Mechanism

of Cycloheximide

in a Cell-free

System

Inhibition

Prepared

from

of Protein
Rat

Synthesis

Liver*
(Keceived for pltblication,

1%.

s.

IhLIGA,

:I.

w.

AS-I)

H.

s.

I\IC-sRO

Chemistry LabolatoGes,
Departme?lt
Cambridge, Massachusetts
02139

SUMMARY
Sites of cycloheximide
action on protein synthesis were
examined
using a cell-free system prepared from rat liver.
If all amino acids or aminoacyl transfer RNA were present
at the start of incubation,
the system appeared to incorporate W-leucine
mainly by elongation
of peptide chains.
Under these conditions,
high dose levels of cycloheximide
were necessary in order to inhibit incorporation
extensively.
The inhibition
could be prevented by raising the glutathione
content of the reaction mixture, and particularly
by preliminary incubation
of a mixture
of transferase
I and II with
high concentrations
of glutathione
before adding
these
enzymes to the system.
Other sulfhydryl
compounds were
also effective in protecting
against cycloheximide.
It has
been concluded
that the inhibitory
action of cycloheximide
on peptide chain elongation
involves inactivation
of transferase II, an enzyme known to have a sulfhydryl requirement.
High concentrations
of glutathione
were also found to prevent the inhibition
of cell-free protein synthesis caused by
streptovitacin
A, a derivative
of cycloheximide,
but not
inhibition
caused by emetine or sparsomycin.
If the protein-synthesizing
system was first incubated
without amino acids or aminoacyl-tRNA,
polysomes present
at the start of incubation
underwent
disaggregation.
On
addition of amino acids at this point, the polysomes became
reaggregated
and incorporation
of 14C-leucine was stimulated, probably by a process involving chain initiation.
The
response of polysome aggregation
and coincident 14C-leucine
uptake could be inhibited
by low doses of cycloheximide.
Furthermore,
this inhibitory
action of cycloheximide
could
not be prevented
by raising the glutathione
content of the
medium.
This suggests that the action of cycloheximide
on
polysome aggregation
differs from its effect on peptide chain
elongation.

(~~clolicsimitle
(.1ctitliolle),
a11 antibiotic
1)roducetl
1))
(1) to inhibit
Sfrepfo-~/yes griwtrs, W;IS first shown by Kerridge
syllthcsis ill :I >-east. Its iuhibitory action h:ls since been
lxotoill
* This investigation
Gratlt

\~as srqlported

1)~ Public

Ilralth

Service

(A-08893-03.
4480

of Nutrifiotz

aud Food Science,

S1assachusett.s

repeatedly confirmed in mammalian cells (2-F).

Using cell-free
s;!-stems, several authors (%ll)
have obtained data suggesting
that the charging of transfer RNA with amino acids is not influenced by the inhibitor, which thus must act at some subsequent
stage in protein synthesis.
Recent studies suggest that cyclohcsimitlc may affect protein synthesis at more than one l)oint.
Lin, Mosteller,
and Hardesty (12) obtained evidence from esperimellts on reticulocytes
that the antibiotic
inhibits the intiation of new peptide chains aud the elongation of nascent peptides on ribosomes by different mechanisms.
In the studies reported below, we have attempted to localize
the sites of action of this antibiotic in a cell-free system for protein synthwis prrl,ared from rat liver (13). The system used
consisted of liver polyaomes, activating and transferring enz~mcs,
and cofactors.
Since the enzyme fractions were treated to rcmove frcxe a11d tRK.&bound
alnino acids, the system is consequentl>. dependent on an exogwous supply of amino acids. If
incubated n-ith all 20 amino acids, it will incorporate mainly 1,~
chain elongation.
If, however, it is incubated without amino
acids, the polysomes disaggregate and amino acid incorporation
is mininlal; if at this point amino acids arc added to the incubntion medium, the polysomes can be reaggrcgatcd
and labeled
amino acids are once more incorporated
into l)eptide chain<.
The nature of this action of delayed amino a&d supl)lementation
is not fully understood, but may be dependent on chain initiation.
This system has been used by us to study the actions of cyclehesimidc and some other antibiotics
on elongation of peptitle
chains and on the phenomenon of polysome aggregation in response to delayed amino acid supplementation.
With this SJ-Stern, KC have now shown that chain elongation and pol\,some
aggregation are inhibited by different concentrations
of cyclehesimidc.
The action of the antibiotic
on chain elongation
aplxws to be directed against aminoacyltransferase
II and can
be prewnted
b,v high concentrations
of sulfh\-dry1 compounda,
whereas the inhibitory
action of cycloheximide
on polysome
aggregation due to addition of amino acids cannot be 1)revtntcd
by increasing the glutathione
content of the nwdium.
*\ preliminary report of the action of t,his antibiotic 011 chain elonpa(14).
tion has :~p~~~rd

Rengejzts and I/e&n-The

amino ackk uwtl n-crc obt;tincd
Othrr chemicals \VCW rwgcllt
from Sutritional
l<iochemicals.
grade. Rat lircr tRS.1 stril)lw(\ of amino acsid< n-:ts ~~~ucl~awtl

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

From the Physiological

Institute
of Technology,

~OSCZUG,

December 23, 196s)

Issue of August 25, 1969

B. X. Baliga,

A. W. Prommk,

4431

p*g of :Irniiio:LcSltranxfcrnsc
protein, 500 g of polysomc twotcin,
and 20,000 cpm of 14C-aminoacyl-tRN-1
in a final volurrrc~ of 1
ml. Inrubations
were carried out at 37, and radioactivity
incorporated into protein was measured as described above.
Hydrolysis of GTl by the protein-synthesizing
system wts
determined by measuring radioactive inorganic phosphorw
released from (y-32P-GTl
as described by Cor~wny
and Lipmann
(I 8). The reaction mixture was similar to that used for 14Carninoacyl transfer to lxptides, except that, only ZC-aminoacyltRN=\ was present and the GTP used WIS labeled with 821 in the
terminal phosphate (11,000 cpm per ml of rrartion
rnixtuw).
The reaction mixture was incubated at 3T for 30 mill a11tl the
reaction was terminated by adding equal amounts of 0.2 RI silicotungstic acid in 0.02 N I-180, and 1 ml of 0.001 M pot,assium t)hosl,hatc (pH 6.8) as carrier.
The extent of GTP hydrolysis was
measured by extraction of the released inorganic l)hosl)h:rte as
the phosphomolybdate
complex into isobutyl alcohol; the extract
was counted for radioactivity
using t,hc Suclcar-Chic,:rgo
gas
flow

COUllk~.

Polysome Prqliles-IIlcubatioll
mist,urcs for redimcntation
analysis of polysome profiles were first diluted with 0.7 \.olume
of 0.01 11 Tris-HCl buffer (pH i.6), thcll layered over :I lilrear
gradient of 10 lo 40 7; sucrose in IKJI buffer.
The gradient xas
crntrifugcd at 38,000 rl)rn in the Sly-50 rotor of the Spinco model
L2 ultracentrifuge
for $0 min. The absorl)tion 1)rofilr at 260 rnp
was recorded automatically
\\-ith a flow cell device in :I Gilford
nlodcl 2000 spectrol)hotollleter.
Radioactivity
on thtx gradient
was nwasured on fractions of 12 droljs; the lxotein \va> I)rrcipitated with carrier albumin and the hot t rirhloracetic
avitl-illsolfilter for rolnrting
ublc lnwipitate
was collected on :I Millil)orc
as drsrribed above.
kstimation of Protein-The
protein content of the c11zyllles,
pol,wornes, and gradient fracl ions was dctcwnincd by thv nwthod
of Lowry et al. (19) using bovine serum albunlin :lb the ,~t;~lrtl:wd.

14&i
J worpoIYJkct of Cycloheximide Concentration on .lkno
ration-The
action of cyclohesimide was ,\tuditJd in a cell-1rw txoteia-synthesizing
system consisting of liyc:r pal>-somes, act i\-:rting
and transferring
enzymes together with cofdctors, alltl Wleucine.
The system had been prepared depleted of frw anlino
acids (see under Materials
and Methods)
and wxs thus tlc~xwdcnt on csogenous amino arids. To onr set of tubes, a caonll)lete
mixture of amino acids was added at thr st,art of incubation;
to
arids wrrc addctl other than 14(~loucine.
the other set,, no amino
Fig. ICCshows that, whwl amino acids w-cre present, inc~wasing
levels of r\-cloheximidc
cawed l)xgrc,wire
inhibition
of 4Cleucinc uptake into l)rotein.
-1dditiolr of 0.1 pg per ml hat1 a
slight action on 14C-lcurinc
_
incorporation;
in order to :irliiere
75%) inhibition,
it was necessary to add 1 mg of inhibitor tw ml
of incubation
medium, a level coml~:w:rble to t,hnt u-cltl by
Rcttstein,
Noll, and Icnman (10) in their cell-frw sy-tcnl to
retard ribosome movement along the mcsseager stra11cl. The
much smaller residual incorporation
of Vleucine
obtained in
the absence of added amino acids (Fig. 16) xas little affwt cd by
cycloheximide at any concentration.
It has previously been shown by us (13) with such a -\-stem
that, after 20 min of incubation wilhout amino acids, incorlwration of 14C-leucine tenses altogether but can be restarted by
a complete mixture of amino acids. Fig. lc shows the effect of
two levels of cycloherimide
on this response to delayed amino

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

Cyclohesimide
was provided by
from General Biochemicals.
ZUdrirh, and puromycin
dihydrochloride
was obtained
from
Nutritional
Biochemicals.
Streptovitacin
A was a ljroduct of
Ultjohn, and cmctinc was provided by the S. B. Penick Company,
Scn- York, Sew York.
Sparsomycin was kindly supplied as
a gift by Dr. I. H. Goldberg of the Harvard Medical School.
The
uniformly
labeled 14C-L-leucine (specific activity, 250 mCi per
mmole) and a mixture of %-amino
acids (1 &i per mg of uniformly lab&xl amino acid mixture) were purchased from sew
England Suclear.
International
Chemical and nuclcar Corporatioll, Irvine, California, provided (+*P-GTP
(specific radioactivity, 22 mCi per pmole), which n-as further purified by chromatography on DEhE-cellulose.
Most of the media used were
matlc ul, in TK(1\1 buffer (0.05 M Tris-HCl, pH 7.6, 0.025 M K(1,
and 0.005 nr ;\IgCIZ) as described by Wcttstein, Staehclin, and
so11 (15).
Preparation 0jPolysomes and Cell Sap ~nzyl,ies-Pol~aomes
(Cribosomes) were obtained from the livers of fasting 150-g rats by
the procedure described by Baliga, lronczuk, and Munro (13).
To obtain activating and transferring cnzymcs, cell sap was prel):nwl :IJKI denuded, first, of free amino acids by dialysi.s, and
second, of tRr\-1 by the lxotamiire sulfate treatme
as described
from this frac1,. IMiga et nl. (13). lrotein n-as precipitated
tioll b&n-een 30 a11d iO7; saturation with (SH,)$O,
to yield a
mixture of activating
and transferring
enzymes with Illinirnal
tontent of free or tRNX-attached
amino acids; t,he l~w~~:tration
was uwd for the system incorporating
free amino acids illto 1x1,tides. For incorl)oration
of amino acids from ami~wacyl-tRr\A
free of
int 0 pal>-.qome?, lillft~ac~tioiiated alllii,o:ic3-ltlaIlsfer:l.~e~
activating enzymes n-we prepared Irolrr the cell salI :rftcxr lrentnwnt with Sephatlcs G-25. The nrtivating
rnzymw \vcrc renlrthod of lwc*il)itation
at 1111 5, :IJ~ a.
moved by the traditional
mixture of transfcrascs I and II was ~~relwwl from the su~)cr~~atarot fraction as dcscribed bJ- Gasior :uld Molda\-r
(I 6). U.ith
cwvh batch of reaction misturc, the optimal amount< of transfernsc fraction alld (when required) of trnllsfcrase and arti\-sting
enzyrue
fxaction wcrc established.
I+-eparation oj C-arrlinoacyl~tR~~~ I -For
the prelwation
of
radionctire
aiilino:rc~l~tRr\TX,
the lxocedure
dcwribed
by
Rat, liver cell sap WM ndjwtcd to l,II
l\Ioltl:lre (I T) \r:w wvtl.
5 to bring dowi :I precipitate containing tRX.1 ai~d amino aridThis precipitate ~1s thrn ret&sol\-rd
and
acti\-sting ellzymw.
allon-cd to rract n-ith the C-amino acid nlisture in the tnwcnce
of 0.01 JI -iTI-, 0.01 hI lIgCls, 0.01 hf GSII, and 0.1 &I lris-IICl
(pI1 7.6). The aminoacyl-tRiSA
was then isolated according to
the method of Moldave (17). The final product had :I specific
act i\-it). of 350,000 cpm lwr mg of RXA.
In&&ion-The
incubation mixture for incorporation
of free
amino witis into protein contained 50 mhl Tris-HCI buffer ($I
7.6), 1 ml1 GTP, X0 rnhZ NHICl, 2 nor 1211, 5 nix AIg(l?, 4 rnl\f
GSII, 0.5 FCi of unilormlg labeled 14C-L-leurine, 500 pug of mixed
artivating
and lransfcrring
enzyme lnvtein, and 500 ,ug of l)olyIhrst amount:: of enzymes
som(x lxotein in 1 ml of final volume.
and 1)oly,wmw have bern shown to bc ol)timal for l)romotilg
incorporation
of X~l(~ucine into protein (13). Incubntioils were
incorporated
into 1)rotein
carried out at, 3i , and radioacti&g
gas flo~v counter on the
wxs measured wit,h ;I Nuclear-Chicago
rexiduc left after treatment with hot trichloracetic
acid (13).
The incubation
mixture for transfer of 14C-nminoar~l-tR~~~
to l~olyaon~~s contained 50 m&I Tris-HCI buffer (1~1~ 7.6), either
4 or 20 111~ GSH, 0.2 mnf GTP, 5 rnlf 1\2&12, 80 mAI XI-T,Cl, 100

and H. N. Munro

Cycloheximide Action on Protein Synthesis

4482

Vol. 244, Wo. 16

Supplementation
fAA

of cycloheximide
on
were incubated
with
protein-synthesizing
peptide
was measured
of cycloheximide

(Cycle.).
a, with all amino acids (AA)
present
throughout
incub, without
addition
of other amino acids to the medium;
c,
bation;
when the medium
was supplemented
with all amino acids and with
The data are the mean
cycloheximide
after
20-min
incubation.
results
from two to four experiments.
acid

_.....

,....._._._._..

22

,ncc/bofjon

20
f2

-AA

Jd
11

-AA
fAA

+0.1+/000Jlg

6% LINEAR

SUCROSE

GRADIENT

Cyd0.

> 40%

FIG. 2. Inhibition
of polysome
resynthesis
by cycloheximide
(Cycle.).
Liver polysomes
were incubated
for 20 min in the amino
acid-dependent
protein-synthesizing
system
containing
4 mM
GSH without
added
amino
acids
(AA),
and then the complete
mixture
of 20 amino
acids was added either
alone (-)
or in the
presence
of 0.1 to 1000 rg of cycloheximide
(---).
Incubation
A control
sample was incubated
was continued
for another
2 min.
for a similar
total period
of 22 min without
added
amino
acids
(-----).
All three
samples
were
separated
simultaneously
on
sucrose
gradients
and the polysome
profiles
were measured
by
ultraviolet
absorption.
These experiments
were replicated
four
times.

supplementation.

In

this

case,

0.1 pg of inhibitor

per

ml

reduced the extra incorporation

after amino acid addition by
75%, and 1 mg per ml reduced it by 95%. Consequently, sensitivity to cycloheximide is much greater when the amino acids
are added 20 min after the start of incubation
than when the
amino acids are present throughout
incubation
(Fig. la).
Eject of Cycloheximide on Polysome Aggregation-When
the
cell-free protein-synthesizing
system is incubated for 20 min in
the absence of amino acids as described above, the polysome
pattern shows considerable loss of large aggregates and an accuWe have previmulation of monosomes and other oligosomes.
ously shown that the polysome aggregates can be regenerated
within 2 min after adding a complete amino acid mixture to the
cell-free system (13). This coincides with the increased incorporation of Gleucine
shown in Fig. lc. When cycloheximide
was added to the medium along with the supplementary
amino
acids, it prevented this reaggregation
at all dose levels from 0.1
and
pg to 1 mg per ml (Fig. 2). Thus, polysome regeneration
the increased uptake of r4C-leucine in response to delayed amino
acid supplementation
each show a similar degree of sensitivity
to cycloheximide.
When incubation of the reaggregated polysomes is continued
in the presence of amino acids, there is subsequent breakdown of
polysomes (Fig. 3a). As shown in our earlier paper (13), the
stimulus of delayed amino acid addition of YXeucine
incorporation is not, in fact, linear (as shown in Fig. lc), but causes an
initial rapid burst of uptake, followed by diminishing increments
as time of incubation
progresses.
Consequently,
the detailed
picture for 14C-leucine uptake after amino acid addition coincides
closely with the aggregation
and then disaggregation
of the
polysomes.
Since it has been established that high concentrations of cycloheximide can stabilize polysomes in vitro (lo), we
examined the effect of various dose levels of cycloheximide on the
subsequent disaggregation
of polysomes that had been reaggreThe polysome system was first
gated by amino acid addition.
incubated for 20 min without exogenous amino acids. Amino
acids were then added, followed 2 min later by various levels of
cycloheximide,
and incubation
was terminated
at 3 min after
addition of the inhibitor.
Fig. 3b shows that the polysome

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

MINUTES
1. Effect
of various
concentrations
Liver
polysomes
amino acid incorporation.
4C-leucine
in an amino
acid-dependent
system
for 40 min and incorporation
into
in the presence
of different
concentrations
FIG.

Issue of August

25, 1969

B. X. Baliga,

A. W. Prone&,
B

and H. N. Munro

4483

TABLE
I
of preliminary
incubation
of transferases
with suljhydryl
compounds on inhibitory
action
of cycloheximide on protein
synthesis in cell-free system
The incubation mixture for transfer of W-aminoacyl-tRNA
to
polysomes contained 50 mM Tris-HCI buffer (pH 7.6)) 0.2 mM GTP,
5 mM MgC12,80 mM NH&l, 100 rg of aminoacyltransferase
protein,
500 pg of polysome protein, and 20,000 cpm of W-aminoacyl-tRNA
in a final volume of 1 ml. The amounts of sulfhydryl compounds
present in the incubation medium are as shown in the table. Incubations were carried out at 37 for 35 min. In most experiments, as indicated, the transferases were incubated at 37 for 5
min in 0.5 ml of buffer before adding the polysomes, GTP, and
W-aminoacyl-tRNA.
Glutathione,
dithiothreitol,
mercaptoethanol, and cycloheximide (1 mg per tube) were added either during preliminary
incubation or during incubation, as indicated in
the table. The data shown are the average of three experiments.

E$ect

ZOOlncvbation

/-AA)

3. Liver polysomes were incubated in the amino acic ldependent protein-synthesizing

system for 20 min without amino
acids (AA) and then a mixture of all amino acids was added and
incubation was continued.
a, control samples (changes in polysome profile after amino acid supplementation).
After 2 min
(-)
and 5 min (- - -) following amino acid addition, a portion
of the mixture was layered onto a sucrose gradient and the polysome profiles were obtained.
A sample incubated for a total
period of 22 min without amino acids (-----) was also run. b,
effect of cycloheximide
on polysome profiles after amino acid
supplementation.
After 2 min of incubation
as above in the
presence of amino acids, cycloheximide in amounts of 1 pg (-----),
100 pg (- - -), or 1000 fig (-)
was added to the incubation mixture and after an additional 3 min of incubation the polysome
profiles were examined.
aggregate was well preserved in the presence of 1 mg per ml of
cycloheximide,
but at lower concentrations
breakdown
was
considerable.
In the samples incubated without
addition
of
cycloheximide (Fig. 3a), the ratio of polysomes to monosomes
was 6.7 at 2 min after addition of amino acids, and 3.4 at 5 min
after addition, which is compatible with disaggregation
seen in
the figure.
For samples receiving the inhibitor 2 min after the
amino acids (Fig. 3b), the ratio of polysomes to monosomes at
5 min of incubation is 4.1 for 1 pg of cycloheximide,
5.0 for 100
pg, and 6.2 for 1000 pg, the latter figure being close to the 2 min
figure. Thus, the level of cycloheximide
needed to prevent
reaggregation of polysomes after amino acid addition (0.1 pg per
ml) is much lower than the level necessary to stabilize the polysome aggregates so formed (1 mg per ml).
E$ect of Cycloheximide on Transfer of Amino Acids from tRNA
to Polysomes-It
has been reported that cycloheximide has no
effect on amino acid activation and binding to tRNh (8). In
order to simplify the protein-synthesizing
system, we therefore
prepared aminoacyl-tRNA
charged with a mixture of 14C-amino
acids and added it to an incorporation
system consisting of liver
polysomes, partly purified transferases, GTP, and GSH. Such a
system incorporated
the labeled amino acids efficiently into protein. Table I shows the mean results obtained from three such
studies with this system. Addition
of large amounts of cycloheximide at the beginning of incubation caused a 50% inhibition
of this transfer of label (tube 1 versus 2). It will be noted that,
in order to achieve this degree of inhibition,
a concentration
of
1 mg of cycloheximide per ml was necessary.
This lack of sensitivity is comparable to that found above for W-leucine
incorporation in the presence of abundant free amino acids in the
system (Fig. la). The inhibitory
action of the antibiotic could

Components
previously
incubated

FIG.

Additions

for incubation

GTP,

Tube

POlYsomes,
MC-

-SH
compounds

amino-

y
-SH
compounds

XYl-

tRNA
lmmles

1
2
3
4
5
6
7
8
9
10
11
12
13

Non e
Non e

+
+
+
+
+
+
+
+
+
+
+

None
None
4 GSH
20 GSH
4 GSH
20 GSH
4 GSH
20 GSH
10 DTT=
20 Mercaptoethanol

_
pmles

Non
Non
+
+
+
+
-

+
+
+
+
+
+
+
+
+
+
+
+
+

IllCOP
poration

mide
(1 m&T,
tube)

4
4
4
4
20

cfim

GSH
GSH
GSH
GSH
GSH
-

+
+
+
+
+

5500
2600
5350
1100
2300
1900
2200
5570
5900
1800
3500
3800
4100

(1DTT,

dithiothreitol.

be enhanced if the fraction containing the transferases was incubated beforehand with cycloheximide
(tube 3 versus 4). It
has been suggested that cycloheximide
inhibits transferase II
(II), and this enzyme is known to be sulfhydryl-dependent
(20).
Consequently, it occurred to us that cycloheximide may retard
peptide elongation by inactivating
the sulfhydryl groups of this
enzyme and that high concentrations
of sulfhydryl compounds
might compete effectively with the inhibitor
and prevent its
action.
Accordingly,
the GSH content of the medium during
incubation
was raised from 4 pmoles to 20 pmoles.
Table I
shows partial protection against inhibitory
action of the previously added cycloheximide (tube 4 versus 5). The action of cycloheximide was also diminished by adding 4 pmoles of GSH
during preliminary
incubation
(tube 4 versus 6), but no better
protective effect was obtained if 20 pmoles were added during
prior incubation along with cycloheximide than if the GSH was
added later during incubation
(tube 5 versus 7). A series of
incubations
was therefore carried out in which the transferase
fraction was incubated with various sulfhydryl compounds before
addition of cycloheximide
(tubes 9 to 13). Increasing the glu-

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

-I

4454
tathionc

Cycloheximide
lcwl

to 20 ~nwlcs

@cm
in thr abwwe
incubation
\vith
thi-:

did

not

affect

incorl)oration

of the inhibitor
(tube
level
of glutathionc

inhibitory
action
of cyclohesimidc
added
IO oersus 11).
Even
more effective
protection
imidc
was obtained
by adding
dithiothreitol

Action
by

8 VBTSUS 9), but

greatly
reduced

on Pwtein

the
prior
the

subscquentl?(tube
against
cgclohrsor ~nerc:tptocthanol

5000
b
z
B2

Vol. 244, Ko. 16

I

I
,ZOmM

control

GSH

4000-

4 mM
q

GSH + cyclohexlmlde

,oLn

IO

20

30
40
MINUTES

50

60

FIG. 5. The effect of glutathione

level dllring
preliminary
incubation
on the
subsequent
action
of cycloheximide
during
Transfer
of 14C-akino
acids from aminosubsequent
incubation.
acyl-tRNA
to peptides
was examined
during
differcut
times of incubation
in the presence
or absence
of cycloheximide.
The system
consisted
of liver
polysomes,
GTP,
MgZ+,
C-arninoac~l-tl~~A,
transferase
enzymes,
and buffer.
The transferase
fraction
was
previouslv
incubated
at 37 for 5 min with either
4 or 20 mM gluThe other constitllellts
of the reactathione
in 0.5 ml of buffer.
tion were then added to a final volume
of 1 ml and cycloheximide
(1 mg) was added to some of the tubes.
Tncubatioll
was continued
at 37 for varying
periods
of time.
The figllre shows incorporation
withollt
cyrloheximide
losing enzyme
previously
illrllbated
at 20
rnM GSH
(A-A)
and 4 rn%f GSH (O-O),
and illcorporat
ion in
presence
of cycloheximide
using enzyme
previollsly
incllbatrdwith
20 rnM GSH (O-O)
and 4 rn>q GSH
(O-0).
The data are the
average
of two kxperiments.

TABLE

II

Effect of preliminary
incubalion
of washed polysomes
compoluztls
on inhibitory
action
of cycloheximide
synthesis
in cell-free
system

with s~tlfhhyclryl
on protein

The incubation
mixture
was similar
to that described
in Table I.
In most experiments,
the polgsomes
\vere pre+iollsly
illcllbated
at
37 for 5 min in 0.5 ml of buffer before
addition
of the trallsferases,
GTP, and 14C-aminoacyl-tRNA.
Glutathione
and c>-clohrximide
(1 mg per tube) were added either
during
preliminary
incrlhat,ion
or incubation,
as indicated
in the table.
The data shown
are
the average
of three experiments.
I

Components
previously
0

5
,umoles

IO
of -SH

15
Compound

Additions
incubation

incubated

for
I

20

Fro. 4. The effect of cycloheximide

on aminoacyl
transferases
previously
incltbated
wit,h -SII
compounds.
The effect of cycloheximide
on transfer
of C-amino
acids from
aminoacy-tIlNA
to peptides
was examined
using a system
consisting
of liver polysomes, lJC-aminoacyl-tl<NA,
a mixture
of transferases
I and II,
GTP,
lIg2f,
and buffer.
The transferase
fraction
in this system
was iucubated
at 37 for 5 min with various
amomits
of ghltathione
(-----),
mercaptoethanol
(- - -), or dithiothreitol
(---)
in a total
volume
of 0.5 ml of buffer.
The other
constitllents
of the reaction were then added to give a final volume
of 1 ml and incubation
was contin1led
for 30 min at 37.
Cycloheximide
(1 mg) XT-as
added to half of the samples
at the end of preliminary
incubation.
The figllre
represent,s
the mean values
obtained
in three experiments.

Polysomes

Glutathione

Cycloheximide

GTP,
q;aminoXyl-

tRNA:
transferaw

CYCb

Cluta- heximide
thione

j.moles

None
None
+
+
+
+
+
+

None
None
-

20
20

Incorporation

+
+

cpm
zioo
2400
5350
2000
5GOO
2350
5700
2900

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

to thr cnzynw
during
previous
incubation
(tubes
12 and 13).
The effwts
01 p;climinary
incubation
of the transferase
preparation
with
\-arious
levels
of GSH,
dit,hiothrcitol,
or rncrcaptoethanol
arc shown
in Fig. 4. This shows that
transfer
of 14Camino
acids in the absence
of cyclohesimide
was not significantly
influenced
by different
levels
of the sulfhgdryl
compounds.
However,
with
increasing
concentration
of these
sulfhydryl
agents
the inhibitory
action
of cyclohesirnide
diminished.
It is
interesting
to note t,hat dithiothreitol,
which
has two sulfhydryl
groups,
K:W maximally
effective
at a level of 10 pmoles
per tube,
whereas
a similar
dcgrec
of protection
against
inhibition
by the
other two con~pounds
required
20 Fmoles.
Since Suttcr
and Moldavc
(20) have shown
that prior
incubation
of transfcrase
II with glutathione
accelerates
particularly
the initial
rata of 14C-aminoacyl
transfer
t,o ribosomal
peptide,
we examined
t,hc effect of GSH level on both the initial
trallsfcr
rate and the final plateau
attained
in the presence
of c\-clohcxirnitl(a.
Fig. 6 shows that,
in absence
of the illhibitor,
previous
incuhtiolr
or the transferasc
fraction
with
20 pmoles
of GSII
rcsullcd
iti :L marginally
grcatcr
initial
incorporation
rate and
also a slightly
higher
final
plateau
than were obtained
in the
lwesence
01 4 pnloles
of GSII.
When
cyclohesimide
\VR~ added
to the reaction
mixture
follolT-ing
prior
incubation
JI-ith 4 pmoles
of GSH,
it had an extensive
inhibitory
action
both on the initial
ratca of rwction
alltl 011 the fillal ljlateau
attained.
Preliminary
incubation
with 20 pmoles
of GSH
protected
against
cyclohesi-

Synthesis

Issue of August

25, 19G9

B. X. Baliga,

A. W. P~mzcxulc,

bated

with

4 pmoles

of GSH

were

diluted

similarly,

but

4483

pmoles.
Thus, llrior treatment with a high level of GSII has a
protective action against the inhibitor.
EJect oj Cycloheximide
on Release oJ Peptides by PuromycinPuromycin releases incomplete peptidc chains from ribosomes by
substituting
for aminoacyl-tRNh
at the acceptor site on the
ribosome and subsequently
reacting to form a peptide bond
(22-24).
It has been shown that cyclohcximidc
retards this rehccordingly,
the capacity
leasing action of puromycin
(II).
of GSH to reverse this effect of cycloheximide was evaluated.
Polysomes were incubated for 5 min with C-sminoac~l-tRnX,
GSH, GTP, and the transfcrase fraction.
The polysomes were
then harrcsted on a sucrose gradient, and the incorporation
of
W activitr into peptides was examined at various points on the
gradient

(Fig.

7a).

Most,

of the

incorporated

radio:Lctivit,y

was

enough

GSH w:ks then added to maintain

the final concentration

at 4
mhr.
Cycloheximide
was added
to some of the tubes at the beginning of incubation.
Fig. 6 shows that enzyme previously
incubated

with

c\-clohesimide

20 pmoles

of GSH

was

much

than was enz?-me previously

less

inhibited

incubated

by

with

/+tJ
I

IO

MINUTES

FIG.
ferases

amino

6. The

effect

of preliminary

lvith cycloheximide
The
acid transfer.

incubation

and glutathione

of nminoacyltrans-

on initial

rate of

system
used transferred
K-amino
acids from aminoacyl-tRNA
to peptides
and consisted
of a mixed
transferase
fraction,
liver polysomes,
GTP, Mgz+,
*C-aminoacyltRNA,
and bluffer.
The transferase
fraction
was incubated
at 37
for 10 min with 4 or 20pmoles
of GSH in 0.5 ml of buffer.
It was
then adjusted
by dilution
to provide
the same amount
of enzyme
and 4 pmoles
of GSH in all samples.
The other
constituents
of
the reaction
\vere then added to give a final volume
of 2 ml.
Cycloheximide
(1 mg) was added at this point to some of the tubes.
Samples
were taken
at different
time intervals
for assay of incorporation
into peptidcs.
The dat,a are the average
of two experiments.

4 5 6
FRACTIONS

: :
:
:
i

:
4 5 6
7 8
9 I
23-d56789
FRACllONS
FRKTIONS

,L!

FIG. 7. Effect
of cycloheximide
and puromycin
on distribution
of labeled
polypeptide
on polysome
profile.
The system
used
transferred
~4C-aminoacyl-tlLNA
to peptides
and consisted
of a
mixed
transferase
fraction,
liver
polysomes,
GTP,
11g2-, GSH,
~4C-aminoacyl-tltNA,
and buffer
to give a final volume
of 1 ml.
Following
incubation
at 37, the polysomes
were separated
on a
sucrose
gradient
and the profile
was recorded
(---).
Fractions
were taken
from the gradient
and 14C-incorporation
into peptides
was measured
(---).
In the case of tubes cl, e, and f, the transferase fraction
in 0.5 ml of buffer was given preliminary
incubation
for 5 min with the amolmt
of GSH described
below.
In addition,
in some tubes
the amount
of GSH was increased
from 4 to 20
Mmolcs per tube, and puromycin
(200 ,~g) or cycloheximide
(1 mg)
or both
were
added
to some samples.
The
gradients
shown
represent
the following
conditions
of incubation:
a, incubation
for 5 min lvithollt
inhibitors
or preliminary
incubation;
b, similar
to preceding
conditions
with addition
of puromycin
after 3 min of
incubation;
c, incubation
with
cycloheximide
for first 3 min of
incubation,
followed
by an additional
2 min with puromycin;
cl,
preliminary
incubation
of transferases
for 5 min with
4 pmoles
of GSH, followed
by 5-min incubation
alone, then incubation
Tvith
cycloheximide
for 5 min, followed
by 5 min with
puromycin;
e,
preliminary
incubation
of trnnsferases
with
20 pmoles
of GSH,
t,hen 5-min incubation
alone, then incubation
with cycloheximide
for 5 min, followed
by 5 mill with puromycin;
f, preliminary
incubation
of transferases
m-ith 20 pmoles of GSH, followed
by IO-min
incubation,
and finally
5 min with puromycin.
The experiment
was replicated
t\vice.

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

This eliminates t,he possibilmide at all stages of the reaction.

ity that, the inhibitory
action of cycloheximide is directed only
against the initial rate of aminoacyl transfer, which is particularly subject to GSII stimulation
according to Hutter and Noldave (20).
In addition, experiments were performed in which the polyHomes instead of the transferases were previously incubated with
cyclohesimidc or GSH. Table II shows that prior incubation of
the polysomes with cycloheximide potentiatcd
the action of the
inhibitor very little, and preliminary incubation of the polysomes
with 20 pmoles of GSH did not confer any additional protective
effect against cyclohcximidc
over and above that obtained by
adding the Same amount of GSH during incubation.
It is to be
noted that the polysomcs used in these studies were washed with
KH,Cl and would thus not be expected to carry aminoacyltrwnsfera.sc II (21). From these various experiments, it can be
concluded that the maximal protective effect of GSH against
c)-clohcxirnidc
is obtained when the transferase preparation
is
previously incubated with the sulfhydrgl
compounds and the
cyclohesimide is added subsequently.
Since maximal protection
by sulfhydryl
compounds is obtaincd by prior incubation with transfcrases, an experiment was
carried out, in which 20 pmoles of GSH were added to 0.5 ml of
trnnsfrrase solution, and they mere incubated together for 10
min. Then the GSH concentration
was lowered by dilution to
4 m&I before adding the rest of the system for transferring amino
iiicuacids from aminoacyl-tRiYA.
Control samples previously

and H. N. Jlunro

4486

Cycloheximide

Action

,0:
Minus
_.---------

Minus

AA-

tRNA

-----_

Ribosomes

Minutes

Fro. 8. Ribosome
and aminoacyl-tRNA
requirements
for
GTPase action. Hydrolysis of GTP was examined in a cell-free
system n-hich transferred amino acids (AA) from aminoacylIt contained rat liver polysomes, the transtRNA to peptides.
ferase enzyme fraction. ,..(Y-~~P)-GTP. aminoacvl-tRNA.
and other
components as described under Materials
and Methods.
The
time course is shown for the complete system and in the absence of
added aminoacyl-tRNA
or polysomal ribosomes.

Synthesis

Vol. 244, No.

16

prevents release of peptide chains by puromycin, an action of

cycloheximide that can be prevented by previous incubation of
the transferase enzyme fraction with large amounts of GSH.
According to Skogerson and Moldave
(25), puromycin reacts
more extensively with peptidyl-tRNA4
when it is at the peptidyl
site on the ribosomes; its presence at that site is dependent on
transferase II and GTP, thus explaining inhibition
of the puromycin reaction by cycloheximide.
Egect of Cycloheximide and Other Inhibitors on GTP Hydrolysis
and Amino Acid Incorporation
in Presence of DiJerenf Concentrations of GSH-From
the preceding evidence, the inhibitory
action of cycloheximide on chain elongation appears to be directed against transferase II, which is the only sulfhydryl-dependent enzyme involved in aminoacyl transfer (20). Nishizuka
and Lipmann (26) have found that the GTP hydrolysis reaction
required for chain elongation in a bacterial protein-synthesizing
system also involves a sulfhydryl-dependent
enzyme. This enzyme (translocase) may be identical with transferase II (25).
We (14) have recently shown that cycloheximide inhibits GTP
hydrolysis in our mammalian system. The effective concentrations of cycloheximide are similar to those required for inhibition
of W-aminoacyl
transfer to peptide chains in the system, and
the action of cycloheximide can be similarly prevented by glutathione (14). This confirms the interrelationship
between cycloheximide and GSH by means of a separate reaction.
In the
present series of studies, we have used the hydrol>-sis of GTP to
follow the action of other inhibitors of protein synthesis in order
to supplement
the information
obtained from amino acid in
corporation.
First, the condit.ions for hydrolysis
of (y-3?P-GTl
xvere
examined using the protein-synthesizing
system for amino acid
transfer from aminoacyl-tRNA
to peptide.
Fig. 8 shows the
amounts of 32P released as inorganic pho+hate
in this cell-free
protein-synthesizing
system when all reagents are present and
also in the absence of aminoacyl-tRNX
or ribosomes.
A small
release is apparent when either of the latter is deleted from the
system, but this reaction terminates after about 5 mm, whereas
the complete system causes more extensive GTP hydrolysis
which proceeds for at least 30 min. Thus , release of 321 in the
complete system is largely dependent on amino acid transfer to
peptides.
The amino acid-dependent,
hydrolysis of GTP was
used to study the action of glutathione
on inhibition
of protein
Samples were also incubated
synthesis by various compounds.
in the same reaction mixture with 14C-atllinoacyl-tRSI
to allow
comparison of their action on amino acid incorporation.
Table
III shows the effect of preliminary incubation of the transferase
enzyme preparation
with either 4 or 20 pmoles of glutathione.
Following
this prior incubation,
the incorporation
system was
completed,
using (Y-~~P)-GTI
or 14C-anlinoac~l-tRS,,
and
inhibitors were added at this point.
In confirmation
of earlier
studies (14), cycloheximide at a concentration
of 1 mg per ml
extensively inhibited bot,h 32P release and i4C-amino acid trailsfer to peptide when the transfcrasc fraction had been previously
incubated with 4 pmoles of glutathione, but inhibition was slight
when the transferase fraction was previously incubated with
20 pmolcs of the sulfhydryl reagent.
Streptovitacin
-1, a hydrosylated
derivative of cyclohesimide,
Teas also added at a
concentration
of 1 mg per ml, since this is known to inhibit l)rotein synthesis effectively in cell-free systems (4). Table HI
shows that it behaved like cycloheximide.
Grollman
(27) has
shown that emetine inhibits the amino acid transfer reaction in

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

found on the polysome aggregates, with little release into the

When puromycin was added during the
supernatant
fraction.
final 2 min of incubation, the activity in the polysomes was much
reduced and the supernatant fraction of the gradient contained
most of the radioactivity
(Fig. 7b). When cycloheximide
was
added 3 min before the puromycin, more act,ivity remained on
the polysorne aggregates and very little was released (Fig. 7~).
Disaggregation
of polysomes to monosomes as a result of puromycin action was also reduced by addition of cycloheximide
($ Fig. 7, b and c). To examine the effect of glutathione
on
this action of cycloheximide,
the transferase fraction was previously incubated in the presence of either 4 or 20 pmoles of GSH
for 5 min; the (:-arnirloac?-l-tRNA
and cofactors were then
added, and incubation was continued for 15 min with addition of
cyclohexirnide and then puromycin as indicated in Fig. 7, d, e,
and f. Fig. 7d confirms that, cycloheximide
reduces total incorporation and prevents release of peptide chains by puromycin
when the transfemse has been previously incubated at the lower
GSH level (cj. Fig. 7b). However, when the enzyme was first
incubated with 20 pmoles of GSH (Fig. 7e), incorporation
of the
Y-amino
acids was very extensive in spite of the presence of the
cyclohcximide,
thus confirming protection against inhibition
by
previous treatment of the transferase fraction with GSH. Furthermore, the puromycin
almost completely released the Wpeptides into the supernatant fraction.
It will also be noted that
the polysome profile under these conditions showed considerable
degradation,
notably the presence of a large monosome peak.
These actions of puromycin were then similar to its effects in the
absence of cycloheximide (Fig. 7f). It will be seen from comparison of Fig. 7.f with Fig. 7b that the presence of high concentrations of GSH actually potentiates the releasing action of puromycin.
These experiments confirm that cycloheximide retards protein
synthesis without releasing the peptide chains (10). It also

on Protein

Issue

of August

B. 8. Baliga,

25, 1969

A. W. Proncxuk,

TABLE
Efect
of preliminary
inhibitory
action
poration
and

aggregation
caused by delayed addition of amino acids to a medium lacking free amino acids could be inhibited by much lower
concentrations
of cycloheximide than those necessary to achieve
Since this suggests a separate
inhibition
of chain elongation.
action of the inhibitor,
we tested the capacity of sulfhydryl
Fig.
compounds to protect against this effect of cycloheximide.
9 shows polysome reaggregation
in response to addition of amino
acids when the GSH concentration
in the medium was raised
from 4 mM to 20 mM. The usual reduction in monosomes and
accumulation
of polysomes was obtained, and the presence of
0.1 pg of cycloheximide was again sufficient to prevent reaggregation (compare Fig. 2). Thus, GSH does not influence this
action
of cycloheximide.
was present in the meIn the same experiments, YXeucine
dium, and incorporation
of radioactivity
into protein was examined after 40 min of incubation.
When no other amino acids

----_-.
-

22

incubation

- AA

{ 2$

incybation

4:

20 incubation
- AA
+2 incubation
+AA and
+O.Ipg
cycloheximide

III

of transferases
with glutathione
on
various
antibiotics
on amino
acid incorGTP
hydrolysis
in cell-free
proteinsynthesizing
system

incubation

of
on

The incubation
mixture
was the same as in Table I, except that
in some experiments
(Y-~~P)-GTP
and Wkminoacyl-tRNA
were
used in order
to study
GTP hydrolysis.
The crude transferase
preparation
was previously
incubated
with glutathione
for 5 min
The remaining
constituent,s
were then added and incubaat 37.
tion was continued
for 30 min.

Prior
incubation

Incubation
MC-

_-

Tube
hzyme

,STP,
IPolY(;hlta- somes,
1thione a mino.-

Antibiotic

t Ri

I moles

1
2
3
4
5
6
7
8
9
10

+
+
+
+
+
+
+
+
+
+

4
20
4
20
4
20
4
20
4
20

AminoacyltRNA
incorporatedl

@m/lube
+
+
+
+
+
+
+
+
+
+

Cycloheximide
Cycloheximide
Streptovitacin
Streptovitadin
Emetine
Emetine
Sparsomycin
Sparsomycin

1000
1200
200
800
210
790
530
530
830
980

4170
4350
1140
3900
830
4000
560
580
660
700

a The value
for 32P released
in tube 1 is equivalent
to 4600
pmoles
of GTP hydrolyzed,
and the value for 14C incorporated
in
tube 1 is equivalent
to 480 pmoles
of amino
acid incorporated.
Conway
and Lipmann
(18) also report
a considerable
discrepancy
between
GTP hydrolysis
and aminoacyl
incorporation.
-

,
J

LINEAR

SUCROSE

GRADIENT

= 40%
FIG. 9. Prevention
by cycloheximide
of polysome
reaggregation
on addition
of amino acids (AA)
in a GSH-rich
medium.
Liver
polysomes
were incubated
for 20 min in an amino acid-dependent
protein-synthesizing
system
similar
to that of Fig. 2 but containing 20 mM GSH without
added amino
acids, and then a complete
mixture
of 20 amino acids w&s added either
alone (-)
or in the
presence
of 0.1 rg of cycloheximide
(-- -).
Incubation
was
continued
for an additional
2 min.
A control
sample
was incubated
for a similar
total
period
of 22 min without
added
amino
acids
(-----).
All three
samples
were separated
simultaneously
on sucrose gradients
and the profiles
were measured
by ultraviolet
absorption.
The experiment
was replicated
twice.
16%

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

cell-free protein-synthesizing
systems, and suggests that this
may occur because of its structural similarity to cycloheximide.
We therefore added 100 pg of emetine per ml to our transfer system, a concentration
shown by Grollman to be within the effective range, and confirmed that both amino acid incorporation
and a2P release were extensively inhibited.
However, unlike
cycloheximide,
neither reaction could be protected against inhibition when the glutathione
concentration
was raised. Grollman (28) has recently found that emetine differs from cycloheximide in its action on HeLa cells. On suspending the cells
in fresh medium, the inhibitory action of cycloheximide could be
reversed, whereas that of emetine could not; however, in Grollmans system the action of streptovitacin
A was also found to be
irreversible.
Finally, the action of sparsomycin was studied in
our system at a concentration
of 2 pg per ml, which is known to
inhibit protein synthesis in mammalian
cell-free systems (29).
Table III shows that inhibition
of amino acid incorporation
was
largely suppressed by this concentration,
and that raising the
glutathione concentration did not alleviate this effect. However,
This result,
inhibition of 32P release by sparsomycin was slight.
which implies an unexpected dissociation between the two requirements of peptide bond formation on ribosomes, has been
confirmed on several occasions and may indicate uncoupling by
sparsomycin of GTP hydrolysis from other events in protein
synthesis.
E$ect of Cycloheximide on Polysome Reaggregation in Presence
of Extra GSH--It
was demonstrated
above that polysome re-

4487

and H. N. Munro

44%

Cyclohc:cimide Action on Protein S7y~lhesis

chain ittitiation atid oti (*lt:titt c~loirg:tlioti.

\ilieti the\- ittcrtbated
intact rcticulocytcs
with S:iF xnd cyclolwsintitlc,
both conpomtds itthibitcd ch:titt initiation, but c~~c~1oIic~sittiitlc:ilso aftfccted
elong:~tiori of nt~swtt1 Iwl)titIc~ c~haitts;. Orir wsults 1)rovidc direct cvidcrtce for such :I dUiLl wliotr lltttlw c*otttlitioits of cell-lace
protcitt syrtthwis.
We have cmltloyc~d :L sys~c~rn ~II \vhich the
presettw 01 amino xitls at the start of ittc~u1J;itiott wsults mainly
in elottgntion of ~woviously esi~tiug 1JrIJtitlc chaitis ott the ribocomes. 011 the other hand, 1Jrclitnittar~- ittcubation of the system
n-ithout tttttitio :tcitls wsrtlts itt tlixiggwgxtion
of the IJol~sotnes
awl, \~lion:ttrtiiio:icitls
arc sulJsecIuctrtly :ttltlttl, the 1tolyso11ics reaggrcgatc~ 1)~ a 1Jrowss that ~Jrwttttlabl\- xquircs ittitiation
of
pq)tide
chains. Ihcb c*ottc*c~tili~:ttioti of c:\-~lollc~sittlitlt! iteedcd
for effcctivc itihilJitiotr of chairi c~lottg:ilioii iii this syslcni is 10
r0tnplct.r:
1Jolysome
times greater Ihan 1h:tt tic&d
lo ~wvc~it
reconstitution
on adding amino acidh. lhis is the rcvwe of Ihe
finding of Stanners (30), w110 estrmittcd t,ht: ac*tiott of cyclohcsimide 011 hamster ctnbryonic
cells iti t issuc: c~ultrrix~ and found
that low doses inhibited movcmcttt of ribosontw along the messenger, 1Jut had Icss xtion 011 rc;ttt;t~httlc~ttt. 01 free ribosonlcs to
the mcssettger. Our cell-flee system appwrs to have :I limited
cnIwcil,y for polyson~e re:~ggw,g:rtio~i, ant1 this might well make
it; more sensitive to inhibition
thrn tlw wmc 1~rtJwss in the
whole ~11. The scw~nd source of c~\~itlcttw of :t tlwtl site of
ac+otr of cyclohesimidc is sltowt by the I~~~c~vc~n~iotl
of its actioll
by sulfhydryl COII~IJO~II~S. lhcse cor~l~Jou~~tls c:m estcttsivcly
reduw the inhiljitory actiotl of c~yclohesitltide on t:h:tin clo~tgltion
but 1101.on amino acid-induced pol~.wntc ~.c~a~gt.cg:~tiott, t~vcn at
the \-cry low c:otic:c:til,ri~tio~i of itihibilor irwdrtl to I~i~~vcttt rcaggfrgatiou.
Olw silo of :&on
01 c~~lolt~siti~itlc oti IJrotcitt synthesis has
been Itrovisiottall,v itlwttificd with tr;~nsle~xsc 11. Itrvolvcment
of tlatt5~cr:rscs \v:ts origitially srtggwtcttl 1~)~Sicgcl :ititl Sislcr (8)
on the groutttls that ~yclohcsiniitlc: (lit1 t101 :r~J~Jcw to ha\-c an
effect oti amino :icitl~:rctiT.;ltiti~ c~~zyniw. Prlicetti,
(olott~bo,
and ILgliotii
(11) .sho\vctl that c?clollcsifltidc
itrhiljits rolense of
peIJtitlc chains front I~ol~~otucs lJ\- 1Jttroiiiy~iti :uid cwtic~lridctl that
tr:insfcr:tsc II is 1hc site of ttc~tioii ol 111~iiihibitot~.
011 lhc other
hand, l\vo other grouljs of ill\-cstigxtors
(23, 31) Iailcd to obtain
an inhil)itory
c~ffcc~l.of cyc~lohcsitttitlc on I~urottt~~in-tlc~~)(~tt(Ic~~t
rcleasc ol 1x1)1 itlw Ii~otn rc~ticuloc\-tc I~olyson~cs. I(otli ol these
grotiI)s uswl 20 rtt>l (:817 in tlitlir inculxttion s~slci~is, \\hcreas
Feliwtti
e/ nl. (11) uwtl 2 iinr GSH. \\c hvc t~ji~fii~nwd the
inhibition
of I)lu~otrtyc~itt wllcn Ihc GSTT cottwtttr:tliou
of the medium is 4 11111. Oltr tl:11a Irtrtllrr tirnlotls;lr:lte lhat Ihis :tt*1ion of

16

cyclohcsimitlc
cxn IJc 1)wvctttctl IJ~ IJrior ittc~rtljttt iott of tlrc c*rutlc
tra.nsferase Ira~t,iotl with higher lcvcls ol (:Sll (III) to 20 ~tnolcs).
These ol~scrvatiotts ctitl&sizc
the int1Jortattc~c~ ol MI1 wtwctitration in the tnctlium whet1 stutlyittg the: actiott ol itihibitot~d of
transfcrwc
II. Itthiljitiott
of rclctrsc of 1w1J1itlw 1)~. 1Jtttwniyc.itt
occurs bcc:Lusc, \vll(~ll llwvestcvl, rliost 0T Ihc: Iwl)titlr is :ttl:tc:hcd
to tRS,L a1 the :ttttitto:tcyl site ott the riljosorttw
(25). Sittce
purom~-citt rnlist bitid to this ri1Josottic site: Iwloi~~ Iotxtitig the
pcptitle bottd, the 1JqJtidylLt1iS.i
has first to IJc ~txttslowtcd
to
the IJqJtitlyl site. lhis :twJunts for 1hc ittltiljitor~~ actiott of
cyc~loliesitnitle oti I)urotitycin rclwsc a11t1 for Itic, 51inlttl:iitl c,ffcct,
of GSH.
Our es1wittwitts
tlii~on. sonic light on tlicx ttaturc~ of the ;iction
of c~!-clohcsitltitlt~ ou 1ra1tslcrnsc I I. Huttw ant1 ~Ioltl:t\-c (20)
clctnonstt~:t1(~(1 with Ititrified IJrc]J:w:ttiotts ol 1x1 liver (rttttsiwtse
II thttt the t~~tzynlc gives m;lsinlaI incoqjotxt iott \vhctt 1)wviousl~
itrcubatctl with \atious sulfI~ytli~~-1 cotri~~outttls , gwatwt :ictivit!
being :rchic5wI 1J\- I)tior iiicu~J:itioii I\-ilh ~otrcctttt,:tt,iotis of GKI-I
of 20 nt~ or higlw.
It, :~IJ~jc:ns that, I)urifiic~tl ~1x11sCw:tst~I I is
readily itt:tc:t iwt.ed Wough its suIIhytIry1 g~~rt~w :rtrtl that it twt
be regctiwttcd
1Jy sulfhydi~yl tlonot~s. Ill th? (:I( ol Ihc t:1wde
trnnsferasc IJrc:I):ltat,iotis usctl I)y us, whi&
iitt~ludc l)olh ctizymcs I and II, it is ~~robablc that transfctxw 1I is less uitst:ible
(20). Ihis is srtIJIwt?,c~tl by the tlata itt Eig. 4 \vliic.h show that,
varying the levels ol (%I1 ant1 other suIfliytl~~~l t~~n~I~otutd~ dtw
ing prclimitiar)ittculJ:ttion did Itot :t1J1JiwialJl~- xffcct ltxiisfcrusc
nctivit,y tlrtt~itig sul~stquent iticulJ:itioti.
Kcwrthelcss,
although
in our unI~ut~ifictl systc>ni the ctizytiic she\\-s t10 great itictwsc in
:tctiritJ- :ts ;t wsult 0E Ijtwious ittculJatioti \vith liigh Icvc+ of
GSH, thcsc high c:ottcetttt,ations ga\~: adtlcd 1xo1 cction xgtinst
subsequc~nt. atldition of c)-clohesimidc (Fig, 4). Ihis is IIOI due
to the high GSII concctttr;~tion during suljsequct~t ittculJ:rtion,~
since the 1trotedivc c,ffcc:t Ijcrsists cvcn if the (XI
c~otitcnt is
Io\verrtI lwforc the c:yclohesitrtidc :tn(l otlicr w:id:iitts
arc ;itltlrd
at the rtitl of IJrcIittiitt:w~~ iitculx~tiou of the twzytnc (Fig. 6).
Ihis agrws with the fitttling ol Sut tcr ;~td RIoltl;tvc (20) that IJrior
trctttmwt
of purified tr:insfelasc I I \vith high l~~vc~lsof (iSI iiicrettsrs subsc~~ucnt :rmitio:icyI ttxtrslcr :tt :L lonc~ GSIJ t~onccnIration.
Our qJwitncttt
~II n-hich the (XII ~ottc,cntt,:ttion W:IS
lowcretl bclorc adtlittg c~?-c~lohc,sitnitlc (Fig. ti) tlt~ntottsttxlcs that.
nlercaIJ1:ttt :iiitl tht, itihilJitot~ (10 ttot ui~tlcrgo tliiw~1 t~lit~tiiiwl
rcnction.
lrotectiori by WIT against, itthiljitiott
of tr:ttts(etxsc I I :tllows
one to lost, othc~ :u~~il~ioiics in ortlcr to tlrtertttittc wht~thw thq
act in the sanic way :LS t:~clo~lc~r;inlitlc. Ihc :rc:liott or high (X313
concctitr:rtiotis
ott th(t itthibitioii
01 ~Jrotcitt sgill,lic~sis l)y slixljtovilacin .I is cxtctly similar to th:lt of c:\-cloltc,sittritlc, to \vhich it
is structurally
rc:latc~l (lablc I I I). I {Ctttctittc \v:ts thought by
Grolltr~an (27) to be attothw :m:dogtte of c,!-c~loltcsir~lidc, hut in
our es]Jwitww its itihiljitot~y :t(*tiori is riot 1Jrcvcitl(~l 1)~ itic~twsitig
thr GSII c~oti~ctilr:ttiott in the cell-lrw systctn. (~rollm:~~~ (28)
found that Ijrotcitt syttthcsis iti 1-1~1~ ~11s \V:IS itthilJilc~1 IJy c:y~lohesinlitlc, stt,cpto~it:tc:itt, tint1 emclittc, l~tit wliw Ihc alla wrrc
stis1tctid~~l in Itwli tit(~tliuttt llic itihiljiliott
\v:is rctno\-cd oiily in
the with 01 c:~c:Iolic~sittiicle. Itl UJlltlXSl~, 0111(l~llil SllOW Ill:11 the
iuhibitory
aciion or etnetirw catt lx distittguishctl
IIQI~I lllaf, of
strcptorit:ic*in
lJy its ittscitsitivily
to GSII.
Iltis tlow itot, of
couwe, c~litnittatc t txttsltwse TT :IS 1110 site, 01 :tclion of cmc+itw.
Fin:dly, sIxirsom~-(*iit \v:ts csanlitrctl, sittw il, wI)iwt~ttts :riiotlici
antibiotic \~hosc I)rcrisc: site ol action on IwlJtitlc lwtttl IOIVII:L~ion
rem:tins rtttcc~r1:iiti (32). .\tt ittt*rc:isc iii GSl I c~otrc~c~ti1t~:t1iot~
:tlso

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

mw :~tltlctl to the tirc~tlium, 2270 rpm lwr tr11Jc~were ittcorIJoratct1.

\\?lcll the: roml)lctc atllillo acitl ttlisturt evils atldctl altcr 20 nlin
of ittcttbatiott, ittc~orIJot~:ttiott row to 6250 cljtii 1Jer tube. If,
ho\x-cvc~i~,0.1 piz; of c~c:lohc~sitttitIc \~a: tttltlctl along with the amino
acid mist ttre, incoqjorat ion wits i,h(~tt rcclu~cd to 3010 clam per
tube; that is, niorc thiiti 80:; of the stitiiul~tttl, actioti of the anliuo
acids was c~limittatctl.
In thcic cs~wrimr~tits, lhc cottc~c~tilr:~tion
of GSll was 20 111~ Ihroughout
incubation,
IJut lhc itthilJitor\
effect 0E cyclohcsimide \vas sitniliir itt tlcgwc to that oljtaitted ill
the ~~rcscr~cc ol 4 I~IM GSII (Fig. lc).
Ihcsc studies Iwovitle furthw c\-itlcttcc that the actioti of cyclohcsimitlc on rc:rggwg:ttiott :iiid on 1wlJtitl~ t~lotlgxtioti is tlue to
diffrrcttt trtcch:tttisttts.

Vol. 244, No.

Issue of August

B. S. Baliga, A. W. Proncmlc, and H. N. Muwo

25, 1969

failed to prevent the inhibition

caused bg this antibiotic.
An
interesting feature of these inhibitors was that cgclohcsimide
and streptovitacin
inhibited GTI hydrolysis to the same extent
as

amino

acid

transfer

from

aminoacyl-tRNA.

Emetine

also

inhibited GTP hydrolysis fairly strongly, but sparsomynin was

almost without effect on this reaction while causing extensive
inhibition
of amino acid incorporation.
This demonstrates a
considerable uncoupling of Gll hydrolysis from peptide bond
formation

when

this

antibiotic

is lxesent.

I>., J. Gen. Microbial.,

19, 497 (1958).
C. W., ROBINSON,
I. F., AND S:UXOR,
B., Biochenr.
Phar&col.,
12, 855 (1963).
COLOMIIO.
B.. FEI~CETTI.
I,.. .\NU
BAGLIONI.
C.. Uiochem.

Biophys.
Res. Commun.,ll,
989 (1965).
COLOMBO,
B., FELICETTI,
L., AND B.\GLIONI,
C., Riochim.
Biophys.
Acta, 119, 109 (1966).
TRIKATELLIS,
A. C., MONTJ,~R,
M., AND AXELROD,
A. E.,
Biochemistry,
4, 2065 (1965).
KORNER,
A., Biochem.
J., 101,627
(1966).
GODCH.~UX.
W.. ADAMSON,
S. II., IIND HERBERT,
E., J. Mol.
Biol.,
27; 57 11967).
SIEGEL,
M. It., AND SISI~ER,
H. l).,
Nature,
200, 675 (IOG3);
Biochim.
Biophys.
Acta, 87, 83 (1964).
ENKIS,
H. L., AND LUBIN,
M., Fed. Proc., 23, 269 (1964).
WETTSTEIN,
F. O., NOLL,
H., )IND PENMAN,
S., Biochim.
Biophys.
Acta, 87, 525 (1964).
FELICETTI.
L., COLOM~O,
B., ,\NI) B.IGLIONI,
C., Xochim.
Biophys:
A&,
119, 120 (196G).
LIN.
S. Y.. MOSLELLER.
R. D.. AXI) I~.II<DES~Y.
B., J. Uol. BioZ..
Zi, 51 (i966).

B.ILIG.\,
B. S., PRONCZUK,
A. W., AND MUNRO,
H. N., J. Mol.
Biol.,
34, 199 (1968).

1. KERIUDQE,
2. YOUNG,
3.
4.

5.
6.

7.
8.
9.

10.
11.
12.
13.

14. MUNRO,
13. N., BAI,IG.\,
B. S., .\AX PI~ONC~UK,
A. W., Nature,
219, 944 (1968).
15. WErrsrEIiv,
F. O., S.~.\EHELII\,
T., AND NOLL,
H., Nature,
197, 430 (1963).
16. GASIOIL,
E., AND MOLD~VE,
Ii., J. Biol.
Chem., 240, 3346 (1965).
K., in S. P. COLOXICK
AND N. 0. KAPLAN
(Editors),
17. MOLD.ZVE,
Methods
in enzvmoloau,
Vol. VI. Academic
Press. New York.
1963, p. 757.
__
__
18. CONI~~Y,
W. T., .\ND
LIPMINN,
F., 11-0~. Nat.
Acad.
Sci.
77. S. A., 62, 1462 (1964).
19. LOUXY,
0. H., Ro~EB&J&,
N. J., FARI~, A. L., AND RANDALL,
R. J., J. Biol. Chem., 193, 265 (1951).
20. SUTTER.
It. P.. .\ND MOI,D.\VE,
K.. J. Biol. Chem..
241, 1698
(196Gj.
21. SKOGERSON,
L., :IND MOLD.\VE,
K., Eochem.
Biophys.
Res.
Commun..
27, 568 (1967).
22. ALLEN,
11. k., .~NI) &ME~NIK,
P. C., Biochim.
Biophys.
Acta,
66, 8G5 (1962).
23. WILLUMSON,
A. I< ., .INI) SCHU-EET,
R., J. Mol.
Biol.,
11,
358 (1965).
24. NATHANS,
I>., Proc.
A-at. Acad. Sci. U. S. A., 61, 585 (1964).
25. SKOGERSON,
I,., AND MOLDAVE,
K., Arch. Biochem.
Biophys.,
126, 497 (1968).
26. NISHIZUIU,
Y., .\NI) LIPMANN,
F., Arch.
Biochem.
Biophys.,
116, 344 (1966).
27. GROLLMIN,
A. P., Proc. Nat. Acad.
Sci. U. S. A., 66, 1867
(1966).
28. GROLLMAN,
A. P., J. Biol. Chem., 243,4089
(1968).
29. TRAKATELLIS,
A. C., Proc. Nat. Acad. Sci. U. S. A., 69, 854
(1968).
C. P., Biochem.
Biophys.
ILes. Commun.,
24, 758
30. STANNEHS,
(1966).
31. CLARIS,
J. M., JR., AND CH.~XG, A. Y., J. Biol.
Chem.,
240,
4734 (1965).
J., .\ND GOLDBERG,
I. H., Biochemistry,
7, 418
32. JAYAR~IMAN,
(1968).

Downloaded from www.jbc.org at NATIONAL INSTITUTE OF SCIENCE EDUCATION & RESEARCH, on April 9, 2012

REFERENCES

4489