modifications at other sites of the molecule were well
tolerated with respect to binding activity (relative affinity; 0.13- 0.61 as compared to folic
acid). From these data it can be hypothesized that the N-3 and the 4-oxo part of the
quinazolinone ring are most important for high affinity binding to the active site site of
the receptor, probably through H-bond interactions.

Figure l. Chemical structures of quinazolinone-based antifolate inhibitors of thymidylate synthase.

Compound X RL R2 RJ R4 Ar Rs code name

1 N 0 CH3 H -C:CH benzoyl Glu ICI-198583

2 N 0 CH3 H -CH3 thiophene Glu ICI-D1694

3 N 0 CH3 CH3 -C=CH benzoyl Glu 7-CHr198583

4 N 0 CH3 Cl -CH3 benzoyl Glu 7-Cl/10CH3 198583
5 c 0 CH3 H -C=CH benzoyl Glu 3-deaza 198583
6 N H CH3 H -C::CH benzoyl Glu 4-H 198583
7 N OCH3 CH3 H -C=CH benzoyl Glu 4-methoxy 198583
8 N 0 CH 2F H -CH3 thiophene Glu 2-CH2F D1694
9 N 0 NH 2 H -CH3 thiophene Glu 2-NH2 D1694
10 N 0 CH3 H -C:CH thiophene Glu N10-propargyl-Dl694
11 N 0 CH3 H -CH3 thiazole Glu thiazole-01694
12 N 0 CH3 H -C=CH benzoyl Val Glu --• Val198583
13 N 0 CH 3 H -C=CH benzoyl Suberate Glu ---t>Sub 198583

768
 Table 1. Characteristics of quinazolinone-based antifolate inhibitors of thymidylate synthase; substrate affinity for RFC and mFBP, substrate affinity
for FPGS, inhibition of TS, and growth inhibitory effects.

Compound nr Inhibition Affinity Affinity Affinity ICso (nM)d

TS FPGS• RFCb mFBPc
(IC5 ~ ~M) RFC+ mFBP+ mFBP+
(1 nM LV) (20 nM FA)

Folic acid + 505 1.0

MTX + 10.8 0.008 8 19 1400

ICI-198,583 1 0.05 ++ 2 0.30 17 0.3 74

ICI-D1694 2 0.65 ++++ 2 0.68 3.8 0.2 118
7-CH3-198,583 3 0.028 - 7 0.36 243 3.8 1855
7-CL/10-CH3-198,583 4 0.066
.
- 2 0.33 695 90 19,000
3-Deaza-198,583 5 6.3 N.D. 1.3 0.0009 >100,000 39,000 >50,000
4-H-198,583 6 4.0 N.D. 3.1 0.019 680 42 7500
4-0CH3-198,583 7 2.8 N.D. 3.1 0.028 46 1.6 N.D.
2-CH2F-D1694 8 1.4 ++++' 5 0.16 9.6 1.2 1585
2-NH2-D1694 9 0.44 +++' 39 1.20 115 1.2 17
N10propargyl-D 1694 10 0.44 +++ 1.6 0.61 17 1.3 N.D.
10-CH3-thiazole-D1694 11 0.42 ++++ 5 0.13 6.7 1.5 1015
Glu --• Val 198,583 12 0.06 N.D. 15 0.34 4400 435 >25,000
Glu --•suberate-198583 13 0.018 N.D. 11 0.27 910 38 5050

• Substrate affinity (K.,) for FPGS is indicated as follows; ++++ (K., < 5 ~M), +++ (K., 5-25 ~M), ++ (K., 25-100 ~M), + (K., >100 ~M),
- (no affinity). '>Determined by cross-resistance studies in the L1210:MB3 line that fails to polyglutamate antifolates. Substrate data by Rick Moran.
b Inhibition of RFC-mediated influx of [3H]-MTX by folate analogue inhibitors of TS. The concentration of drug required to inhibit [3H]-MTX
influx in CEM-7A cells by 50% is depicted. Extracellular concentration of [3H]-MTX; 5 ~M. Influx buffer; HEPES buffered saline pH 7.4.
c Relative affinity of MFR for folate analogue inhibitors of TS. The inverse molar ratio of drug required to displace 50% of [3H)folic acid from
the receptor is given as relative affinity. The relative affinity for folic acid is set to 1.
d IC50 is defined as the drug concentration required to inhibit cell growth by 50% as compared to controls.
N.D.; Not determined, LV; leucovorin, FA; folic acid.
$
Regardless of efficient transport via RFC and/or mFBP, compounds which can not be
polyglutamated (J., ,4, 12, 13) or are poor inhibitors of TS (~, Q) were less potent cell
growth inhibitors than compounds which were efficiently polyglutamated (Table 1).
Under conditions of low extracellular concentrations of folates (e.g. 1 nM leucovorin),
mFBP-mediated uptake of compounds 1,2,7-11 seemed to be involved in the potent
growth inhibitory effects. However, in the presence of 20 nM folic, which mimicks a near
physiological concentration of serum folate, only the compounds for which mFBP has a
high affinity or were efficiently polyglutamated (1, 2 and 9) retained their growth
inhibitory potential against mFBP expressing leukemia cells.
In conclusion, a number of antifolate compounds have been identified which may use
either the RFC (MTX), mFBP (2) or both (1,2,1.~,10,11) routes for cellular uptake.

ACKNOWLEDGEMENTS
G. Jansen is a recipient of a fellowship from the Royal Netherlands Academy of Arts and
Sciences.

REFERENCES
1. P.R. Marsham, L.R. Hughes, A.L. Jackman, A.J. Hayter, J. Oldfield, J.M.
Wardleworth, J.A.M. Bishop, B.M. O'Connor, and H.A. Calvert, J. Med. Chern.
34:1594 (1991).
2. R.L. Hagan, D. Duch, G.K. Smith, M.H. Hanlon, B. Shane, J.H. Freisheim, and
J.B. Hynes, Biochem. Pharmacal., 41:781 (1991).
3. A. Rosowsky, R.A. Forsch, R.G. Moran, and J.H. Freisheim, J. Med. Chern., 34:
227 (1991).
4. P.M. Sirotnak, Cancer Res., 45:3992 (1985).
5. A.C. Antony, Blood, 79:2807 (1992).
6. S.D. Weitman, A.G. Weinberg, L.R. Coney, V.R. Zurawski, D.S. Jennings, and
B.A. Kamen, Cancer Res., 52:6708 (1992).
7. I.G. Campbell, T.A. Jones, W.D. Foulkes, and J. Trowsdale, Cancer Res., 51:5329
(1991).
8. G. Jansen, G.R. Westerhof, M.J.A. Jarmuszewski, I. Kathmann, G. Rijksen, and
J.H. Schornagel, J. Bioi. Chern., 265:18272 (1990).
9. G. Jansen, I. Kathmann, B.C. Rademaker, B.J.M. Braakhuis, G.R. Westerhof, G.
Rijksen, and J.H. Schornagel, Cancer Res., 49:1959 (1989).
10. G. Jansen, G.R. Westerhof, I. Kathmann, B.C. Rademaker, G. Rijksen, and J.H.
Schornagel, Cancer Res., 49:2455 (1989).
11. A.L. Jackman, D.R. Newell, W. Gibson, D.I. Jodrell, G.A. Taylor, .T.A. Bishop,
L.R. Hughes, and A.H. Calvert, Biochem. Pharmacal., 42:1885 (1991).
12. A.L. Jackman, G.A. Taylor, W. Gibson, R. Kimbell, M. Brown, A.H. Calvert, I.R.
Judson, and L.R. Hughes, Cancer Res., 51:5579 (1991).
13. F.T. Boyle, A.J. Barker, L.R. Hughes, P.R. Marsham, J.M. Wardleworth, T.C.
Stephens, and A.L. Jackamn, Proc. 7th EORTC/NCI symposium on new drugs in
cancer chemotherapy, Amsterdam 1992 (Abstract 115).

770
IDENTIFICATION OF A REDUCED FOLATE/METHOTREXATE CARRIER IN
HUMAN KB-CELLS EXPRESSING IDGH LEVELS OF MEMBRANE ASSOCIATED
FOLATE BINDING PROTEIN

G. Robbin Westerhof1, Jan H. SchornageF, Simon Rijnboutt\ Herbert M.

Pinedo 1•2 , and Gerrit Jansen 1

Dept. of Oncology
1 Dept. of Internal Medicine
2

Free University Hospital Netherlands Cancer Institute

de Boelelaan 1117 Plesmanlaan 121
1081 HV Amsterdam 1066 ex Amsterdam
The Netherlands The Netherlands

INTRODUCTION

Membrane transport is an essential step for the delivery of antifolate drugs to their
intracellular targets. At least two structurally unrelated transport systems have been
characterized for their role in the uptake of reduced folate cofactors and antifolate compounds;
the reduced folate/methotrexate carrier (RF/MTX-carrier) (1) and a membrane-associated
folate binding protein (mFBP) (reviewed in 2, 3, and 4). Recent studies have shown that
both proteins can be expressed within the same cell (5).
Based on the high levels of expression of mFBP, human nasopharyngeal KB-cells have
been extensively used as a model system to study the biochemical characteristics of mFBP
and to assess it's potential role in (anti)-folate transport (1, 4, 6, 7). During investigations
with KB cells we were intrigued by observations that these cells were sensitive to growth
inhibition by antifolate compounds for which mFBP has a low afftnity (e.g. the dihydrofolate
reductase (DHFR) inhibitors methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM)
whereas the cells were quite resistant to antifolates for which the mFBP has a high affinity
(e.g. the quinazoline-based inhibitor of thymidylate synthase (TS), N10-propargyl-5,8-
dideazafolic acid (CB3717)) (8). In order to explain this discrepancy we have carried out
a series of experiments which support the hypothesis that KB cells, in addition to high amounts
of mFBP, express functional levels of the RF/MTX-carrier protein.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 771
RESULTS AND DISCUSSION

KB cells have been grown under two conditions; (a) in standard RP:MI medium containing
2 ~-tM folic acid (FA) supplemented with 10% fetal calf serum (FCS), further referred to
as KB cells, and in folate free RP:MI medium supplemented with 10% dialyzed FCS and
1 nM leucovorin (LV) as the sole folate source, further designated as KB-LF cells. Under
both conditions the amount of mFBP did not differ significantly (300-500 pmol [3H]-folic
acid binding/107 cells) except that in contrast to KB-LF cells, all of the folate receptors in
KB cells were saturated with FA. For KB cells and KB-LF cells we have tested the growth-
inhibitory effects of MTX, 10-EdAM and CB3717 (Table 1).

IC 50-values (nM) for growth inhibitionbl

KB KB-LF
Relative Affinity•l RPMI; 10% FCS RPMI-FA; 10% Dial FCS

RFC mFBP I 2p.M FA 1 nMLV 2 p.M LV 2 p.M FA

MTX 10.8 0.038 16.6 4.8 835 17

10-EdAM 1.6 0.006 2.6 1.6 187 3.17

CB3717 57 0.95 628 4.7 nt nt

FA 505

LV 4.5 0.1

•J Relative affinities of the RFC for the (anti)-folate compounds are presented as the concentration
(p.M) necessary to inhibit rHJ-MTX-uptake (extracellular concentration 5 p.M) by 50%. Relative
affinities of the mFBP for these compounds are presented as the inverse molar ratio of compound
that inhibits [3H]-folic acid binding by 50%.
bJ Determined after 72 hrs of continuous exposure to the drug.

The rationale for selecting these compounds is that mFBP has a poor binding affinity
for MTX and 10-EdAM, and a high binding affinity for CB3717, whereas the substrate
affinities of the RF/MTX-carrier for these compounds is just the reverse, a low affinity
for CB3717 and high affinities for MTX and 10-EdAM (Table 1). Therefore, if a high affmity
for either of the two transport systems correlates with drug sensitivity and a poor affinity
with drug resistance, it can be expected that KB cells grown in the presence of 2 ~-tM FA
will be resistent to MTX and 10-EdAM if mFBP is the major transport route for these
compounds (9). In the absence of competing folates for the receptor, however, like for KB-LF
cells, mFBP-mediated uptake of drugs may be more efficient and can lead to growth inhibition.
The results from Table 1 are not consistant with the concept that mFBP is the only route
for antifolate uptake in KB cells, KB cells were fully sensitive to growth inhibition by MTX
and 10-EdAM and resistant to CB3717, an observation which is compatible with RF/MTX-
carrier-mediated drug uptake rather than uptake via the mFBP.
Some additional experiments provide further evidence that KB cells may express functional

772
levels of RF/MTX-canier; (A) Table 1 demonstrates that protection against MTX- and 10-
EdAM-induced cytotoxicity can be achieved by LV, a good substrate for RFC, rather than
by FA for which mFBP has the highest affmity; (B) influx rates and steady state levels for
uptake of rH]-10-EdAM in KB-LF cells were 4-fold higher than for rHJ-MTX and were
much higher than expected if transport was to proceed solely via the mFBP (results not shown);
(C) this uptake was inhibited by LV (Figure 1) or an N-hydroxysuccinimide (NHS)-ester
ofMTX which irreveribly blocks the RF/MTX-carrier (Figure 2). In contrast, uptake was
not inhibited by FA or an NHS-ester of FA (which blocks mFBP but not the RF/MTX-carrier);
(D) polyglutamate forms of fH]-MTX and rHJ-10-EdAM (up to tri-glutamate) were identified
shortly ( < 30 min) after drug incubation (not shown) which is consistant with rapid drug
accumulation via the RF/MTX-carrier, and fmally (E) labeling ofKB-LF cells with an 1251-
labeled photoaffmity analogue ofMTX resulted in the identification of two labeled membrane
proteins of which the molecular weights, 40 kD and 75-85 kD, were in agreement with
those reported for human mFBP and RF/MTX-carrier proteins (10, 11) (not shown).

~ e 100
c c
0
g
- "
Q

0 0 7$

.c..
~

.
so

"'
"
.
~ ~s .; '25

"'• "
.>:
~

~ 0 ii.
:::>
('H)-MTX ('H ]-10EdAM ('H J-MTX {'HJ-1 OEdAM

- 2 v MFA ~ 100vMFA EJ 1 00~M LV - Co n1rol ~ NHS-FA I2J NHS-MTX

Fig.llnhibitionoffH]-MTXandfH]-10-EdAM
uptake by FA and LV. KB-LF-cells grown in 9
em petri-dishes on RPMI-1640 medium without
FA, 10% dialyzed fetal calf serum (80 % confluen-
cy) were incubated with 1 J!M fH]-MTX or f H]-
10-EdAM with or without an excess of 100 f.tM
FA or LV. After 4 hrs of incubation at 37°C. in
a humidified atmosphere containing 5% C02 , the
cells were washed twice with Hepes buffered
saline solution (HBSS: 20 mM HEPES, 107 mM
NaCI, 26.2mMNaHC03, 5.3 mMKCl, 1.9mM
CaC12 , 1 mM MgC12 and 7mM D-Glucose, pH
7.4 with NaOH), washed once with HBSS pH
3.5 (same as HBSS but without NaHC03, pH 3.5
with HCl) to release extracellular bound ligand,
treated with 0. 25% trypsin in phosphate buffered
saline (PBS) to harvest the cells after which the
samples were analyzed for radioactivity.
Fig.21nhibitionoffH]-MTXandfH]-JO-EdAM
uptake by NHS-esters ofMTXand FA. N-Hydroxy-
Succinimide (NHS) esters of MTX and FA were
synthesized as described by Henderson et al. (I2).
KB-LF-cells grown in petri-dishes (see legend
Fig. 1) were washed twice with HBSS pH 7.4,
and incubated with either I f.tM NHS-MTX and
2 f.tM FA or 0.3 f.tM NHS-FA and 5 f.tM 10-
EdAM to specifically label the RF/MTX-carrier
or the mFBP respectively. After 10 min. of
incubation at 25°C. the cells were washed twice
again with HBSS pH 7.4 and incubated with I
f.tM [lH]-MTXor rH]-10-EdAMfor2 hrs. After
this, the cells were washed, harvested and
analyzed for radioactivity as described the legend
of Fig. 1.

These results demontrate the presence of at least two functional transport routes for
antifolates in KB cells, the mFBP and the RF/MTX-carrier. The relative contribution of
either one of these routes in (anti)-folate transport is dependent on the medium folate status
and the affmity of these proteins for (anti) ·folate compounds.

773
ACKNOWLEDGEMENTS.

This study was supported by Dutch Cancer Society-Grants IKA- 89-34 and NKI 92-43.
G. Jansen is a recipient of a fellowship from the Royal Netherlands Academy of Arts and
Sciences

REFERENCES

1. F.M. Sirotnak, Obligatt: genetic expression in tumor cells of a fetal membrane property mediating
"folate" transport: Biological significance and implications for improved therapy of human
cancer. Cancer Res., 45: 3992 (1985).
2. G.B. Henderson, Folate binding proteins. Ann.Rev.Nutr., 10: 319 (1990).
3. M. Ratnam, and J.H. Freisheim, Proteins involved in the transport of folates and antifolates by normal
and neoplastic cells, in "Folic Acid Metabolism", Wiley-Liss, Inc., 91 (1990).
4. A. C. Antony, The biological chemistry of folate receptors. Blood, 79: 2807 (1992).
5. G.R. Westerhof, G. Jansen, N van Emmerik, I. Kathmann, G. Rijksen, A.L. Jackman, and J.H.
Schomagel, Membrane transport of natural and antifolate compounds in murine L1210 leukemia
cells: Role of carrier- and receptor-mediated transport systems. Cancer Res., 51: 5507 (1991).
6. J.C. Deutsch, P.C. Elwood, R.M. Portillo, M.G. Macey, and J.F. Kolhouse, Role of the membrane-
associated folate binding protein (folate receptor) in methotrexate transport by human KB cells.
Arch.Biochem.Biophys., 274: 327 (1989).
7. P.C. Elwood, M.A. Kane, R.M. Portillo, and J.F. Kolhouse, The isolation, characterisation, and
comparison of the membrane- associated and soluble folate-binding proteins from human KB
Cells. J.Biol.Chem., 261: 15416 (1986).
8. T.R. Jones, A.H. Calvert, A.L. Jackman, S.J. Brown, M. Jones, and K.H. Harrap, A potent
antitumor quinazoline inhibitor of thymidylate synthetase: synthesis, biological properties and
therapeutic results in mice. Eur.J.Cancer, 17: 11 (1981).
9. G. Jansen, J.H. Schomagel, G.R. Westerhof, G. Rijksen, D.R. Newell, and A.L. Jackman, Multiple
membrane transport systems for the uptake of folate-based thymidylate synthetase inhibitors.
Cancer Res., 50: 7544 (1990).
10. M. Ratnam, H. Marquardt, J.L. Duhring, and J.H. Freisheim, Homologous membrane folate binding
proteins in human placenta: Cloning and sequence of a eDNA. Biochemistry, 28: 8249 (1989).
11. J.H. Freisheim, M. Ratnam, T.P. McAlinden, K.M.R. Prasad, F.E. Williams, G.R. Westerhof,
J.H. Schomagel, and G. Jansen, Molecular events in the membrane transport of methotrexate in
human CCRF-CEM leukemia cells. Adv.Enz.Regul., 32: 17 (1992).
12. G.B. Henderson, and E.M. Zevely, Affinity labeling of the 5-methyltetrahydrofolate/methotrexate
transport protein of L1210 cells by treatment with an N-hydroxysuccimide ester of rH]metho-
trexate. J.Biol. Chem., 259, 4558-4562 (1984).

774
ALTERED TRANSPORT OF FOLIC ACID AND ANTIFOLATES THROUGH
THE CARRIER MEDIATED REDUCED FOLATE TRANSPORT SYSTEM IN A
HUMAN LEUKEMIA CELL LINE RESISTANT TO (6R) 5,10-
DIDEAZATETRAHYDROFOLIC ACID (DDATHF)

Mirjana Pavlovic, Janine J. Leffert, Orsola Russello, Marlene A. Bunni,

G. Peter Beardsley, David G. Priest and Giuseppe Pizzomo
Departments of Internal Medicine and Pediatrics, Yale University School of
Medicine, New Haven, Cf 06510 and Department of Biochemistry and Molecular
Biology, Medical University of South Carolina, Charleston, SC 29425

INTRODUCTION
5,10-Dideazatetrahydrofolic acid is a potent antiproliferative agent in vitro and in
vivo, arresting or delaying the proliferation of tumor cells in a number of murine and human
xenografts models 1,2. In contrast to classical antifolates, (dihydrofolate reductase
inhibitors), DDATHF blocks de novo purine biosynthesis as the result ofa potent inhibition
of both glycinarnide ribonucleotide (GAR) and arninoimidazole carboxarnide ribonucleotide
(AICAR) transformylases.
DDATHF has been described to be an excellent substrate for folylpolyglutamate
synthetase (FPGS), which readily converts DDATHF to higher polyglutamates that are
retained intracellularly 3. Polyglutamylation is recognized to be an essential step in the
activation of DDATHF. Decreased formation of polyglutamates reduces the effectiveness of
DDATHF, since the poly glutamate forms are more potent inhibitors of both GAR and
AICAR transformylases than the monoglutamate form 4.
DDATHF shares the reduced folate transport system with natural folates and classical
antifolates and also utilizes the high affinity low capacity folate binding protein 5. A number
of DDATHF resistant cell lines were developed by continuous exposure of CCRF-CEM
human T-lymphoblastic leukemia cells to increasing concentration of the antifolate. We
describe in this report a DDATHF resistant subpopulation of CCRF-CEM cells able to
proliferate in the presence of 1 f..LM DDATHF. We define the multimodal mechanism of
resistance to DDATHF including impaired polyglutamylation, altered transport and increased
reduced folate pools.

RESULTS and DISCUSSION

The DDATHF resistant cell line CCRF-CEM R16 presented an EDso for
(6R)DDATHF of 2.6 f..LM compared to 0.012 f..LM of the parental cell line. The resistance was
maintained for more then a year without the presence of (6R)DDATHF in the medium.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 775
 3
CCRF-CEM
CCRF-CEM Rl6

....
c
81

0
Glu 1 Glu 2 Glu 3 Glu 4 Glu 5 Glu 6 Glu 7

Fig .1. Polyglutamates accumulation after a 24 hour incubation with l!J.M (6R)DDATHF. Cells were
incubated with l!J.M [3H]-(6R)DDATHF, after 24 hrs. cells were harvested and washed twice in
PBS. The HPLC analysis was perforfermed as described in Reference 3.

No significative difference was observed in the activity and kinetic properties of

several folate related enzymes including the two targets GAR and AICAR transformylases.
However, CCRF-CEM R16 cells showed a dramatic reduction in their capacity to
accumulate (6R) DDATHF polyglutamate forms (Fig.l) and this phenomenon correlated
with decreased FPGS activity whether DDATHF, aminopterin, or tetrahydrofolic acid were
the substrates for the enzyme (Table 1). The effect of DDATHF on the expression of FPGS
gene was examined by Northern blot analysis in sensitive and resistant CCRF-CEM cell
lines. No significant difference in expression was detected between sensitive and resistant
cells (data not shown) suggesting that the cause for this impaired mechanism was not at the
transcriptional level.
The reduction in polyglutamate retention was also investigated through the
distribution and retention of endogenous folates. The polyglutamate chain length moved
from glu6 and glu7 with some glu5 in the sensitive cell line to mostly glu4, glu5 and glu3 in
the resistant cell line (Fig. 2).

Table 1. Activity of FPGS for different folate and antifolate substrates.

Cell Line (6R)DDATHF Aminopterin Tetrahydrofolate

(pmol/h/mg prot.)

CCRF-CEM 750 1280 595

CCRF-CEM R16 352 447 276

FPGS activity was assayed using the method described in Reference 3.

776
 1000
CCRF-CEM CCRF-CEMR16
600 +
600
• • 6 7 8
E
+
400 + + + + + + ++
Q.
u + + + • 7 •

200

0
0 10 20 30 40 so 60 70 80 0 10 20 30 40 so 60 70 80

Fraction Number

Fig.2. Folate polyglutamates distribution in sensitive and resistant CCRF-CEM cells. Folate depleted
cells were incubated for a 24 hour period in 2.2 J1M [3H]-Folic acid. Cells were then harvested,
washed and analyzed as described in Reference 6.

Surprisingly, together with this noticeable shift toward shorter polyglutamate chain
lengths in the resistant cells, we observed a 3-4 fold increase in the total accumulation of
reduced folates. The analysis of individual reduced folate pools revealed that the overall
increase was almost entirely due to the accumulation of 10-formyl-tetrahydrofolate in the
resistant cell line (Fig. 3). This reduced folate is the natural substrate for both GAR and
AICAR transformylases and increases in its intracellular level could certainly contribute to
deazatetrahydrofolate resistance.
This data led us to carefully investigate the transport characteristics of the resistant
cell line compared to the parental CCRF-CEM cells. As shown in Table 2 the resistant
subpopulation CCRF-CEM R16 presented a slightly increased Km for the diffe;rent substrate
used. However there was a 3-4 fold elevation in Vmax for folic acid and a 2 fold increase in

20 ~ lHF
II 10 formyl TiiF
D 5 formyl TiiF
0 15 ~ Folic Acid
>
Iii 5 methyl TiiF
~
<.J
10
-2-
0
e
Q,
5

0
CCRF-CEM CCRF-CEM Rl6

Fig. 3. Reduced folates formation after a 24 hour incubation with [3H]-Folic Acid. Folate depleted cells were
incubated in 2.2 JlM [3H]-Folic acid, after 24 hrs. cells were harvested. washed and ana!Y2ed as
described in Reference 7.

777
 Table 2. Transport parameters of different folates and antifolates in CCRF-CEM cells
sensitive and resistant to (6R)DDATHF.

Cell Lines (6R)DDATHF Methotrexate Folic Acid (6R)DDATHF

binding sites
Kt Vmax Kt Vmax Kt Vmax
uM pmol/min/107 cells uM pmoVmin/107 cells uM pmoVmin/W cells pmoVW cells

CCRF-CEM 1.3 4.8 3.1 6.9 28.9 2.6 0.68

CCRF-CEM R16 2.2 10.4 5.8 10.6 34.0 8.6 1.20

Transport and binding sites detenninations are described in Reference 5.

Vmax for both (6R)DDATHF and methotrexate.The increment in Vmax was associated with
a 2 fold increase in the number of binding sites for the resistant cells.
The possible contribution to folate and (6R)DDATHF incorporation via potocytosis
was investigated in both cell lines and did not reveal any significant involvement.
We conclude that resistance to (6R)DDATHF in CCRF-CEM R16 cells is due to a
series of different mechanisms including impaired polyglutamylation due to diminished
FPGS activity and accumulation of reduced folates, especially 10-formyl-tetrahydrofolate,
possibly related to an altered reduced folate transport system with different kinetic properties
toward both folates and antifolates.

REFERENCES
1. R.G. Moran, E.C. Taylor, and G.P. Beardsley. 5:10-Dideaza-5,6.7,8-Tetrahydrofolate:
a potent antifolate inhibitory to de novo purine synthesis, Proc. Am. Assoc. Cancer
Res. 26:231 (1985)
2. G.P. Beardsley, E.C. Taylor, B.A. Moroson, and R.G. Moran. 5,10-Dideaza- 5,6,7,8-
Tetrahydrotolate: an exceptionally potent inhibitor of de novo purine synthesis. J.
Bioi. Chern. 264:328-333 (1989)
3. G. Pizzorno, J.A. Sokoloski, A.R. Cashmore, B.A. Moroson, A.D. Cross and G.P.
Beardsley: Intracellular metabolism of 5,10-Dideazatetrahydrofolic Acid in human
leukemia cell lines. Mol. Pharm. 39:85-89 (1990)
4. G. Pizzorno, 0. Russello, A.R. Cashmore, B.A. Moroson, A.D. Cross, M. Coronnello,
and G.P. Beardsley. Polyglutamylation: an essential step in the activation of 5,10-
Dideazatetrahydrofolic Acid. Proc. Am. Assoc. Cancer Res. 31: 2005 (1990)
5. G. Pizzorno, A.R. Cashmore, B.A. Moroson, A.D. Cross, A.K. Smith, M. Marling-
eason, B.A. Kamen and G. P. Beardsley: 5,10-Dideazatetrahydrofolic Acid
(DDATHF) transport in CCRF-CEM and MA104 cell lines J. Biol. Chern.
268:1017-1023 (1993)
6. C.J. Allegra, R.L. Fine, J.C. Drake and B.A. Chabner. The effect of methotrexate on
intracellular folates pools in human MCF-7 breast cancer cells: evidence for direct
inhibition of purine synthesis. J. Bioi. Chern. 261:6478-6485 (1986)
7. G. Pizzorno, E. Mini, M. Coronnello, J.J. McGuire, B.A. Moroson, A.R. Cashmore,
R.N. Dreyer, J.T. Lin, T. Mazzei, P. Periti and J.R. Bertino. Impaired
polyglutamylation of methotrexate as a cause of resistance in CCRF-CEM cells after
short-term, high-dose treatment with this drug. Cancer Res. 48:2149-2155 (1988)

778
TRANSFORMATION OF AN L-CELL LINE WITH THE DNA CODING FOR
THE REDUCED-FOlATE/METHOTREXATE TRANSPORTER PROTEIN
FROM A CCRF-CEM HUMAN LEUKEMIA CELL LINE

Frederick E. Williams\ Manohar Ratnam\ Terence P. McAlinden\

Gerrit Jansen2, Jan H. SchornageP, and James H. Freisheim1

1Department of Biochemistry and Molecular Biology

Medical College of Ohio

Toledo, OH 43699-0008 USA

2Department of Oncology

Free University Hospital

1007 MB Amsterdam
The Netherlands

Department of Internal Medicine

3

Netherlands Cancer Institute

1066 ex Amsterdam
The Netherlands

INTRODUCTION

The reduced-folate/methotrexate transporter represents the major avenue of

methotrexate uptake into mammalian cells 1·3• This transport system has a high affinity
for methotrexate (MTX) (K, = 1-3 JLM) and reduced folates with substitutions at
position 5 (3-26 JLM)\ but a correspondingly low affinity for folic acid (K, = 200-400
JLMY. Considerable progress has been made towards elucidation of MTX transport.
The development of folate analogs containing chemically or photodynamically
activatable groups has allowed the identification of specific proteins that could be
involved in the carrier-mediated transport of MTX in both murine and human
systems5-6. Our laboratory has attempted to study the membrane component of the
transporter protein from human leukemic CCRF-CEM cells by cotransfection of
mouse Ltk cells with the herpesvirus thymidine kinase gene and genomic DNA from
the CCRF-CEM-7A cell line. The resulting mousehuman cell lines may be able to be
used to study the protein's regulation. This paper will discuss the creation and partial
characterization of the hybrid cell lines.

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 779
 RESULTS AND DISCUSSION

Transfection of Ltk Cells

Murine fibroblast Ltk cells were cotransfected with the plasmid pDG510
containing the herpesvirus thymidine kinase gene and genomic DNA from CCRF-
CEM-7A cells. HAT selection was carried out in folate free IMDM media with 10
nM folinic acid as the only source of folate. As shown in Table 1, out of a possible
75,000 clones on 75 plates screened, 5 cell clones were identified that were capable of
growing under the selection pressures. This could be considered a high transfection
frequency if compared to other published frequencies using Ltk cells7, and could
represent a situation where multiple copies of the gene are present in CCRF-CEM-
7A cells. The 5 established cell clones (L!7A 1-5) are capable of growing at
concentrations of folinic acid that would be lethal to the parent Ltk cells.

Table 1. Transfection frequency of DNA mediated transfer of function.

Cells Growing in 10 nM
Transfection Number Positive PlatesLiotal FHJ IX+ Cells
1 1/15 1/15,000
2 1/15 1/15,000
3 1/20 1/20,000
4 1/25 2/25,000

Characterization of U7A cell Oones

The cell clones were then partially characterized by Southern blot to establish
that human DNA had been transferred, by uptake assays with 3H-MTX to show that
there was an increase in reduced transporter function, and by labelling studies with an
iodinated photoprobe (APA-1251-ASA-lys) to see if a protein of similar size to the
human reduced folate/MTX transporter was being produced by the new cell clones.

The genomic DNA from the L/7A cell clone, L/7A1, was digested with EcoRI
and run on a 0.8% agarose gel along with equally treated genomic DNA from human
spleen, CCRF-CEM-7A cells, L1210 cells, and Ltk cells. The DNA was blotted by
method of Southern, and probed with the labelled human Alu sequence probe,
pBLUR2. The resultant autoradiogram indicated that the L/7A1 cell clone did have
human DNA in its genome (Figure 1).

HUMAN SPLEEN/ECORI
CCRF-CEM-7A/ECORI
L121 0/ECORI
U7A1 /ECORI
Ltk-/ECORI

Ftgure 1. Autoradiogram or a Southern blot showing introduction o[ human DNA into L{IAl clone. Genomic DNAs were
cut with EcoRI. The probe [or the Southern blot was pBLUR2 containing human Alu sequences. Lane 1, human spleen
DNA; Lane 2, CCRF-CEM-7A DNA; Lane 3, Ll210 DNA; Lane 4, L{IAl DNA; Lane 5, Ltk- DNA

780
 The L/7Al and L/7A3 clones were tested for uptake of 3H-MTX and compared
with Ltk cells and L1210R81 cells. Cells were harvested, washed and resuspended in
HBSS buffer, and given 3H-MTX for 5 minutes at 37°C. The cells were then
centrifuged, washed, and resuspended in scintillation cocktail. The data in Table 2
suggest that the L/7A clones exhibit 1.5-2X the uptake found in Ltk" cells. Also
shown is that L1210R81 cells and a no cell control exhibited no 3H-MTX uptake in
these experiments.

Table 2 3H-MTX uptake in L/7A cell clones.

Uptake of 3H-MTX
Cell Line I!molL107 cellsLS min. % of Wildty~

Ltk 20 100
LnAt 30 150
LnAJ 33 165
L1210R81 0.7 <5
no cells 0.9 <5

Cells from the L/7Al cell clone were exposed to the iodinated photoprobe APA-
1251-ASA-lys both in the presence and absence of 1 mM cold MTX in order to identify
any proteins that the probe would bind to specifically. As shown in Figure 2, a
protein of an approximate molecular weight of 75-80 kDa was labelled by the
photoprobe in L/7Al cells. This is approximately the correct molecular weight for the
human reduced folate carrier, whereas the mouse protein is approximately 46 kDa in
size. The 75-80 kDa protein was protected from labelling by incubation with 1 mM
cold MTX. This protein was not labelled in the parent Ltk cells (data not shown).

205

-
97
69
46

30

23

Figure 2. A 75-80 kDa protem from L/7Al cells bmds to the photoprobe APA-125!-ASA-lys L/7Al cells were exposed to the
photoprobe m the presence and absence of 1 mM cold MTX at 4°C Membrane protems were prepared, run on SDS-PAGE,
and subjected to autoradiography Arrow pomts to a protem ol 75 80 kDa that IS labelled m L/7Al cells (Lane 1) and
protected from labellmg With 1 mM cold MTX (Lane 2)

781
 Taken together this data suggests that we have established mouse cell lines that
express a human protein that is approximately 75-80 kDa in size that is responsible
for increased uptake of 3H-MTX and viability in nM concentrations of folinic acid as
the only source of folates. Each piece of data supports this point. The Southern blot
shows a transfer of human DNA to the cell clones. The increase in 3H-MTX uptake,
although not great could be explained by a doubling of the amount of the reduced
folate carrier. This possibility is supported by the iodinated photoprobe binding and
identification of a 75-80 kDa protein that is specifically bound by the photoprobe that
is not seen in Ltk cells. This is the postulated size of the human reduced folate/MTX
carrier protein leading us to believe that we have isolated this protein away from the
CCRF-CEM-7A cells. Using these cell clones, and after further characterization, we
may be able to answer some preliminary questions about the regulation of this
protein.

REFERENCES

1. F.M. Sirotnak, Obligate genetic expression in tumor cells of a fetal membrane

property mediating "folate" transport: Biological significance and
implications for improved therapy for human cancer, Cancer Research
45:3992-4000 (1985).
2. J.H. Freisheim, E.M. Price, and M. Ratnam, Folate coenzyme and antifolate
transport proteins in normal and neoplastic cells, Advances in Enzyme
Regulation 29:13-26 (1989).
3. I.D. Goldman and L.H. Matherly, The cellular pharmacology of methotrexate,
Pharmaco 1ogy and Therapeutics 28:77-102 (1985).
4. G. Jansen, J.H. Schornagel, G.R. Westerhof, G. Rijksen, D.R. Newell, and A.L.
Jackman, Multiple membrane transport systems for the uptake of folate-
based thymidylate synthase inhibitors, Cancer Research 50:7544-7548
(1990).
5. E.M. Price and J.H. Freisheim, Photoaffinity analogues of methotrexate as
folate antagonist binding probes. 2. Transport studies, photoaffinity
labelling, and identification of the membrane carrier protein fro
methotrexate from murine L1210 cells, Biochemistry 26:4757-4763
(1987).
6. J.H. Freisheim, M. Ratnam, T.P. McAlinden, KM.R. Prasad, F.E. Williams,
G.R. Westerhof, J.H. Schornagel, and G. Jansen, Molecular events in the
membrane transport of methotrexate in human CCRF-CEM leukemia cell
lines, Advances in Enzyme Regulation 32:17-31 (1992).
7. C. Mendelsohn, B. Johnson, KA. Lionetti, P. Nobis, E. Wimmer, and V.R.
Racaniello, Transformation of a human poliovirus receptor gene into
mouse cells, Proceedings of the National Academy of Sciences U.S.A.
83:7845-7849 (1986).

782
DETERMINANTS OF THE DISPARATE ANTITUMOR EFFECTS OF (6R)5,10-
DIDEAZA-5,6, 7,8-TETRAHYDROFOLATE AND METHOTREXATE TOWARD
METHOTREXATE RESISTANT CCRF-CEM CELLS, CHARACTERIZED BY
SEVERELY IMPAIRED ANTIFOLATE MEMBRANE TRANSPORT

Larry H. Matherly and Susan M. Angeles

Developmental Therapeutics Program

Michigan Cancer Foundation
Detroit, MI 48201

INTRODUCTION

The antifolate, (6R) 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF), derives its

antitumor effects by inhibiting 5'-phosphoribosylglycinamide (GAR) transformylase 1, the
first folate-dependent enzyme in the de novo purine nucleotide biosynthetic pathway.
Although DDATHF uptake by mammalian cells is generally mediated by the "classical"
membrane carrier for methotrexate (MTX) and reduced folates2 , with transport impaired
MTX resistant cells, cross resistance is often not observed3•4 • In this study, we assess
the roles of membrane transport and polyglutamylation in the disparate pharmacologic
activities of MTX and (6R) DDATHF toward a highly MTX resistant CCRF-CEM cell
line. Impaired MTX transport in this variant is associated with the synthesis of a
structurally modified isoform of the MTX transporter.

MATERIALS AND METHODS

Chemicals. Unlabelled and (benzoyl carboxyl- 14C) ( 6R)D D ATHF ( 13.01 p. Ci/ mg) were
provided by Lilly Research Laboratories. Unlabelled MTX and (6R,S)5-formyltetra-
hydrofolate were obtained from the Drug Development Branch, National Cancer Institute.
[3' ,5' ,7-3H]MTX was purchased from Moravek Biochemicals.

Cell Culture. The CCRF-CEM human T -cell leukemia and a MTX resistant sub line
(CEM/MTX6) were gifts from Dr. Andre Rosowsky. Cells were cultured as described
earlier. Drug cytotoxicity assays were detailed in our earlier report5•

Assays for Antifolate Transport and Polyglutamylation. Transport experiments

with 3H-MTX and 14C-DDATHF were performed as described previously 6 • Kinetic
constants (i.e., Kt and Vmax, and Ki values) for assorted (anti) folate substrates were
calculated from Lineweaver-Burke and Dixon plots, respectively. For analysis of intra-
cellular MTX and DDATHF polyglutamates, saline-washed cell pellets were suspended
in 50 mM sodium phosphate, pH 6.0, containing 100 mM 2-mercaptoethanol and boiled

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling eta/., Plenum Press, New York, 1993 783
(5 min). Following centrifugation, the supernatants were fractionated by reversed phase
HPLC on a 5 ~tm octadecylsilyl column (4.5 x 250 mm), using a gradient of 0-15%
acetonitrile in 0.1 M sodium acetate buffer (pH 5.5) over 20 min, followed by a 5 min
isocratic elution at 15% acetonitrile. The flow rate was 2 mllmin; 0.3 min fractions
were assayed for radioactivity. Tritiated MTX polyglutamates were identified by
comparison of elution times to those of unlabelled standards7· DDATHF polyglutamates
were confirmed by digestions with hog kidney conjugase7 •

RESULTS

Although CEM/MTX cells were highly resistant to MTX (243-fold), they were only
3.6-fold resistant to (6R)DDATHF (IC50s of 16.2 nM and 59 nM for parental and
CEM/MTX cells, respectively). The possibility that differences in membrane transport
between DDATHF and MTX might account for the disparate sensitivities of the
CEM/MTX line to these agents was examined. As depicted in Figure 1 (left panel),
initial uptakes of both 3H-MTX and 14C-DDATHF (at 2 ~tM) were significantly impaired
(95-97%) in CEM/MTX cells. For MTX, this was reflected in a reduced Vmax (0.78
versus 4.56 pmol/mg/min for the parent) and an increased Kt (12.57 versus 3.86 ~tM).
However, decreased DDATHF uptake was entirely due to a reduced Vmax (0.17 versus
3.22 pmol/mg/min). Interestingly, the affinity of the CEM/MTX carrier for DDATHF
was higher (Kt=0.16 ~tM) than in parental cells (Kt=0.47 ~tM).
Uptake of 14C-DDATHF in parental cells was inhibited by MTX (Ki = 4.7 ~tM),
(6R,S)5-formyl tetrahydrofolate (Ki = 2.2 ~tM), and folic acid (Ki = 148 ~tM). While
14C-DDATHF uptake into CEM/MTX cells was likewise inhibited, significant differences

were measured not only in the binding of MTX, as described above, but also for folic
acid (Ki=8.9~tM) and (6R,S)5-formyl tetrahydrofolate (Ki=0.73~tM). The MTX Ki was
11.9 ~tM, approximating the Kt for 3H-MTX uptake in CEM/MTX cells.
Unlike MTX, 14C-DDATHF continued to accumulate in CEM/MTX cells over an
extended interval (Fig. 1, right panel). After 4 h, the intracellular DDATHF level
was 62% of that in parental cells. Total DDATHF accumulations by this time exceeded
those for MTX by 5.4- (parent) and 7.9-fold (CEM/MTX).
For both lines, the intracellular drug forms from 14C-DDATHF were mostly
polyglutamates. Five DDATHF polyglutamates (PG1-5 in Table 1) could be resolved
by HPLC. This metabolism was extensive and nearly identical between the lines (Fig.
2, main graph). After 4 hours, 53% (parent) and 71% (CEM/MTX) of the intracellular

32 ~----·----·----·

.f
./
24 J -~-~-------0
16 ----~6----
~,,"'
8 ...Li • :&
(J /::,. /::,.••• -----/::,.
0 6:/::,.--·- -------- -------
0 60 120 180 240 300 0 2 4

Seconds Hours

Figure 1. Uptake of 14C-DDATHF and 3H-MTX in Parental CCRF-CEM (solid lines) and CEM/MTX
Cells (broken lines). The left panel shows the initial drug influx whereas the right panel illustrates drug
uptake over a 4 hour interval. The symbols are as follows: MTX, triangles; DDATHF, circles.

784
 '%-a 0
4

2 ......-·---::-~,.......~~i
" .• «•"
Q ......... ..& ••••• ~-•••·•

0 4
24

s
Ol

"0
sP. 16

0 2 3 4

Hours

Figure 2. Time Course of Accumulation of Antifolate Mono- and Poly-

glutamates. Parental (solid lines) and CEM/MTX (broken lines) cells
were incubated with 3H-MTX (inset) or 14C-DDATHF (main graph). Intra-
cellular monoglutamate (open symbols) and polyglutamate (solid symbols)
forms were quantitated as described in Materials and Methods.

drug was polyglutamyl DDATHF. The distributions of the individual DDATHF forms
at 4 h are compared in Table 1. Slightly different polyglutamate distributions were
found, with PG4 predominating in parental cells (29.5% of the total drug), and PG5 the
major form in CEM/MTX cells (51% of the total drug).ThreeMTXpolyglutamates(di-
through tetraglutamates) were detected in parent cells incubated 4 h with 2 ~M MTX;
30% of the intracellular MTX was polyglutamylated (Figure 2, inset). For CEM/MTX
cells, 2.5% of the intracellular radiolabel was polyglutamyl MTX (diglutamate only).
After 4 h, net accumulations of DDATHF polyglutamates in parental and CEM/MTX
lines exceeded those for MTX by 8.4- and 237-fold, respectively,

DISCUSSION

The inhibitions of 14C-DDATHF uptake by assorted (anti) folates closely resemble

those for MTX and establish a shared high affinity transport system for DDATHF,
MTX, and 5-formyl tetrahydrofolate in parental CCRF-CEM cells. Competition for
uptake was also observed in the CEM/MTX line, however, binding affinities for these
compounds were substantially changed. This likely reflects the structural alterations in
the classical membrane carrier in this line5 • In any case, transport of both MTX and
DDATHF was severely impaired, indicating that the disparate sensitivities of CEM/MTX
cells to these antifolates were not due to an effect at this level.
In contrast to MTX, the excellent substrate properties of DDATHF for
folylpolyglutamate synthetase1 facilitate its extensive polyglutamylation, irrespective of
the intracellular level of monoglutamyl antifolate. An apparent consequence of the
different intracellular levels of competing monoglutamyl DDATHF in parental and
CEM/MTX lines is the accumulation of longer chain length polyglutamates in the latter.
Total polyglutamate levels (after 4 h) in the two lines were nearly identical. The
pharmacologic significance ofDDATHF polyglutamate synthesis involves the conversion
of monoglutamyl antifolate to a highly retained form 8 capable of exerting up to a 100-
fold greater inhibition at GAR transformylase than unmetabolized DDATHf9.
Hence, even for tumor cells with severely impaired antifolate transport, the exten-
sive conversion of DDATHF to polyglutamyl forms required for GAR transformylase

785
 Table 1. Distribution of DDATHF Polyglutamates in Parental and CEM/MTX Cells.

Cell Line Total DDATHF POl P02 P03 P04 P05 Total POl-5
(All fQIIJJS)
pmol/mg pmol/mg

Parent 32.68 16.40 0.51 1.29 2.19 9.63 2.69 16.31

CEM/MTX 20.17 5.44 0.11 0.21 0.69 3.47 10.22 14.70

Cells were incubated for 4h with 2~M radiolabeled DDATHF. Drug quantitation and HPLC
analysis are described in Materials and Methods. PO 1·5 correspond to the DDATHF polyglutamates
with increasing glutamate chain' lengths. HPLC retention times are as follows: POl (15.3 min);
P02 (13.5 min); P03 (12 min); P04 (11.1 min); P05 (10.2 min); DDATHF (18.3 min.)

preserves high levels of antitumor activity. An analogous effect might be expected for
other antifolates which are polyglutamylated to high levels, and for other tranport-
impaired cells with adequate drug influx to sustain polyglutamate synthesis, and for
which folylpolyglutamate synthesis is largely intact.

ACKNOWLEDGEl\.fENT

We thank Dr. Andre Rosowsky for the gifts of the CCRF-CEM and CEM/MTX
lines and Drs. Chuan Shih and Gerald Grindey for providing the unlabelled and 14C -
labelled (6R)DDATHF. This work was supported by grants from the Public Health
Service (CA-53535) and the American Cancer Society (DH-30C). L.H. Matherly is a
recipient of a Scholar Award from the Leukemia Society of America, Inc.

REFERENCES

1. R.G. Moran, S.W. Baldwin, B.C. Taylor, and C.J. Shih, J. Biol. Chern. 264:21047
(1989).
2. G. Pizzorno, A.R. Cashmore, B.A. Moroson, A.D. Gross, A.K. Smith, M.
Marling-Cason, B.A. Kamen, and G.P. Beardsley, J. Biol. Chern. 268:1017 (1993).
3. F.M. Sirotnak, G.M. Otter, J.R. Piper, and J.I. DeGraw, Biochem. Pharmacol. 37:
4775 (1988).
4. G. Jansen, G.R. Westerhof, I. Kathmann, G. Rijksen, and J.H. Schornagel, Cancer
Chern. Phannacol. 28: 115 (1991).
5. L.H. Matherly, S.M. Angeles, and C.A. Czajkowski, J. Bioi. Chern. 267: 23253
(1992).
6. A. Rosowsky, H. Lazarus, G.C. Yuan, W.R. Beltz, L. Mangini, H.T. Abelson,
E.J. Modest, and E. Frei, Biochem. Phannacol. 29: 648 (1980).
7. D.W. Fry, J.C. Yalowich, and I.D. Goldman, J. Biol. Chern. 257: 1890 (1982).
8. G. Pizzorno, J.A. Sokoloski, A.R. Cashmore, B.A. Moroson, A.D. Cross, and G.P.
Beardsley, Mol. Phannacol. 39: 85 (1991).
9. S.W. Baldwin, A. Tse, L.S. Gossett, E. C. Taylor, A. Rosowsky, C. Shih, and R.G.
Moran, Biochemistry 30: 1997 (1991).

786
UP-REGULATED TRANSPORT OF METHOTREXATE AND 5-METHYL-
TETRAHYDROFOLATE IN A HUMAN BREAST CANCER CELL LINE

Frederika Mandelbaum-Shavit

Department of Bacteriology
Hebrew University-Hadassah Medical School
Jerusalem, Israel

INTRODUCTION

In a majority of tumor cells, a high affinity/low capacity transport system

mediates the uptake of 5-substituted reduced folates and the analog methotrexate
(MTX), 1' 3 an important chemotherapeutic agent. The relatively low levels of the MTX
carrier prompted studies on selecting variants able to up-regulate this system. Such
strategy was first introduced by Sirotnak4, who by cultivating L1210 cells in
progressively decreasing and growth limiting concentrations of (6R,S)-5-
formyltetrahydrofolate (5-HCO-H4PteGlu), selected cells with increased transport
capacities for this compound. A similar approach was used to obtain a variant of
human erythroleukemia line that exhibited elevated influx Vmax for MTX and 5-
HCO-H4PteGlu relative to the parental cells'. Up-regulation of the reduced folate
carrier was also found in human CCRF-CEM leukemia cells adapted to grow on <
1 nM 5-HCO-H4PteGJu," whereas cultivation in the presence of low concentrations
of folate induced an increased level of a membrane-associated folate-binding
protein7'8 •
The purpose of the present study was to select MCF-7 cells capable to grow
in the presence of physiological (nM) concentrations of 5-HCO-H4PteGlu
approaching the range usually found in human serum". Studies of transport of the
reduced folates and the analog, MTX, as well as examination of the susceptibility of
these cells to the latter, might provide data more reflecting the conditions in vivo than
investigation of cells in media with standard (p.M) concentrations of folates.

RESULTS AND DISCUSSION

Selection and Transport Characteristics of MCF-7 Cell Variants Adapted to Grow

in the Presence of nM Concentrations of S-HCO-H4PteGlu

MCF cells were cultured in folate-free RPMI1640 medium supplemented with

10% dialyzed fetal bovine serum and stepwise decreasing concentrations of 5-HCO-

Chemistry and Biology of Pteridines and Folates, Edited by

J.E. Ayling et al., Plenum Press, New York, 1993 787
H 4PteGlu as the sole folate source. This procedure resulted in isolation of a variant,
MCF-7FLN, capable to grow in the presence of 1 nM 5-HCO-H 4PteGlu at a rate
similar to that of the parental strain in the standard medium.
Studies of the time course of influx of MTX or 5-methyltetrahydro-folate
(5-CH3-H4PteGlu) at a concentration of 3 J.i.M exhibited a 10-12 fold higher uptake
capacity (steady-state accumulation) of the variant (not shown) than that of the
parentalline10• Kinetic constants for transport of MTX and 5-CH3-H4PteGlu in both
lines were calculated by Lineweaver-Burk analysis of initial uptake velocities
measured during the first min of exposure. As summarized in Table 1, the apparent
influx Km values for MTX and 5-CH3-H4PteGlu in parental and variant cells showed
only minor differences. However, striking differences were found in the Vmax values.
Thus the markedly elevated uptake capacity for MTX and 5-CH3-H 4PteGlu in the
MCF-7FLN cells appears to be due to a 26- and 21-fold elevated Vmax for these
compounds, respectively. The apparent Ki for5-HCO-H 4PteGlu measured with [3H]
MTX in the presence of 5 J.i.M 5-HCO-H4PteGlu was 3.52 ± 0.30 J.i.M in the parental
line and almost the same in the variant. The affinity for influx of folate was markedly
lower; the apparent Km value was 185 ± 2.58 J.i.M for the parental line and not
significantly different in the variant (data not shown).
Similar phenomena of increased transport capacity for the reduced folates and
MTX due to elevated Vmax in cells adapted to grow in the presence of physiological
concentrations of 5-HCO-H4PteGlu were previously found in human erythroleukemia
cells', variants of L12104,1 1 and human CCRF-CEM leukemia".

Table 1. Kinetics of transport of 5-CH3-H 4PteGlu and MTX in MCF-7 and

MCF-7FLN cells.

MCF-7 MCF-7FLN
Compound Km (J.i.M) Vmax (pmol/min Km (J.i.M) Vmax(p1mVmin
mg protein) mg protein)

5-CH3- H 4PteGlu 3.25±0.28" 11.60±0.91 3.10±0.26 243.63±21.61

MTX 7.85±0.61 6.55±0.59 7.91±0.65 170.31±15.12

Exponential cultures at almost confluency in 35x10 mm dishes were washed with a transport buffer
(HBSS) 4 containing: 107 mM NaC!, 20 mM Hepes, 26.2 mM NaHCo 3. 5.3 mM KC!, 1.9 mM CaCI,,
1.0 mM MgC1 2 and 7.0 mM glucose, pH 7.2 with NaOH. ['HJ MTX, sodium salt, sp. act. 250
mCi/mmol and 5-[ 14 CJ-methyltetrahydrofolate, barium salt, sp. act. 58 mCi/mmol, both from
Amersham, England, were purified and quantitated by spectrophotometryl 2• The labeled compound
was added in 1 ml of transport buffer and the dishes were incubated at 37" C. The uptake was
terminated by rapid washes with an ice-cold phosphate-buffered saline. Further cell processing for
radio-activity and protein quantitation was as described previously 13• Initial uptake kinetics were
determined in cells incubated for 1 min with various concentrations of the compound examined (at
a range from 0.5 to 20 ~M).
" Mean ± S.E. of three experiments in triplicate.

Down-Regulation of the Transport Capacity for Reduced Folates and MTX in

MCF-7FLN Cells

Propagation of the variant in a standard medium containing 2.2 J.i.M of folate

788
reversed the uptake capacity to that of the parental cells (not shown). A markedly
decreased transport capacity was also achieved by relatively short incubation (2 h) of
the MCF-7FLN cells in a medium with 50 nM 5-HCO-H4PteGlu. As depicted in Fig.
1, the accumulation of MTX at the steady state decreased 4.6-fold in these cells as
compared to the untreated controls. Similar phenomenon was also found in human
CCRF-CEM leukemia cells6•

400
c
·a;
0
Q.
Ol
300
_§
0
EQ.

200
Q)
c;;
X
~
0
.r;
Q) 100
::;;;

30

Figure 1. Down-regulation of transport of MTX. 5-HCO-H 4PteGlu was added, up to a concentration

of 50 nM, to exponential cultures at almost confluency (35x10 mm dishes) growing in RPM! medium
with 1 nM of the reduced folate. After incubation of the cultures for 2 hat 37°C, the medium wa>
removed and the cell layers were washed twice with the transport buffer. Cells incubated in medium
with 1 nM 5-HCO-H 4PteGlu (controls) were treated similarly. eHJ MTX (3 1-LM) in 1 ml of tran>port
buffer was added and the cells were incubated at 37°C. The uptake was terminated at the indicated
time intervals and further procedures were as described in the legend of Table I. MCF-7FLN cell;,
preincubated with 1 nM (0) and 50 nM 5-HCO-H 4PteGlu (I). Point> represent the mean value of
three experiments in triplicate with S.E. within the symbols.

Susceptibility of MCF-7 and MCF-7FLN Cells to MTX

The increased capacity of the variant to accumulate MTX prompted us to examine

the susceptibility of these cells to the drug. Continuous exposure (96 h) of MCF-7
cells and the variant to increasing concentrations of MTX caused inhibition of growth
with IC50 values of 26.90±1.80 and 7.50+0.64 nM respectively (Fig. 2). The 3.6-fold
higher susceptibility of MCF-7FLN than that of the parental cells partially correlates
with the increased uptake of the drug by this variant.
The presented study describes a reversible induction of a high capacity
transport system for the reduced folates and MTX in a human breast cancer cell line.
Enhanced transport of MTX and its increased cytotoxicity in tumor cells adapted to
grow at nM concentrations of a 5-substituted reduced folate, conditions reflecting
those existing in vivo, appears to be of importance in determination of protocols for
chemotherapy with antifolates.

789
 ec
0
(.)

10 100 1000
Methotrexate ( nM )

Figure 2. Inhibition of growth ofMCF-7 and MCF-7FLN cells by MTX. MCF-7 and MCF-7FLN cells
were cultivated in a standard RPMI 1640 and a folate-free medium containing 1 nM 5-HCO-
H4PteGlu, respectively. Cells (3x10 3) plated in 96-well plates were incubated for 24 h in a humidified
5% C0 2 atmosphere at 37° C. MTX was added in 20 ~1 medium and the cultures were incubated for
additional 72 h. Cell growth was measured hy a MTT colorimetric assay 14 • MCF-7 cells (I), MCF-
7FLN cells (0). Points represent the mean value of three experiments in triplicate with S.E. within
the symbols.

REFERENCES

1. I.D. Goldman, Ann. NY Acad. Sci. 186:400 (1971).

2. F.M. Sirotnak, Cancer Res. 45:3992 (1985).
3. M. Ratnam, and J.H. Freisheim in: Folic Acid Metabolism in Health and
Disease, pp. 91-120. Wiley-Liss, Inc., New York (1990).
4. F. Sirotnak, D.M. Moccio, and C.-H. Yang, J. Bioi. Chern. 259:13139
(1984).
5. L.H. Matherly, C.A. Czajkowski, and S.M. Angeles, Cancer Res. 51:3420
(1991).
6. G. Jansen, G.R. Westerhof, M.J.A. Jarmuszewski, I. Kathmann, G. Rijksen,
and J.H. Schornagel, J. Bioi. Chern. 265:18272 (1990).
7. G.B. Henderson, J.M. Tsuji, and H.P. Kumar, J. Membr. Bioi. 101:247
(1988).
8. G. Jansen, G.R. Westerhof, I. Kathmann, B.C. Rademaker, G. Rijksen, and
J.H. Schornagel, Cancer Res. 49:2455 (1989).
9. V. Herbert, A.R. Larrabee, and J.M. Buchanan, J. Clin. Invest. 41:1134
(1962).
10. F. Mandelbaum-Shavit, in: Chemistry and Biology of Pteridines, pp. 1150-
1153. C.H. Curtius, S. Ghisla, and N. Blau, eds., Walter de Gruyter, Berlin
(1990).
11. C.-H. Yang, J. Pain, and F.M. Sirotnak, J. Bioi. Chern. 267:6628 (1992).
12. F. Mandelbaum-Shavit, and N. Grossowicz, J. Bacterial. 104:1 (1970).
13. F. Mandelbaum-Shavit, in: Chemistry and Biology of Pteridines, pp. 855-
859, B.A. Cooper, and V.M. Whitehead, eds., Walter de Gruyter, Berlin
(1986).
14. T. Mosmann, J. Immunol. Meth. 65:55 (1983).

790
PARTICIPANTS

Dr. Ann Abraham Dr. Steven Bailey

Dept. of Biochemistry Dept. of Pharmacology
University of South Alabama College of Medicine
College of Medicine University of South Alabama
Mobile, AL 36688 Mobile, AL 36688
USA USA
FAX: 1-205-460-6127 FAX: 1-205-460-6798
Phone: 1-205-460-6848 Phone: 1-205-460-6787
Dr. Panagiotis Anastasiadis Dr. Mesbaheddin Balaghi
William T. Gossett Neurology Labs. Dept. of Biochemistry
of Henry Ford Hospital Vanderbilt Univ. Medical School
Metropolitan Center for High Technology 865 Bellevue Road #B-3
2727 Second Avenue, Room 302 Nashville, TN 37221
Detroit, MI 48201 USA
USA FAX: 1-615-343-2704
FAX: 1-313-963-4021 Phone: 1-615-382-6345
Phone: 1-313-963-4020
Dr. Shanker Balasubramanian
Dr. Wilfred L. F. Armarego Dept. of Chemistry
Div. of Biochem. & Molec. Biology Pennsylvania State Univ.
John Curtin School of Med. Res. 415 Wartik Laboratory
The Australian Nat. University University Park, PA 16803
P.O. Box 334 USA
Canberra City, A.C.T. 2601 FAX: 1-814-865-2973
Australia Phone: 1-814-865-9508
FAX: 61-6-249-0415
Phone: 61-6-249-2598 Dr. Malgorzata Balinska
Nencki Inst. of Exp. Biol.
Dr. Akira Ashida 3 Pasteur Street
Dept. of Medical Chemistry PL-02-093 Warszawa
Osaka Medical College Poland
2-7 Daigakumachi FAX: 48-22-22-53-43
Takatsuki, Osaka 569 Phone: 48-2-659-85-71 ext. 328
Japan
FAX: 81 726 81 3723 Dr. Barry Barclay
Phone: 81 726 83 1221 ext. 2453 Dept. of Genetics
University of Alberta
Dr. June E. Ayling Edmonton, Alberta
Dept. of Pharmacology T6G 2E9
College of Medicine Canada
University of South Alabama FAX: 1-403-492-1903
Mobile, AL 36688 Phone: 1-403-492-5376
USA
FAX: 1-205-460-6798
Phone: 1-205-460-6128

791
 Dr. Charles Barnett Dr. Timothy R. Billiar
Lilly Research Laboratories University of Pittsburgh
Eli Lilly and Company 497 Scaife Hall
Lilly Corporate Center Pittsburgh, PA 15261
Indianapolis, IN 46285-4813 USA
USA FAX: 1-412-648-9551
FAX: l-317-276-4507 Phone: 1-412-648-9862
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Dr. Raymond L. Blakley
Dr. Gertrud Cremer-Bartels Dept Biochem & Clin. Pharm.
Augenklinik St. Jude Children's Res Hospital
Westflilische Wilhelms Universitat 332 North Lauderdale, PO Box 318
Domagkstrasse 15 Memphis, TN 38101-0318
D-4400 Miinster USA
Germany FAX: 1-901-521-1668
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Dr. Nenad Blau
Dr. Charles M. Baugh Univ. of Ziirich, Dept. of Ped.
College of Medicine Division of Clin. Chemistry
University of South Alabama Steinwiesstrasse 75
Mobile, AL 36688 CH-8032 Ziirich
USA Switzerland
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Vassilios Bavetsias Ben Blount

Institute of Cancer Research Molecular and Cellular Biology
Drug Development, CRC Laboratories Univ. of Califorina, Berkely
15 Cotswold Road 416 Barker Hall
Sutton, Surrey SM2 5NG Berkeley, CA 94720
United Kingdom USA
FAX: 44-81-770-7899 FAX: l-510-643-7935
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Dr. G. Peter Beardsley Dr. Andrew Bognar
Dept. of Pediatrics University of Toronto
Yale University Department of Microbiology
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New Haven, CT 06510 Toronto, MSS lAl, Ontario
USA Canada
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Patricia Benkovic Teodoro Bottiglieri
The Pennsylvania State Univ. Baylor Univ. Med. Center
415 Wartik Laboratory 3812 Elm Street
Dept. of Chemistry Dallas, TX 75226
University Park, PA 16802 USA
USA FAX: 1-214-820-4952
FAX: 1-814-865-2973 Phone: 1-214-820-2687
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Dr. Peter H. Boyle
Dr. Stephen J. Benkovic Department of Chemistry
Department of Chemistry Trinity College
Pennsylvania State Univ. Dublin 2
415 Wartik Laboratory Ireland
University Park, PA 16802 FAX: 353-1-712826
USA Phone: 353-1-7021423
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Phone: 1-814-865-2882 Dr. F. Thomas Boyle
Zeneca Pharmaceuticals
Dr. Joseph R. Bertino Mereside, Alderley Park
Sloan Kettering Cancer Center 601RL Macclesfield SK10 4TG
430 E. 67th Street United Kingdom
New York, NY 10021 FAX: 44 625 583074
USA Phone: 44 625 513330
FAX: 1-212-639-2767
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792
Michael P. Brand
Institute of Child Health
University of London
30 Guilford Street
London WC1N 1EH
United Kingdom
FAX: 44 71 831 0488
Phone: 44 71 242 9789
Dr. Claudette M. Briand
Lab. de Physique Pharmaceutique
Faculte de Pharmacie
27 bd Jean Moulin
F-13385 Marseille Cedex 5
France
FAX: 33 91 80 26 12
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Dr. Gene Brown
Department of Biology 16-512C
Massachusetts Inst of Tech.
77 Massachusetts Ave.
Cambridge, MA 02139
USA
FAX: 1-617-253-8699
Phone: 1-617-253-0882
Dr. Marlene Bunni
Dept. of Biochemistry
Medical Univ. of South Carolina
171 Ashley Ave
Charleston, SC 29425
USA
FAX: 1-803-792-4322
Phone: 1-803-792-2331
Dr. Hilary Calvert
Division of Oncology
Newcastle General Hospital
Westgate Road
Newcastle Upon Tyne NE4 6BE
United Kingdom
FAX: 44-91-226-1170
Phone: 44-91-273-8811 ext.22655
Robert Carr
Department of Chemistry
Pennsy~vania State Univ
415 Wartik Laboratory
University Park, PA 16802
USA
FAX: 1-814-865-2973
Phone: 1-814-865-2882
Linda H. Chen
Univ. of California, Berkeley
119 Morgan Hall
Berkeley, CA 94720
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Phone: 1-510-642-0750
Yun-jung Choi
Univ. of California, Berkeley
119 Morgan Hall
Berkeley, CA 94720
USA
FAX: 1-510-642-0535
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Dr. Srinivas Chunduru
Dept. of Pharmacology
St. Jude Children's Res. Hosp.
332 N. Lauderdale
Memphis, TN 38105
USA
FAX: 1-901-521-1668
Phone: 1-901-522-0449
Dr. Joanna Ciesla
New York State Dept. of Health
Wadsworth Ctr. for Labs & Res.
Albany, NY 12201-0509
USA
FAX: 1-518-473-2900
Phone: 1-518-486-2567
Dr. Stephen Clarke
15 Cotswold Rd, Block E
Belmont, Sutton
Surrey SM2 5NG
United Kingdom
FAX: 44 81 770 7885
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Dr. Vivian Cody
Medical Foundation of Buffalo
73 High Street
Buffalo, NY 14203
USA
FAX: 1-716-852-4846
Phone: 1-716-856-9600
Dr. Robert J. Cook
Dept. of Biochemistry
Vanderbilt University Med. School
Nashville, TN 37232
USA
FAX: 1-615-322-4349
Phone: 1-615-322-6330
Dr. Bernard Cooper
Hematology Department
Royal Victoria Hospital, Rm. A 202
687 Pine Ave. West
Montreal, Quebec H3A 1A1
Canada
FAX: 1-514-842-8459
Phone: 1-514-843-1558
Dr. Edwin A. Cossins
Department of Botany
University of Alberta
B414 Biological Sci. Ctr.
Edmonton, Alberta T6G 2E9
Canada
FAX: 1-403-492-9457
Phone: 1-403-492-3991
Dr. Richard G.H. Cotton
The Murdoch Institute
Royal Children's Hospital
Flemington Rd., Parkville
Melbourne, Victoria 3052
Australia
FAX: 61-3-348-1391
Phone: 61-3-345-5045
793
 Dr. Gale Craviso Dr. John Donlon
Phannacology Dept. Department of Biochemistry
Univ. of Nevada Sch. of Med. University College Galway
Howard Bldg., Rm. 219 National Univ. of Ireland
Reno, NV 89557 Galway
USA Ireland
FAX: 1-702-784-1620 FAX: 353-91-25700
Phone: 1-702-784-4118 Phone: 353-91-24411 ext. 2412
Dr. H.-Ch. Curtius James T. Drummond
Univ. of Ziirich, Dept. of Ped. University of Michigan
Division of Clin. Chemistry Biophysics Research Division
Steinwiesstrasse 75 1240 IST
CH-8032 Ziirich 2200 Bonisteel Blvd.
Switzerland Ann Arbor, M1 48109
FAX: 41 1 266 7171 USA
Phone: 41 1 266 7544 FAX: 1-313-764-3323
Phone: 1-313-747-1829
Dr. Colette Daubner
Dept. of Biochemistry/Biophysics Dr. David Duch
Texas A&M University Burroughs Wellcome Company
College Station, Texas 77843-2128 The Wellcome Res. Labs.
USA 3030 Cornwallis Road
FAX: 1-409-845-9274 Research Triangle Park, NC 27709
Phone: 1-409-845-6832 USA
FAX: 1-919-248-8375
Dr. Thomas J. Delia Phone: 1-919-248-4363
Dept. of Chemistry
Central Michigan University Michelle Duffourc
Mt. Pleasant, M1 48859 University of South Alabama
USA Phannacology Dept., MSB 3130
FAX: 1-517-774-7106 College of Medicine
Phone: 1-517-774-3981 Mobile, AL 36688
USA
Anjali Desai FAX: 1-205-460-6798
Department of Biochemistry Phone: 1-205-460-6063
College of Medicine
University of South Alabama Dr. Jolanta Dzik
Mobile, AL 36688 Nencki Inst. of Exp. Biology
USA 3 Pasteur St.
FAX: 1-205-460-6127 PL-02-093 Warszawa
Phone: Poland
FAX: 48-22-22-53-42
Dr. Inderjit Dev Phone: 48-2-659-85-71 ext.297
Wellcome Research Labs.
3030 Corwallis Road Dr. Patrick Elwood
Research Triangle Park, NC 27709 Medicine Branch
USA National Cancer Institute
FAX: 1-919-248-8375 Bldg. 10
Phone: 1-919-248-4314 Bethesda, MD 20892
USA
Rajesh Devraj FAX: 1-301-402-0172
Duquesne University Phone: 1-301-496-6035
School of Phannacy
Pittsburgh, PA 15282 Todd Engle
USA Baylor Univ. Med. Center
FAX: 1-412-434-5130 3812 Elm Street
Phone: 1-412-434-5006 Dallas, TX 75226
USA
Shirley Dillard FAX: 1-214-820-4952
Dept. of Phannacology Phone: 1-214-820-2687
College of Medicine
University of South Alabama Emine Ercikan
Mobile, AL 36688 Memorial Sloan-Kettering Ctr.
USA Mol. Phann. and Therapeutics
FAX: 1-205-460-6798 1275 York Ave., Box 78
Phone: 1-205-460-6063 New York, NY 10021
USA
FAX: 1-212-639-2767
Phone: 1-212-639-8235

794
Dr. G. Werner-Febnayer Laura Gahn
Inst. Fiir Med. Chemie und Biochemie Dept. of Biochem. & Mol. Biology
Universitiit Innsbruck LSU Medical Center
Fritz Pregl Str. 3 1100 Florida Ave.
A-6020 Innsbruck New Orleans, LA 70119
Austria USA
FAX: 43-512-507-2279 FAX: 1-504-942-8341
Phone: 43-512-507-2298 Phone: 1-504-948-8568

Robert Perone Dr. Aleem Gangjee

Burroughs Wellcome Co. Dusquesne University
Molecular Gen. & Microbiol. School of Pharmacy
3030 Cornwallis Road Pittsburgh, PA 15282
Res. Triangle Park, NC 27709 USA
USA FAX: 1-412-434-5130
FAX: 1-919-248-8375 Phone: 1-412-434-6070
Phone: 1-919-248-4313
Dr. Robert Garrett
Dr. Victoria Finnerty Dept. of Biochemistry
Emory University Duke University Medical Center
Department of Biology Box 3711
Atlanta, GA 30322 Durham, NC 27710
USA USA
FAX: 1-404-727-2880 FAX: 1-919-684-5470
Phone: 1-404-727-4219 Phone: 1-919-684-3120

Dr. Berthold Fischer Dr. Tim Garrow

Anorganisch Chemisches Univ. of California, Berkeley
Institut der Universitiit Dept. of Nutritional Sciences
Ziirich, Winterthurerstr. 190 119 Morgan Hall
CH-8057 Ziirich Berkeley, CA 94720
Switzerland USA
FAX: 411-363-8611 FAX: 1-510-642-0535
Phone: 411-257-4612 Phone: 1-510-642-0750
Dr. Anthony L. Fitzhugh Robert Gilli
Pri I Dyn Corp. Laboratoire de Physique Pharmaceutique '
NCI-FCRDC Faculte De Pharmacie
P.O. Box B, Bldg 325 27 bd Jean Moulin
Frederick, MD 21702-1201 F-13005 Marseille
USA France
FAX: 1-301-846-5866 FAX: 33 91 80 26 12
Phone: 1-301-846-1050 Phone: 33 91 83 55 06

Dr. Paul Fitzpatrick Dr. John Giovanelli

Texas A & M University Lab of Neurochemistry
Dept. of Biochem. & Biophys. NIH - Bldg. 36, Rm. 3D-30
College Station, TX 77843 Bethesda, MD 20892
USA USA
FAX: 1-409-845-9274 FAX: 1-301-496-9935
Phone: 1-409-845-5487 Phone: 1-301-496-6881
Dr. Torgeir Flatmark Dr. Miki Goto
Dept. of Biochemistry Dept. of Chemistry
Univ. of Bergen Gakushuin University
N5009 Bergen Toshirna-ku, Tokyo 171
Norway Japan
FAX: 47 5 206400 FAX: 81 3 5992 1005
Phone: 47 5 206428 Phone: 81 3 5992 1019
Dr. Sally Frost-Mason Dr. Jill E. Gready
Univ. of Kansas The University of Sydney
Dept. of Physiology & Cell Biology Dept. of Biochemistry
Lawrence, Kansas 66045 Sydney, N.S.W. 2006
USA Australia
FAX: 1-913-864-5321 FAX: 61-2-692-4726
Phone: 1-913-864-3296 Phone: 61-2-692-3907

795
 Christopher Greene Dr. Steen Ingemann Hansen
Dept. of Biology Dept. of Clin. Chemistry
Box 870344 Central Hospital Hilleroed
Univ. of Alabama DK-3400 Hilleroed
Tuscaloosa, AL 35487 Denmark
USA FAX: 45 48 24 00 67
FAX: 1-205-348-1786 Phone: 45 42 261500 ext. 2298
Phone: 1-205-348-9810
Dr. Toshie Harada
Dr. Roger Griffm Dept. of Medical Chemistry
Dept. of Chemistry Osaka Medical College
Bedson Building 2-7 Daigakumachi
Univ. of Newcastle upon Tyne Takatsuki, Osaka 569
Newcastle upon Tyne, NE1 7RU Japan
United Kingdom FAX: 81 726 81 3723
FAX: 44-91-222-6929 Phone: 81 726 83 1221 ext. 2453
Phone: 44-91-222-8591
Dr. Larry Hardy
Dr. Steven S. Gross Biotech 2 I Molecular Medicine
William Harvey Research Inst. Univ. of Massachusetts Medical Center
St. Bartholomew's Hospital 373 Plantation St.
Medical College Worcester, MA 01605
Charterhouse Square USA
London EC1M 6BQ FAX: 1-508-856-4900
United Kingdom Phone: 1-508-856-4289
FAX: 44-71-251-1685
Phone: 44-71-982-6119 Dr. Peter G. Hartman
F. Hoffmann-La Roche Ltd.
Dr. Nathan Grossowicz Preclinical Research, Bldg. 70
Dept. Bacteriology CH-4002 Basel
Hebrew Univ. Switzerland
Hadassah Medical School FAX: 41 61 688 2729
Bin Kerem Phone: 41 61 688 7563
Jerusalem 91010
Israel Dr. Hiroyuki F.asegawa
FAX: 972-2-784010 Dept. of Eioscience
Phone: 972-2-42-82-43 Nishi-Tokyo University
2525 Uenohara
Dr. Alfred Gut Yamanashi 409-01
Dr. B. Schircks Laboratories Japan
Buechstrasse 17a FAX: 81 554 63 4431
CH-8645 Jona Phone: 81 554 63 4411
Switzerland
FAX: 41 55 28 29 54 Dr. K. Hatakeyama
Phone: 41 55 28 28 40 Dept. of Medical Chemistry
Osaka Medical College
Dr. Markus Giitlich 2-7 Daigakumachi
Inst. Exp. ffiimatologie der GSF Takatsuki, Osaka 569
Marchioninistr. 25 Japan
D-8000 Miinchen 70 FAX: 81 726 81 3723
Germany Phone: 81 726 83 1221 ext. 2453
FAX: 49 89 7099 200
Phone: 49 89 7099 222 Dr. Simon J.R. Heales
Institute of Child Health
Dr. Jan Haavik University of London
Department of Biochemistry 30 Guilford St.
University of Bergen London WC1N 1EH
Arstadveien 19 Uniteri Kingdom
N-5009 Bergen FAX: 44 71 831 0488
Norway Phone: 44 71 242 9789
FAX: 47-5-206400
Phone: 47-5-206432 Dr. Robert N. Henrie
FMC Corporation
Mary Hanlon Agricultural Chemical Group
Burroughs Wellcome Company PO Box 8
3030 Cornwallis Rd. Princeton, NJ 08543
Research Triangle Park, NC 27709 USA
USA FAX: 1-609-951-3835
FAX: 1-919-248-8375 Phone: 1-609-951-3468
Phone: 1-919-248-4427

796
Joan M. Revel Dr. Donald W. Horne
College of Pharmacy Research Service (151)
University of Michigan VA Medical Center
428 Church Street Nashville, 1N 37212
Ann Arbor, MI 48109-1065 USA
USA FAX: 1-615-321-6305
FAX: 1-313-763-2022 Phone: 1-615-327-4751 ext. 5474
Phone: 1-313-747-2221
Dr. Masaaki Hoshiga
Charlie Hill Dept. of Medical Chemistry
Dept of Biochemistry Osaka Medical College
Medical Univ. of South Carolina 2-7 Daigakumachi
171 Ashley Ave Takatsuki, Osaka 569
Charleston, SC 29425 Japan
USA FAX: 81 726 81 3723
FAX: 1-803-792-4322 Phone: 81 726 83 1221 ext. 2453
Phone: 1-803-792-2331
Dr. Elizabeth Howell
Dr. James Hilton University of Tennessee
Dept. of Biochemistry Department of Biochemistry
Duke University Medical Center M407 Walters Life Bldg.
Box 3711 Knoxville, 1N 37996-0840
Durham, NC 27710 USA
USA FAX: 1-615-974-6306
FAX: 1-919-684-5470 Phone: 1-615-974-4507
Phone: 1-919-684-3120
Dr. Ying Hu
Dr. Richard H. Himes Dept. of Cancer & Biology Res.
University of Kansas Lilly Research Labs.
Department of Biochemistry Lilly Corporate Center
Lawrence, KS 66045-2106 Indianapolis, IN 46285-1523
USA USA
FAX: 1-913-864-5321 FAX:
Phone: 1-913-864-3813 Phone: 1-317-276-1519
Dr. Kei Hirayama Teng Huang
Room 2309 Scott Hall Box 614 MCV Station
Wayne State University Dept. of Biochemistry
540 East Canfield Richmond, VA 23298
Detroit, MI 48201 USA
USA FAX: 1-804-786-1473
FAX: 1-313-993-4269 Phone: 1-804-786-9482
Phone: 1-313-577-5632
Dr. Keith Hyland
Dr. George H. Hitchings Baylor Univ. Med. Center
Burroughs Wellcome Co. 3812 Elm Street
3030 Cornwallis Road Dallas, TX 75226
Res. Triangle Park, NC 27709 USA
USA FAX: 1-214-820-4952
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Dr. John B. Hynes
Dr. Bernhard Hoffmann Pharmaceutical Sciences
Milupa Scientific Dept Medical University of S.C.
Bahnstr. 14-30 171 Ashley Ave.
D-6382 Friedrichsdorf!faunus Charleston, SC 29425
Germany USA
FAX: 49 6172 99 1595 FAX: 1-803-792-0759
Phone: 49 6172 99 0 Phone: 1-803-792-8642
Dr. Jan Holm Dr. Teruhiko Iino
Dept. of Clinical Chemistry Dept. General Education
Central Hospital Nihon University
Nykobing Falster Sakurajosui 3-25-40, Setagayaku
DK-4800 Tokyo 156
Denmark Japan
FAX: 45 54 8533 94 FAX: 81-3-3303-9899
Phone: 45 54 8530 33 Phone: 81-3-3329-1151 ext. 368

797
 Dr. Takuji Imamura Dr. Georg Johnen
Osaka City Univ. Med. School National Inst. of Mental Health
Dept. of Pediatrics Bldg. 36., Room 3D-30
1-5-7 Asahimachi, Abeno-Ku 9000 Rockville Pike
Osaka 545 Bethesda, MD 20892
Japan USA
FAX: 81 6 646 5862 FAX: 1-301-496-9935
Phone: 81 6 645 2126 Phone: 1-301-496-6881

Dr. Takafumi Ishihara Dr. Jean L. Johnson

Suntory lnst. for Biomed. Research Department of Biochemistry
1-1-1, Wakayamadai Duke Univ. Medical Center
Shimamoto-Cho, Mishima-Gun Durham, NC 27710
Osaka 618 USA
Japan FAX: 1-919-684-8919
FAX: 81-75-962-6448 Phone: 1-919-684-3120
Phone: 81-75-962-8476
Cheryl Johnson
Dr. Seiji Ishii Drug Synthesis Section
Dept. of Medical Chemistry Southern Research Institute
Osaka Medical College 2000 Ninth Ave. S., PO Box 55305
2-7 Daigakumachi Birmingham, AL 35255-5305
Takatsuki, Osaka 569 USA
Japan FAX: 1-205-581-2877
FAX: 81-726-81-3723 Phone: 1-205-581-2213
Phone: 81-726-83-1221
Dr. Jacques Jolivet
Michael Ivery Institut du Cancer de Montreal
Department of Biochemistry 1560 Sherbrooke Street, East
University of Sydney Montreal, Quebec H2L 4M1
Sydney NSW 2006 Canada
Australia FAX: 1-514-876-5476
FAX: 61-2-692-4726 Phone: 1-514-876-7078
Phone: 61-2-692-3907
Dr. John A. Joule
Dr. Ann Jackman Department of Chemistry
Institute of Cancer Research University of Manchester
Drug Development, Block E Oxford Road
15 Cotswold Road Manchester M13 9PL
Sutton, Surrey United Kingdom
United Kingdom FAX: 44 61 275 4598
FAX: 44-81-770-7885 Phone: 44 61 275 4633
Phone: 44-81-643-8901 ext. 4319
Dr. Thomas Kalman
Dr. Robert C. Jackson State Univ. of N.Y. at Buffalo
Agouron Pharmaceuticals Dept. Medicinal Chemistry
3565 General Atomics Court 457 Cooke Hall
San Diego, CA 92121 Buffalo, NY 14260
USA USA
FAX: 1-619-622-3299 FAX: 1-716-645-2393
Phone: 1-619-622-3000 Phone: 1-716-645-2850

Dr. Gerrit Jansen Dr. Un Jung Kang

Dept. of Oncology, Univ. Hosp. Dept. of Neurology
Free Univ. of Amsterdam Univ. of Chicago
P.O. Box 7057 5841 South Maryland Ave.
NL-1007 MB Amsterdam Chicago, IL 60637
The Netherlands USA
FAX: 31-20-548-64-44 FAX: 1-312-702-9076
Phone: 31-20-548-64-32 Phone: 1-312-702-6389

Soon-Seog Jeong Dr. Gregory Kapatos

Department of Biochemistry Rm 2309 Scott Hall
University of Sydney Psychiatry Dept.
Sydney NSW 2006 Wayne State University
Australia 540 East Canfield
FAX: 61-2-692-4726 Detroit, MI 48201
Phone: 61-2-692-3907 USA
FAX: 1-313-993-4269
Phone: 1-313-577-5949

798
Dr. Setsuko Katoh Dr. Devanand Kowlessur
Department of Biochemistry National Inst. of Mental Health
Meikai Univ. School of Dentistry Bldg. 36., Room 3D-30
1-1 Keyakidai 9000 Rockville Pike
Sakado. Saitama 350-02 Bethesda, MD 20892
Japan USA
FAX: 81-492-87-6657 FAX: 1-301-496-9935
Phone: 81-492-85-5511 ext. 457 Phone: 1-301-496-6881

Dr. Seymour Kaufman Heidi L. Kruschwitz

Lab. of Neurochemistry Box 614 MCV Station
Nat. Inst. of Mental Health Dept. of Biochemistry
Bethesda, MD 20892 Richmond, VA 23298
USA USA
FAX: 1-301-496-9935 FAX: 1-804-786-1473
Phone: 1-301-496-3579 Phone: 1-804-786-9482

Dr Gerard Kennealey Dr. Gen Kudo

Zeneca Pharmaceuticals Drug Metabol. & Anal. Chern. Ctr.
1800 Concord Pike Daiichi Pharmaceutical Co., Ltd.
Wilmington, Delware 19897 16-13 Kita-Kasai 1-Chome
USA Edogawa-Ku
FAX: 1-302-886-2765 Tokyo 134
Phone: 1-302-866-2540 Japan
FAX: 81 3-5696-8332
Dr. Rosemary Kimbell Phone: 81 3-5696-8298
Institute of Cancer Research
Drug Development, Block E Dr. Donald Kuhn
15 Cotswold Road Professor of Psychiatry
Sutton, Surrey Wayne State University
United Kingdom Metropolitan Center for High Technology
FAX: 44-81-770-7885 2727 Second Avenue, Room 319
Phone: 44-81-643-8901 ext. 4218 Detroit, MI 48201
USA
Dr. Roy L. Kisliuk FAX: 1-313-963-4021
Dept. of Biochem. & Pharmcol. Phone: 1-313-963-4020
Tufts Univ. School of Medicine
136 Harrison Ave. Martin Kussman
Boston, MA 02111 Universitilt Konstanz
USA Fakultilt fiir Chemie
FAX: 1-617-956-6409 Postfach 5560
Phone: 1-617-956-6869 D-7750 Konstanz I
Germany
Dr. Roger Klein FAX: 49-7531-88-3097
lnstitut Curie Phone: 49-7531-88-2497
Laboratoire de Physique et
Chimie Biomoleculaire Dr. Werner Langgut
II, Rue Pierre et Marie Curie Institut Fuer Biochemie/Medizinische Fak.
f-75231 Paris Cedex 05 Fahrstrasse 17
France D-8520 Erlangen
FAX: 33 I 40 51 06 36 Germany
Phone: 33 I 40 51 67 95 FAX: 49 91 31 854605
Phone:
Per M. Knappskog
Dept. of Medical Genetics Dr. John Ledbetter
Haukeland Hospital Dept. of Biochemistry
5021 Bergen Med. Univ. of South Carolina
Norway 171 Ashley Avenue
FAX: 47 5 97 54 79 Charleston, SC 29425
Phone: 47 5 97 54 75 USA
FAX: 1-803-792-4322
Dr. Ei-ichi Kokue Phone: 1-803-792-4321
Lab. Veterinary Pharmacology
Tokyo Univ. of Agr. & Techno!. Dr. Robert J. Leeming
Fuchu, Tokyo 183 General Hospital of Birmingham
Japan Haematology Dept.
FAX: 81 423-61-0034 Steelhouse Lane
Phone: 81 423-61-3311 ext.361 Birmingham, B4 6NH
United Kingdom
FAX: 44 21 233 3966
Phone: 44 21 236 8611

799
 Stephen Lentz Dr. Frank Maley
Psychiatry Dept. N.Y. State Dept. of Health
Wayne State University Wadsworth Ctr. for Labs & Res.
540 East Canfield Empire State Plaza, Box 509
2309 Scott Hall Albany, NY 12201-0509
Detroit, MI 48201 USA
USA FAX: 1-518-473-2900
FAX: 1-313-993-4269 Phone: 1-518-474-4184
Phone: 1-313-577-5949
Dr. John H. Mangum
Dr. Robert A. Levine 692 WIDB
Director, William T. Gossett Neurology Brigham Young University
Labs. of Henry Ford Hospital Provo, UT 84602
Metropolitan Center for High Technology USA
2727 Second Avenue, Room 302 FAX: 1-801-378-5474
Detroit, MI 48201 Phone: 1-801-378-4114
USA
FAX: 1-313-963-4021 Dr. Fabrizio Marazza
Phone: 1-313-963-4020 c/o SAPEC SA
CH-6903 Lugano-Barbengo
Innina Lewandowska Switzerland
Inst. of Biochemistry & Biophysics FAX: 41 91 556325
Rakowiecka 36 Phone: 41 91 556414
PL-02-532 Warszawa
Poland Dr. Michael Marietta
FAX: 48-22-22-53-43 College of Pharmacy
Phone: 48-2-659-85-71 University of Michigan
428 Church Street
Dr. Walter M. Levenberg Ann Arbor, MI 48109-1065
Marion Merrell Dow Research Institute USA
2110 E. Galbraith Road FAX: 1-313-763-2022
Cincinnati, OH 45215 Phone: 1-313-764-2442
USA
FAX: 1-513-948-7472 Dr. Masahiro Masada
Phone: 1-513-948-7168 Dept. of Agricultural Chemistry
Fac. of Horticulture
Dr. Joseph A. Maddry Chiba Univ. Matsudo 648
Bio-Organic Chemistry Division Chiba Prefecture 271
Southern Research Institute Japan
2000 Ninth A venue, South FAX: 81-473-63-1497
PO Box 55305 Phone: 81-473-63-1221 ext. 346
Birmingham, AL 35255-5305
USA Dr. Larry H. Matherly
FAX: 1-205-581-2877 Michigan Cancer Foundation
Phone: 1-205-581-2748 110 E. Warren Ave.
Detroit, MI 48201
Dr. Josef Maier USA
Inst. Fiir Chemische Pflanzenphysiol. FAX: 1-313-831-8714
Universitat Tiibingen Phone: 1-313-833-0710
Corrensstr. 41
D-7400 Tiibingen 1 Dr. Christopher Mathews
Germany Dept. of Biochemistry & Biophys
FAX: 49 7071 87815 Oregon State University
Phone: 49 7071 296399 2011 AG/Life Sciences Bldg.
Corvallis, OR, 97331-7305
Dr. Gladys Maley USA
N.Y. State Department of Health FAX: 1-503-737-0481
Wadsworth Ctr for Labs & Res. Phone: 1-503-737-1865
Empire State Plaza- Box 509
Albany, NY, 12201-0509 Dr. Rowena G. Matthews
USA Univ. of Michigan
FAX: 1-518-473-2900 Biophysics Research Division
Phone: 1-518-474-9623 2200 Bonisteel Blvd.
Ann Arbor, MI 48109-2099
USA
FAX: 1-313-764-3323
Phone: 1-313-764-9459/8154

800
Farahnaz Mavandadi Dr. Nobuo Nakanishi
Duquesne University Meikai Univ. Sch. of Dentistry
School of Pharmacy Dept. of Biochemistry
Pittsburgh, PA 15282 1-1 Keyaki-Dai
USA Sakado, Saitama 350-02
FAX: 1-412-434-5130 Japan
Phone: 1-412-434-5006 FAX: 81-492-87-6657
Phone: 81-492-85-5511 ext. 280
Dr. John J. McGuire
Roswell Park Memorial Inst. Dr. Takayuki Nakano
666 Elm Street Dept. Biochem & Clin Pharm.
Buffalo, NY 14263 St. Jude Children's Res Hospital
USA 332 North Lauderdale, PO Box 318
Fax: 1-716-845-8857 Memphis, TN 38101-0318
Phone: 1-716-845-8249 USA
FAX: 1-901-521-1668
Dr. Kenneth E. McMartin Phone: 1-901-522-0449
Pharmacology Dept. PO Box 33932
Louisiana State Univ. Med Ctr. Dr. Masahiro Natsuhori
1501 King's Highway Lab. Veterinary Pharmacology
Shreveport, LS 71130-3932 Tokyo Univ. of Agr. & Technol.
USA Fuchu, Tokyo 183
FAX: 1-318-674-7857 Japan
Phone: 1-318-674-7871 FAX: 81 423-61-0034
Phone: 81 423-64-3311 ext.361
Dr. Peter W. Melera
Dept. of Biochemistry Dr. Wendi Neckameyer
School of Medicine Dept. of Pharmacol. & Physiol. Sci.
Univ. of Maryland St. Louis Univ. Sch. of Medicine
108 North Greene St. 1402 S. Grand Blvd.
Baltimore, Maryland 21201 St. Louis, MO 63104
USA USA
FAX: 1-410-706-4037 FAX:
Phone: 1-410-706-7731 Phone:

Dr. Laurane Mendelsohn Dr. L.D. Nord

Eli Lilly & Co. Catholic Medical Center
Biotechnol. Res. Drapcode 0434 Dept. of Cancer Research
Lilly Corporate Center 89-15 Woodhaven Blvd.
Indianapolis, IN 46285 Woodhaven, NY 11421
USA USA
FAX: 1-317-276-1414 FAX: 1-718-849-6552
Phone: 1-317-276-6924 Phone: 1-718-441-1147

Dr. Sheldon Milstien Dr. Janis O'Donnell

Lab of Neurochemistry Dept. of Biology
NIH- Bldg. 36, Rm. 3D-30 Box 870344
Bethesda, MD 20892 Univ. of Alabama
USA Tuscaloosa, AL 35487
FAX: 1-301-402-5025 USA
Phone: 1-301-496-6881 FAX: 1-205-348-1786
Phone: 1-205-348-9810
Dr. Soichi Miwa
Dept. of Pharmacology Dr. Kazuya Oguro
Kyoto Univ. Fac. Med. The Nishi-Tokyo Univ.
Sakyo- Ku 2525 Uenohara
Kyoto 606 Yamanashi, 40901
Japan Japan
FAX: 81-75-753-4402 FAX: 81 554 63 4431
Phone: 81-75-753-4690 Phone: 81 554 63 -•All

Dr. M. Gopal Nair Dr. Satoshi Onozawa

Dept. of Biochemistry Dept. of Biochemistry
College of Medicine Meikai University
University of South Alabama Sch. of Dentistry
Mobile, AL 36688 Sakado Saitama 350-02
USA Japan
FAX: 1-205-460-6127 FAX: 81-492-87-6657
Phone: 1-205-460-6857 Phone: 81-492-85-5511 ext. 280

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