Agricultural and Biological Chemistry

ISSN: 0002-1369 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb19

Characterization of Betaine Aldehyde

Dehydrogenase from Cylindrocarpon didymum
M-1

Nobuhiro Mori, Bunsei Kawakami, Kimiaki Hyakutome, Yoshiki Tani &

Hideaki Yamada

To cite this article: Nobuhiro Mori, Bunsei Kawakami, Kimiaki Hyakutome, Yoshiki Tani
& Hideaki Yamada (1980) Characterization of Betaine Aldehyde Dehydrogenase from
Cylindrocarpon didymum M-1, Agricultural and Biological Chemistry, 44:12, 3015-3016, DOI:
10.1080/00021369.1980.10864450

To link to this article: http://dx.doi.org/10.1080/00021369.1980.10864450

Published online: 09 Sep 2014.

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 Agric. Bioi. Chern., 44 (12), 3015~3016, 1980
3015

Note C. didymum M-I was grown in a medium containing

Characterization of Betaine Aldehyde
Dehydrogenase from Cylindrocarpon
didymum M-l
Nobuhiro MORI,* Bunsei KAWAKAMI,
Kimiaki HYAKUTOME, Yoshiki TAN!
and Hideaki YAMADA
Department of Agricultural Chemistry,
Kyoto University, Kyoto 606 Japan
Received July 21, 1980
In the previous papers, we reported the purification and
characterization of choline oxidase,I,2) dimethylglycine
oxidase') and sarcosine oxidase4 ) which are involved in
choline metabolism by Cylindrocarpon didymum M-l. In
addition, we found betaine aldehyde dehydrogenase in the
cell-free extract of fungus grown on choline medium.
Betaine aldehyde dehydrogenase from rat liverS) was
partially purified and characterized to require NAD+ as
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1.0 g of choline chloride, 0.1 g of meat extract, 0.1 g of
K 2 HPO., 0.1 g of KH 2 PO., 0.1 g of NaCl and 0.05 g of
MgSO. 7H 20 in 100 ml of tap water at pH 7.0. The
cultivation was carried out at 28C for 60 hr with
reciprocal shaking. The harvested cells were suspended in
100 mM potassium phosphate buffer (pH 8.0) containing
0.5 mM dithiothreitol (DTT)*I) and disrupted with an
ultrasonic oscillator. The cell-free extract was added to
solid ammonium sulfate to 30% saturation and the
precipitate was discarded. The precipitate formed by the
further addition of ammonium sulfate to 60% saturation
was collected and dissolved in 40 mM buffer. The enzyme
solution was desalted by Sephadex G-2S equilibrated with
the 40 mM buffer containing 20% glycerol. Then, the
enzyme solution was applied to DEAE-Sephacel column
chromatography. The enzyme which was eluted with a
linear gradient of the buffer concentration from 40 mM to
150 mM. Active fractions were combined and concentrated
by ultrafiltration in a collodion bag apparatus. The
concentrated enzyme solution was applied to a Sephacryl
S-200 column (2.5 x 80 cm) equilibrated with a 100mM
buffer containing 20% glycerol. Repeated gel filtration
gave single and nearly symmetrical peaks of protein and

coenzyme. Highly purified betaine aldehyde dehydro-
genase from Pseudomonas aeruginosa A-166 ) required both
NADP+ and NAD+ as coenzyme. On the other hand,
choline oxidase purified from C. didymum M-I oxidized
betaine aldehyde in addition to choline. 2) The apparent Km
value for betaine aldehyde (5.8 mM) was about four times
the Km for choline (1.3 mM). This was also found in
choline oxidase from Arthrobacter globiformis. 7 ) The
present report deals with the partial purification and
characterization of betaine aldehyde dehydrogenase from
C. didymum M-1.
activity. Result of the purification is summarized in Table
I. The purified enzyme gave many protein bands on
acrylamide gel electrophoresis but one 'predominant band
and one faint band on SDS-acrylamide gel electrophoresis.
The molecular weight of the enzyme was estimated to be
about 220,000 by gel filtration on Sephacryl S-200. The
molecular weight of the subunit of the enzyme was
estimated to be 58,000 by SDS-acrylamide gel elec-
trophoresis. Enzyme activity was completely lost when
dialyzed aginst the 10 mM buffer (pH 8.0). However,
addition of glycerol at a concentration of 20% stabilized

TABLE I. SUMMARY OF PURIFICATION PROCEDURE OF BETAINE ALDEHYDE DEHYDROGENASE

FROM Cylindrocarpon didymum M-I
The reaction system for the determination of betaine aldehyde dehydrogenase activity consisted of 250
limol of potassium phosphate buffer (pH 8.0), llimol of betaine aldehyde, llimol of NAD+ and a suitable
amount of enzyme solution in a total volume of 3.0 m!. The increase of absorbance at 340 nm was measured
spectrophotometrically at 30C. One unit of the enzyme activity was defined as the amount of enzyme which
catalyzed the formation of llimol of NADH per min. Protein concentration was determined by the absorbance
at 280nm, where Ej:;;. value of 10.0 was used. Specific activity was defined as unit per mg protein.

Total Total Specific

Yield
Fraction protein activity activity
(mg) (U) (U/mg) (%)

Cell-free extract 8,564 521 0.06 100

Ammonium sulfate
(30 ~ 60% sat.) 1,666 508 0.30 98
D EAE-Sephacel 42.7 214 5.03 41
Sephacryl S-200 6.6 135 20.5 26

* Present address: Department of Agricultural

Chemistry, Tollori University, Koyama, Tollori 680, Japan. *1) This buffer was used throughout this work.
 3016 N. MORI, B. KAWAKAMI, K. HYAKUTOME, Y. TAN! and H. YAMADA

the activity. The enzyme was most active at pH 8.5 ~ 9.0. -c

-6
The enzyme activity was completely inhibited by Cu 2 +, --,
E

Zn 2 +, Hi' +, p-chloromercuribenzoate, iodoacetate, 0-

phenanthroline and partially by Co2+, IX,IX'-dipyridyl. The
enzyme was highly specific for betaine aldehyde. Aliphatic o 2 4 6
lINAO'(mM")
aldehydes and a variety of aldehydes such as' crotonal- A

dehyde, benzaldehyde and D-glucose were not dehydro-

c1
.-
B
C
genated. The enzyme required NAD+ as coenzyme but .,E o
15
NADP + was inert. Double reciprocal plots of v against , 10
betaine aldehyde gave parallel straight lines for various ->-~

o 5 10 15
-
>
1/Betaine aldehyde(mM-1)
o 2 4 6 8
l/Betaine aldehyde (mM-1}
A FIG. 2. Double Reciprocal Plots of the Reaction
B
Catalyzed by Betaine Aldehyde Dehydrogenase.
-.S 15 C
o
Experimental conditions were the same as those in Fig. 1.
.,-E The concentrations of NAD + were: A, 0.66; B, 0.33; C,
o
,10 0.23; D, 0.17 mM, respectively.
->
choline was oxidized to betaine aldehyde by choline
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oxidase and further oxidized to betaine by NAD-

dependent betaine aldehyde dehydrogenase in C. didymum
M-1.
o 5 10 15
1/NAD+( mW1}
Acknowledgments. We wish to thank Professor
FIG. 1. Double Reciporcal Plots of the Reaction
Yoshio Ichikawa, Tottori University, for his encourage-
Catalyzed by Betaine Aldehyde Dehydrogenase.
ment during the course of this study.
Experimental conditions were described in Table 1. The
concentrations of betaine aldehyde were: A, 0.33; B, REFERENCES
0.23; C, 0.17; D, 0.13 mM, respectively.
I) Y. Tani, N. Mori, K. Ogata and H. Yamada, Agric.
BioI. Chern., 43, 815 (1979).
concentrations of NAD + (Fig. I). Parallel lines were also 2) H. Yamada, N. Mori and Y. Tani, Agric. Bioi.
observed for various concentrations of betaine aldehyde in Chem., 43, 2173 (1979).
double reciprocal plots of v against NAD + concentration 3) N. Mori, B. Kawakami, Y. Tani and H. Yamada,
(Fig. 2). These results suggest that the reaction proceeds by Agric. Bioi. Chern., 44, 1383 (1980).
a "Ping-Pong" mechanism as designated by Cleland. B ) 4) N. Mori, M. Sano, Y. Tani and H. Yamada, Agric.
Michaelis constants for betaine aldehyde and NAD + were, Bioi. Chern., 44, 1391 (1980).
respectively, calculated from secondary plots of the 5) H. A. Rothschild, O. Cori and E. S. G. Barron, J.
intercept of the parallel lines at the ordinate versus the BioI. Chem., 208, 41 (1954).
concentration of NAD + or betaine aldehyde (inserted in 6) T. Nagasawa, Y. Kawabata, Y. Tani and K. Ogata,
Figs. 1 and 2). Km values of 0.31 mM for betaine aldehyde Agric. Bioi. Chem., 40, 1743 (1976).
and 0.38 mM for NAD+ were obtained. Thus, the affinity 7) S. Ikuta, S. Imamura, H. Misaki and Y. Horiuti, J.
for betaine aldehyde of the enzyme was about twenty times Biochem., 82, 1741 (1977).
that of choline oxidase. Therefore, it can be concluded that 8) W. W. Cleland, Ann. Rev. Biochem., 36, 77 (1967).

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