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Mechanism of Cycloheximide Inhibition of Protein Synthesis
in a Cell-free System Prepared from Rat Liver*
(Keceived
for
pltblication, December
23, 196s)
1%. s. IhLIGA, :I. w. ~OSCZUG, AS-I) H. s. I\IC-sRO
From the Physiological Chemistry LabolatoGes, Departme?lt of Nutrifiotz aud Food Science, S1assachusett.s
Institute
of
Technology, Cambridge, Massachusetts 02139
SUMMARY
Sites of cycloheximide action on protein synthes is 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 incor-
porate 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 prelimi-
nary incubation of a mixture of transferase I and II with
high concentrations of glutathione before adding these
enzymes to the system. Other sulf hydryl compounds were
also ef fec tiv e in protecting against cycloheximide. It has
been concluded that the inhibitory action of cycloheximide
on peptide chain elongation involves inactivation of trans-
ferase II , an enzyme known to have a sulfhydryl requirement.
High concentrations of glutathione were also found to pre-
vent the inhibition of cell-free protein synthes is 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 stimu-
lated, probably by a process involving chain initiation.
The
response o f 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 dif fers from its ef fect on peptide chain
elongation.
(‘~~clolicsimitle (.1ctitliolle), a11 antibiotic 1)roducetl 1))
Sfrepfo-~/yes griwtrs, W;IS first shown by
Kerridge (1) to inhibit
lxotoill
syllthcsis ill :I >-east.
Its iuhibitory action h:ls since been
* This investigation
\~as srqlported 1)~ Pub lic Ilralth Service
Gratlt (A-08893-03.
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 influ-
enced by the inhibitor, which thus must act at some subsequent
stage in protein synthesis. Recent studies suggest that cyclo-
hcsimitlc may af fect protein synthesis at more than one l)oint.
Lin, Mosteller, and Hardesty (12) obtained evidence from es-
perimellts on reticulocytes that the antibiotic inhibits the in-
tiation of new peptide chains aud the elongation of nascent pep-
tides 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 pro-
tein synthwis prrl,ared from rat liver (13). The system used
consisted of liver polyaomes, activating and transferring enz~mcs,
and cofactors. Since the enzyme fract ions were treated to rc-
move frcxe a11d tRK.&bound alnino acids, the system is conse-
quentl>. dependent on an exogwous supply of amino acids. I f
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 incubn-
tion medium, the polysomes can be reaggrcgatcd and labeled
amino acids are once more incorporated into l)eptide chain
8/19/2019 Mechanism of Cycloheximide
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Issue of August 25, 1969 B. X. Baliga, A. W. Prommk, and H. N. Munro 4431
from General Biochemicals.
Cyclohesimide was provided by
ZUdrirh, and puromycin dihydrochloride was obtained from
Nutritional Biochemicals. Streptovitacin A was a ljroduct o f
Ultjohn, and cmctinc was provided by the S. B. Penick Company,
Scn- York, Sew York. Sparsomycin was kindly supplied as
a gif t by Dr. I. H. Goldberg of the Harvard Medical School. The
uniformly labeled 14C-L-leucine (specif ic act ’iv ity , 250 mCi per
mmole) and a mixture of %-amino acids (1 &i per mg of uni-
formly lab&xl amino acid mixture) were purchased from sew
England Suclear. International Chemical and n’uclcar Corpora-
tioll, Irvine, California, provided (+*P-GTP (speci fic radioac-
tivi ty , 22 mCi per pmole), which n-as further purified by chroma-
tography on DEhE-cellulose. Most of the media used were
matlc ul, in TK(1\1 buf fer (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 (C-
ribosomes) were obtained from the livers of fasting 150-g rats by
the procedure described by Baliga, l’ronczuk, and Munro (13).
To obtain activating and transferring cnzymcs, cell sap was pre-
l):nwl
: IJKI
denuded, firs t, of free amino acids by dialysi.s, and
second, of tRr\‘-1 by the lxotamiire sulfate treatme as described
1,. IMiga et nl. (13). l’rotein n-as precipitated from this frac-
tioll b&n-een 30 a11d iO7; saturation with (SH,)$O, to yield a
mixture of activating and transferring enzymes with Illinirnal
tontent of free or tRNX-attached amino acids; t,he l~w~~:tration
was uwd for the system incorporating free amino acids illto 1x1,-
tides. For incorl)oration of amino acids from ami~wacyl-tRr\‘A
int 0 pal>-.qome?,
lillft~ac~tioiiated alllii,o:ic3-ltlaIlsfer:l.~e~ free of
activating enzymes n-we prepared I’rolrr the cell salI :rf tcxr lrent-
nwnt with Sephatlcs G-25. The nrtivating rnzymw \vc rc re-
moved by
the
traditional nlrthod of lwc*il)itation at 1111 5,
: IJ~ a.
mixture of transfcrascs I and II was ~~relwwl from the su~)cr~~a-
tarot fraction as dcscribed bJ- Gasior :uld Molda\-r (I 6).
U.ith
cwvh batch o f reaction misturc, the optimal amount< of trans-
fernsc fraction alld (when required) of trnllsfcrase and arti\-sting
enzyrue
fxaction wcrc established.
I+-eparation oj “C-arrlinoacyl~tR~~~ I -For the prelwation of
radionctire aiilino:rc~l~tRr\TX, the lxocedure dcwribed by
l\Ioltl:lre (I T) \r:w wvtl .
Rat, liver cell sap WM ndjwtcd to l,II
5 to bring dowi :I precipitate containing tRX.1
ai~d
amino arid-
acti\-sting ellzymw.
This precipitate ~1s thrn ret&sol\-rd and
allon-cd to rract n-ith the ‘“C-amino acid nlisture in the tnwcnce
of 0.01 JI -iTI-‘, 0.01 hI lIgCls, 0.01 hf GSII, and 0.1 &I l’ris-IICl
(pI1 7.6). The aminoacyl-tRiSA was then isolated according to
the method of Moldave (17). The final product had
:I
specific
act i\-it) . of 350,000 cpm lwr mg of RXA.
In&&ion-The incubation mixture for incorporation of free
amino witis into protein contained 50 mhl Tris-HCI buf fer ($I
7.6), 1 ml1 GTP, X0 rnhZ NHICl, 2 nor 12’1’1’, 5 nix AIg(‘l?, 4 rnl\ f
GSII, 0.5 FCi o f unil’ormlg labeled 14C-L-leurine, 500 pug of mixed
artivating and lransfcrring enzyme lnvtein, and 500 ,ug of l)oly-
som(x lxotein in 1 ml o f final volume.
‘I’hrst amount:: of enzymes
and 1)oly,wmw have bern shown to bc ol)timal for l)romotilg
incorporation of “X~l(~ucine into protein (13). Incubntioils were
carried out a t, 3i”
, and radioacti&g incorporated into 1)rotein
wxs measured wit,h ;I Nuclear-Chicago gas flo~v counter on the
rexiduc lef t 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 rnl f 1\2&12, 80 mAI XI-T,Cl, 100
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 in-
corporated into protein was measured as described above.
Hydrolysis of GTl’ by the protein-synthesizing system wts
determined by measuring radioact ive inorganic phosphorw re-
leased from (y-32P-GTl’ as described by
Cor~wny
and Lipmann
(I 8). The reaction mixture was similar to that used for 14C-
arninoacyl transfer to lxptides, except that, only ‘ZC-aminoacyl-
tRN=\ 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 silico-
tungstic acid in 0.02 N I-180, and 1 ml of 0.001 M pot,assium t)hos-
l,hatc (pH 6.8) as carrier. The extent of GTP hydro lysis 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
f low 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 buf fer (pH i.6), thcll layered over :I lilrear
gradient of 10 lo 40 7; sucrose in ‘I’KJI buf fer . 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. Radioactivi ty on thtx gradient
was nwasured on fractions of 12 droljs; the lxotein \va> I)rrcipi-
tated with carrier albumin and the hot t rirhloracetic avitl-illsol-
ublc lnwipitate was collected on
:I
Millil)orc filter for rolnrting
as drsrribed above.
k’stimation of Protein-The protein content of the c11zyllles,
pol,wornes, and gradient frac l ions was dctcwnincd by thv nwthod
of Lowry et al. (19) using bovine serum albunlin :lb the ,~t;~lrtl:wd.
IYJkct of Cycloheximide Concentration on .lkno 14&i J worpo-
ration-The action of cyclohesimide was ,\tuditJd in a cell-1’rw txo-
teia-synthesizing system consisting of liyc :r pal>-somes, act i\-:rting
and transferring enzymes together with cofdctors, alltl W-
leucine. The sys tem had been prepared depleted of frw anlino
acids (see under “Materials and Methods”) and wxs thus tlc~xwd-
cnt 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
the other set,, no amino arids wrrc addctl other than 14(‘~loucine.
Fig. ICCshows that, whwl amino acids w-cre present, inc~wasing
levels of r\-cloheximidc cawed l)xgrc,wire inhibition of ‘4C-
leucinc uptake into l)rotein. -1dditiolr of 0.1 pg per ml hat1 a
slight action on _4C-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 I’cnman (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, incorlwra-
tion of 14C-leucine tenses altogether but can be restarted by
a complete mixture of amino acids. Fig. lc shows the ef fect of
two levels of cycloherimide on this response to delayed amino
a t N A T I ON A L I N
S T I T
U T E
OF
S C I E N
C E E D
U C A T I ON
& R E
S E A R
C H
, onA
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c .
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a d e d f r om
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/
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4482 Cycloheximide Action on Protein Synthesis
Vol. 244, Wo. 16
Supplementation
fAA
MINUTES
FIG. 1. Effect of various concentrations of cycloheximide on
amino acid incorporation.
Liver polysomes were incubated with
‘4C-leucine in an amino acid-dependent protein-synthesizing
system for 40 min and incorporation into peptide was measured
in the presence of different concentrations of cycloheximide
_ .. . .. , .. .. ._ ._ ._ ._ .. 22 , nc c/ bo fj on -AA
20’ Jd
-AA
f2’ 11 fAA
+0.1+/000Jlg Cyd0.
6%
LINEAR SUCROSE GRADIENT
> 40%
FIG. 2. Inhibition of polysome resynthesis by cycloheximide
(Cycle.). Liver polysom es were incubated for 20 min in the amino
acid-dependent protein-synthesizing system containing 4 mM
GSH without added amino ac ids (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
was continued for another 2 min.
A control sample was incubated
for a similar total period of 22 min without added amino acids
(-----). All three samp les were separated simultaneou sly on
sucrose gradients and the polysome profiles were measured by
ultraviolet absorption. Thes e experiments were replicated four
times.
(Cycle.).
a, with all amino a cids (AA) present throughout incu-
bation;
b,
without addition of other amino a cids to the medium; c,
when the medium was supplemented with all amino acids and with
cycloheximide after 20-min incubation.
The data are the mean
results from two to four exp eriments.
acid suppleme ntation. 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, sen-
sit ivi ty 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).
Ej’ect 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 accu-
mulation of monosomes and other oligosomes.
We have previ-
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 incor-
poration 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
pg to 1 mg per ml (Fig. 2).
Thus, polysome regeneration and
the increased uptake of r4C-leucine in response to delayed amino
acid supplementation each show a similar degree of sensi tivi ty
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 incorpo-
ration 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 concentra-
tions of cycloheximide can stabilize polysomes in vitro (lo), we
examined the ef fect of various dose levels of cycloheximide on the
subsequent disaggregation of polysomes that had been reaggre-
gated by amino acid addition.
The polysome system was first
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
a t N A T I ON A L I N
S T I T
U T E
OF
S C I E N
C E E D
U C A T I ON
& R E
S E A R
C H
, onA
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c .
or g
D ownl o
a d e d f r om
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/
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Issue of August 25, 1969
B. X. Baliga, A. W. Prone&, and H. N. Munro
4483
B
TABLE
I
E$ect of preliminary incubation of transferases with suljhydryl
ZOO’lncvbation /-AA)
compounds
on inhibitory action
of cycloheximide
on protein
synthesis
in cell-free system
The incubation mixture for transfer of W-aminoacyl-tRNA to
-I
polysomes contained 50 mM Tris-HCI buf fer (pH 7.6)) 0.2 mM GTP,
5 mM MgC12,80
m M
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 sulf hydryl compounds
present in the incubation medium are as shown in the table.
In-
cubations were carried out at 37” for 35 min.
In most experi-
ments, as indicated, the transferases were incubated at 37” for 5
min in 0.5 ml of buff er before adding the polysomes, GTP, and
W-aminoacyl-tRNA. Glutathione,
dithiothreitol, mercapto-
ethanol, and cycloheximide (1 mg per tube) were added either dur-
ing preliminary incubation or during incubation, as indicated in
the table. The data shown are the average o f three experiments.
Tube
-
I
IG. 3. Liver polysomes were incubated in the amino acic -
dependent 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 poly-
some 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 poly-
some 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 af ter amino acid
supplementation. After 2 min of incubation as above in the
presence o f amino acids, cycloheximide in amounts of 1 pg (-----),
100 pg (- - -), or 1000 fig (-) was added to the incubation mix-
ture 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 o f
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 o f 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 poly-
some 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 simpli fy 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 eff icient ly into pro-
tein. Table I shows the mean results obtained from three such
studies with this system. Addition of large amounts of cyclo-
heximide 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 sen-
sit ivi ty is comparable to that found above for W-leucine incor-
poration in the presence of abundant free amino acids in the
system (Fig. la). The inhibitory action of the antibiotic could
1
Non
2 Non
3
+
4
+
5
+
6
+
7
+
8
+
9
+
10
+
11
+
12
+
13
+
Components previously
incubated
-
s
e
e
-SH
compounds
lmmles
None
None
-
-
-
4 GSH
20 GSH
4 GSH
20 GSH
4 GSH
20 GSH
10 DTT=
20 Mer-
capto-
ethanol
Non’
Non’
-
+
+
+
+
-
-
-
-
-
Additions for incubation
GTP,
POlY-
somes,
MC-
amino-
XYl-
tRNA
+
+
+
+
+
+
+
+
+
+
+
+
+
-SH
compounds
pmles
4 GSH
4 GSH
4 GSH
4 GSH
20 GSH
-
-
-
-
-
-
-
-
-
_
-
y’
mide
(1 m&T,
tube)
-
+
-
-
-
-
-
-
-
+
+
+
+
IllCOP
poration
cfim
5500
2600
5350
1100
2300
1900
2200
5570
5900
1800
3500
3800
4100
(1DTT, dithiothreitol.
be enhanced if the fraction containing the transferases was in-
cubated 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 sul fhyd ryl groups of this
enzyme and that high concentrations of sulfhydryl compounds
might compete effecti vel y 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 previ-
ously added cycloheximide (tube 4 versus 5). The action o f cy-
cloheximide was also diminished by adding 4 pmoles of GSH
during preliminary incubation (tube 4 versus 6), but no better
protective ef fec t 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 sulf hydryl compounds before
addition of cycloheximide (tubes 9 to 13). Increasing the glu-
a t N A T I ON A L I N
S T I T
U T E
OF
S C I E N
C E E D
U C A T I ON
& R E
S E A R
C H
, onA
p
r i l 9 ,2
0 1 2
www. j b
c .
or g
D ownl o
a d e d f r om
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/
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4454 Cycloheximide Action on Pwtein Synthesis
Vol. 244, Ko. 16
tathionc lcwl to 20 ~nwlcs did not affect incorl)oration by the
@cm in thr abwwe of the inhibitor (tube 8 VBTSU S 9), but prior
incubation \vith thi-: level of glutathionc greatly reduced the
inhibitory action of cyclohe simidc added subscqu entl?- (tube
IO oersus 11). Even more effective protection against cgclohrs-
imidc was obtained by adding d ithiothreitol or ~nerc:tptocthanol
to thr cnzynw during previous incubation (tubes 12 and 13).
The effwts 01’ p;climinary incubation of the transferase prepara-
tion with \-arious levels of GSH, dit,hiothrcitol, or rncrcapto-
ethanol arc shown in Fig. 4. Th is shows that transfer of 14C’-
amino acids in the absence of cyclohesimide was not signif icantly
influenced by different levels of the sulfhgdryl compou nds.
However, with increasing concentration of these sulfhydryl
agents the inhibitory action of cyclohesirnide diminishe d. 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 o f protection against inhibition by the
other two con~pound s required 20 Fmoles.
Since Suttcr and Moldavc (20) have shown that prior incu-
bation 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\-clohcxi-
rnitl(a. Fig. 6 shows that, in absen ce 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
lwesen c’e 01’ 4 pnloles of GSII. When cyclohe simide \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-
0
I I I I
1
5 IO 15 20
,umoles of -SH Co mpound
Fro. 4. The effect of cycloheximide on aminoacyl transferases
previously incltbated wit,h -SII compo unds. The effect of cyclo-
heximide on transfer of ‘“C-amino acids from aminoacy-tIlNA
to peptides was examined using a system cons isting of liver poly-
some s, lJC-aminoacyl-tlq GSH (O-0). The data are the
average of two kxperiments.
TABLE II
Effect of preliminary incuba lion of washed polysomes with s~tlfhhyclryl
compo luztls on inhibitory action of cycloheximide on protein
synthesis in cell-free system
The incubation mixture was similar to that described in Table I.
In most experiments, the polgsom es \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.
Components
previously incubated
I
Additions for
incubation
I
Poly-
Cyclo-
Gluta- heximide
somes thione
None
None
+
+
+
+
+
+
j .moles
None
None
-
-
-
20
20
G T P ,
q; -
amino-
Xy
tRNA:
trans-
feraw
Cluta-
thione
CYCb
Incorpo-
hexi-
ration
mide
cpm
- zioo
+
2400
- 5350
- 2000
- 5GOO
- 2350
- 5700
+ 2900
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Issue of August 25, 19G9 B. X. Baliga, A. W. P~mzcxulc, and H. N. Jlunro
4483
mide at all stages of the reaction.
This eliminates t,he possibil-
ity that, the inhibitory action of cycloheximide is directed only
against the initial rate of aminoacyl transfer, which is particu-
larly subject to GSII stimulation according to Hutter and Nol-
dave (20).
In addition, experiments were performed in which the poly-
Homes instead of the transferases were previously incubated with
cyclohesimidc or GSH. Table II shows that prior incubation of
the polysomes wit’h 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
ef fect 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 aminoacyl-
trwnsfera.sc II (21). From these various experiments, it can be
concluded that the maximal protective ef fect 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 ob-
taincd 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 fo r 10
min. Then the GSH concentration was lowered by dilution to
4 m&I before adding the rest of the system for transferring amino
acids from aminoacyl- tRiYA. Control samples
previously iiicu-
bated with 4 pmoles of GSH were diluted similarly, but enough
GSH w:ks then added to maintain the final concentration at 4
mhr. C’ycloheximide was added to
some of
the t’ubes at the be-
ginning of incubation. Fig. 6 shows
that enzyme previously in-
cubated with 20 pmo les of GSH was much less inhibited by
c\-clohesimide than was enz?-me previously incubated with 4
2 4 6 8 IO
MINUTES
FIG. 6. The effect of preliminary incubation of nminoacyltrans-
ferases lvith cycloheximide and glutathione on initial rate of
amino acid transfer.
The system used transferred ‘K-amino
acid s from aminoacyl-tRNA to peptides and cons isted of a mixed
transferase fraction, liver polysomes , GTP, Mgz+, ‘*C-aminoacyl-
tRNA, and bluffer. The transferase fraction was incubated at 37”
for 10 min w ith 4 or 20pm oles 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 al l samples. The other consti tuents of
the reaction \vere then added to give a final volume of 2 ml. Cy-
cloheximide (1 mg) was added at this point to some of the tubes.
Samp les were taken at different time intervals for assay of in-
corporation into peptidcs . The dat,a are the average of two ex-
periments.
pmoles. Thus, llrior treatment with a high level of GSII has a
protective action against the inhibitor.
EJ’ec t oj” Cycloheximide on Release oJ Peptides by Puromycin-
Puromycin 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 re-
leasing action of puromycin (II) .
hccordingly, the capacity
of GSH to reverse this effe ct of cycloheximide was evaluated.
Polysomes were incubated for 5 min with “C-sminoac~l-tRn’X,
GSH, GTP, and the transfcrase fract ion. The polysomes were
then harrcsted on a sucrose gradient, and the incorporation of
W act ivit r into peptides was examined at various
points on the
gradient (Fig. 7a). Most, of the incorporated radio:Lctivit,y was
/+tJ i : : : : : : i
”
’ :
,L
I 2 3 4 5 6 7 8 9 / 2 3 4 5 6 7 8 9 I 2’3’-d5’6’7’ 8’9
FRACTIONS FRACllONS
FRKTIONS
FIG. 7. Effect of cycloheximide and puromycin on distribution
of labeled polypep tide on polysome profile. The system used
transferred ~4C-aminoacyl-tlLNA to peptides and cons isted 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 trans-
ferase fraction in 0.5 ml of buffer wa s given preliminary incubation
for 5 min with the amolmt of GSH d escribed 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 samp les. The gradients shown
represent the following cond itions of incubation : a, incubation
for 5 min lvithollt inhibitors or preliminary incubation ; b, similar
to preceding cond itions 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 purom ycin; f, preliminary incu-
bation 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.
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4486 Cycloheximide Action on Protein Synthesis Vol. 244, No. 16
found on the polysome aggregates, with little release into the
supernatant fraction.
When puromycin was added during the
final 2 min of incubation, the act ivi ty in the polysomes was much
reduced and the supernat’ant fraction of the gradient contained
most of the radioactivity (Fig. 7b). When cycloheximide was
added 3 min before the puromycin, more act ,iv ity remained on
the polysorne aggregates and very little was released (Fig. 7~).
Disaggregation of polysomes to monosomes as a result of puro-
mycin action was also reduced by addition of cycloheximide
($ Fig. 7, b and c). To examine the ef fect of glutathione on
this action of cycloheximide, the transferase fraction was pre-
viously 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 in-
corporation 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 fir st
incubated with 20 pmoles of GSH (Fig. 7e), incorporation of the
Y-amino acids was very extensive in spite of the presence o f the
cyclohcximide, thus confirming protection against inhibition by
previous treatment of the transferase fraction with GSH. Fur-
thermore, the puromycin almost completely released the W-
peptides into the supernatant fraction. It will also be noted that
the polysome profi le under these conditions showed considerable
degradation, notably the presence of a large monosome peak.
These actions of puromycin were then similar to its eff ect s in the
absence of cycloheximide (Fig. 7f) .
It will be seen from compari-
son of Fig. 7 .f with Fig. 7b that the presence of high concentra-
tions of GSH actually potentiates the releasing action of puro-
mycin.
These experiments confirm that cycloheximide retards protein
synthes is without releasing the peptide chains (10). It also
‘,0°:
Minus AA’- tRNA
_ --------- -----_
Minus Ribosomes
Minutes
Fro. 8. Ribosome and aminoacyl-tRNA requirements for
GTPase action. Hydro lysis of GTP was examined in a cell-free
system n-hich transferred amino acids (AA) from aminoacyl-
tRNA to peptides.
It contained rat liver polysomes, the trans-
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.
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 puro-
mycin reaction by cycloheximide.
Egect of Cycloheximide and Other Inhibitors on GTP Hydro lysis
and Amino Acid Incorporation in Presence of DiJerenf Concen-
trations of GSH-From the preceding evidence, the inhibitory
action of cycloheximide on chain elongation appears to be di-
rected against transferase II, which is the only sulfhydryl-de-
pendent 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 en-
zyme (translocase) may be identical with transferase II (25).
We (14) have recently shown that cycloheximide inhibits GTP
hydro lysis in our mammalian system. The eff ect ive concentra-
tions 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 gluta-
thione (14). This confirms the interrelationship between cy-
cloheximide 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 synthes is 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 hydro lysis
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, hydro lysis of GTP was
used to study the action of glutathione on inhibition of protein
synthesis by various compounds.
Samples were also incubated
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 trails-
fer 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 hy-
drosylated derivative of cyclohesimide, Teas also added at a
concentration of 1 mg per ml, since this is known to inhibit l)ro-
tein synthesis effecti vely 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
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Issue of August 25, 1969
B. 8. Baliga, A. W. Proncxuk, and H. N. Munro
4487
cell-free protein-synthesizing systems, and suggests that this
may occur because of its structural similar ity to cycloheximide.
We therefore added 100 pg of emetine per ml to our transfer sys -
tem, a concentration shown by Grollman to be within the eff ec-
tive range, and confirmed that both amino acid incorporation
and a2P release were extens ively inhibited. However, unlike
cycloheximide, neither reaction could be protected against in-
hibition when the glutathione concentration was raised.
Groll-
man (28) has recently found that emetine dif fers from cyclo-
heximide in its action on HeLa cells. On suspending the cells
in fresh medium, the inhibitory action of cycloheximide could be
reversed, whereas that o f emetine could not; however, in Groll-
man’s sys tem 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 eff ect . However,
inhibition of 32P release by sparsomycin was slight. This result,
which implies an unexpected dissociation between the two re-
quirements 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$ect of Cycloheximide on Polysome Reaggregation in Presence
of Extra GSH--It was demonstrated above that polysome re-
TABLE III
Efect of preliminary incubation of transferases
with
glutathione on
inhibitory action of various antibiotics on amino acid incor-
poration and on GTP hydrolysis in cell-free protein-
synthesizing system
The incubation mixture was the same as in Table I, except that
in some experiments (Y-~~P)-GTP and Wkminoacyl-tRNA were
used in order to study GTP hydrolysis. The crude transferase
preparation was previously incubated with glutathione for 5 min
at 37”.
The remaining constituen t,s were then added and incuba-
tion was continued for 30 min.
aggregation
caused by delayed addition of amino acids to a me-
dium lacking free amino acids could be inhibited by much lower
concentrations of cycloheximide than those necessary to achieve
inhibition of chain elongation.
Since this suggests a separate
action of the inhibitor, we tested the capacity of sulfhyd ryl
compounds to protect against th is ef fect of cycloheximide.
Fig.
9 shows polysome reaggregation in response to addition of amino
acids when the GSH concentration in the medium was raised
from 4
mM
to 20 mM.
The usual reduction in monosomes and
accumulation of polysomes was obtained, and the presence of
0.1 pg of cycloheximide was again suf ficient to prevent reaggre-
gation (compare Fig. 2). Thus, GSH does not influence this
action
of cycloheximide.
In the same experiments,
YXeucine was present in the me-
dium, and incorporation of radioactivity into protein was ex-
amined after 40 min of incubation. When no other amino acids
----_-. 22’ incubation - AA
- { 2$ incybation 4:
20’ incubation - AA
+2’ incubation +AA and
+O.Ipg cycloheximide
-
I
-
I
rior
incubation
Incubation
STP,
PolY-
omes,
mino-
“R’i
+
+
+
+
+
+
+
+
+
+
MC-
Amino-
acyl-
tRNA
hzyme
_-
,
I
s
a
t
.-
-
Tube
;hlta-
thione
incor-
porated’l
(
: 1
I
-
Antibiotic
-
-
Cycloheximide
Cycloheximide
Streptovitacin
Streptovitadin
Emetine
Emetine
Sparsomycin
Sparsomycin
moles
4
20
4
20
4
20
4
20
4
20
@m/lube
1
2
3
4
5
6
7
8
9
10
1000 4170
1200 4350
200 1140
800 3900
210 830
790 4000
530 560
530 580
830 660
980 700
,
J
16%
LINEAR SUCROSE GRADIENT
= 40%
FIG. 9. Prevention by cycloheximide of polysome reaggregation
on addition of amino ac ids (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 contain-
ing 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 samp le w as incu-
bated for a similar total p eriod of 22 min without added amino
acids (-----). All three samp les were separated simultaneou sly
on sucrose gradients and the profiles were measured by ultraviolet
absorption. The experiment was replicated twice.
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 considera ble discrepancy
between G TP hydrolysis and aminoacyl incorporation.
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44% Cyclohc:cimide Action on Protein S7y~lhesis
Vol. 244, No. 16
mw :~tltlc tl to the tirc~tlium, 22’70 rpm lwr tr11Jc~ ere it tcorIJoratct1.
\\?lcll the: roml)lctc atllillo acitl ttlisturt evils atldctl al’tcr 20 nlin
of ittcttbatiott, ittc~orIJot~:ttiott row to 6250 cljtii 1Jer tube. If ,
ho\x-cvc~i~, .1 piz; of c~c:lohc~sitttitIc \~a’: tttl tlct l 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).
‘I’hcsc studies Iwovitle furthw c\-itlcttcc that the actioti of cy-
clohcsimitlc on rc:rggwg:ttiott :iiid on 1wlJtitl~ t~lotlgxtiot i is tlue to
diffrrcttt trtcch:tttisttts.
chain ittitiation atid oti (*lt :tit t c~loirg:tlioti. \ilieti the\- ittcrtbated
intact rcticulocytcs with S:iF xnd cyclolwsintitlc, both con-
pomtds itthibitcd ch:ti tt initiation, but c~~c~1oIic~sittiitlc ilso aft fcc ted
elong:~tiori of nt~swtt1 Iwl)titIc~ c~haitts;. Orir wsults 1)rovidc di-
rect cvidcrtce for such :I
dUiLl
wliotr lltttlw c*otttl itioits 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 1JrIJti tlc chaitis ott the ribo-
comes. 011 the other hand, 1Jrclitnittar~- ittcubation of the system
n-ithout tttt titio :tci tls wsrtlts itt tlixiggwgxtion of the IJol~sotnes
awl, \~lion:ttrtiiio:icitls arc sulJsecIuctrtly :ttltlt tl, the 1tolyso11ics re-
aggrcgatc~ 1)~ a 1Jrowss that ~Jrwttttlabl\- xquircs ittitiation of
pq)tide chains. ‘I’hcb c*ottc*c~ti li~:ttioti of c:\-~lollc~sittlitlt iteedcd
for eff cct ivc itihilJitiotr of chairi c~lottg:ilioii iii this syslcni is 10”
times greater Ihan 1h:t t tic&d lo
~wv c~it r0tnplct.r: 1
Jolysome
reconstitution on adding amino acidh. ‘l’his is the rcvwe of Ihe
finding of’ Stanners (30), w110 estrmittcd t,h t: ac*tiott of cyclohcsi-
mide 011 hamster ctnbryonic cells iti t ssuc: c~ultrrix~ and found
that low doses inhibited movcmcttt of ribosontw along the mes-
senger, 1Jut had Icss xtion 011 rc;t tt;t~httlc~ttt . 01’ free ribosonlcs to
the mcssettger. Our cell-flee system appwrs to have :I limited
cnIwci l,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~iotlf its actioll
by sul fhydryl COII~IJO~II~S. ‘l’hcse cor~l~Jou~~tls c:m estcttsivcly
reduw the inhiljitory actiotl of c~yclohesitltide on t:h:tin clo~tgltion
but 1101. n 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 rcag-
gfrgatiou.
Olw silo of :&on 01’ c~~lolt~siti~itlc oti IJrotcitt synthesis has
been Itrovisiottall, v itlwttificd with tr;~nsl’e~xsc 11. Itrvolvcment
of tlatt5~cr:rscs \ v:t s origitially srtggwtcttl 1~)~Sicgcl :ititl Sislcr (8)
on the groutttls that ~yclohcsinii tlc: (lit1 t101 :r~J~Jcw to ha\-c an
eff ect oti amino :icitl~:rctiT.;ltiti~ c~~zyniw. Prlicetti, (‘olott~bo,
and ILgliotii (11) .sho\vct l that c?clollcsi fltidc 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~ iihibitot~. 011 lhc other
hand, l \vo other grouljs o f ill\-cstigx tors (23, 31) I’ailcd to obtain
an inhil)itory c~f fcc~ l. f cyc~lohcsitt titlc on I~urottt~~in-tlc~~)(~tt(Ic~~t
rcleasc ol 1x1)1 tlw I’i~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~otr tyc~itt wllcn Ihc GSTT cottwtttr: tliou of the me-
dium is 4 11111. Oltr tl:11a I’rtrtllrr tirnlotls;lr: lte lhat Ihis :tt*1ion of
cyclohcsimitlc cxn IJc 1)wvctttctl IJ~ IJrior ittc~rtljttt iott of tlrc c*rutlc
tra.nsferase I’ra~t,iotl with higher lcvcls ol’ (:Sll (III) to 20 ~tnolcs).
These ol~scrvatiotts ctitl&sizc the int1Jortattc~c~ ol’ MI1 wtwcti-
tration in the tnctlium whet1 stut lyit tg 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 :tt tti tto :tcyl site ott the riljosorttw (25). Sittce
purom~-citt rnlist bitid to this ri1Josottic site: Iwl’oi~~ I’otxti tig the
pcptitle bottd, the 1JqJtidylLt1iS. i has first to IJc ~txttslowtcd to
the IJqJtitlyl site. ‘l‘his :twJunts for 1hc ittltiljitor~~ actiott of
cyc~loliesitnitle oti I)urotitycin rclwsc a11t1 for Itic, 51 nlttl:iitl c,f fcc t,
of GSH.
Our es1wittwitts tlii~on. sonic light on tlicx ttaturc~ of the ;iction
of c~ -clohcsitltitlt~ ou 1ra1tsl’crnsc 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 \vhct t 1)wviousl~
itrcubatctl with \at ious sulf’I~ytli~~-1 cotri~~outttls , gwatwt :ict ivit
being :rchic5wI 1J\- I)tior iiicu~J:itioii I\-ilh ~ot rccttt t,:tt ,iotis of GKI-I
of 20 nt~ or higlw. It, :~IJ~jc:n‘s that, I)urifi ic~tl ~1x11sCw:tst~ I is
readily itt:tc :t wt.ed Wough its suII’hytI ry1 g~~rt~w :rtrtl that it twt
be regctiwttcd 1Jy sulfhydi~y l tlonot~s. Ill th? (‘:I’( ol’ Ihc t:1wde
trnnsferasc IJrc:I ):lt’a t,iotis usctl I)y us, whi& iitt~ludc l)olh cti-
zymcs I and II, it is ~~robablc that transfc txw 1 is less uitst:ible
(20). ‘I’his is srtIJIwt?,c~tl by the tlata itt E’ig. 4 \vli ic.h show that,
varying the levels ol (%I1 ant1 other suIfliyt l~~~l t~~n~I~otutd~ dtw
ing prclimitiar)- ittculJ:ttion did Itot :t1J1JiwialJl~- xf fcct ltxiisfcrusc
nct ivi t,y tlrtt~itig sul~stquent iticulJ:i tioti. Kcwrthelcss, although
in our unI~ut~ifictl systc>ni the ctizyt iic she\\-s t10 great itictwsc in
:tctiritJ- :ts ;t wsult 0E Ijtwious ittculJatioti \vith liigh Icvc+ o f
GSH, thcsc high c:ottcet ttt,a tions ga\~: adtlcd 1xo1 cction xgtinst
subsequc~nt. atldition of c)-clohesimidc (Fig, 4). ‘I’his is IIOI due
to the high GSII concctttr;~tion during suljsequct~t ittculJ:rtion,~
since the 1trotedivc c,f fcc :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).
‘I’his agrws with the fitt tling ol Sut tcr ;~td RIol tl; tvc (20) that IJrior
trctttmwt of purified tr:insfelasc I I \vith high l~~vc~lsof (iSI iii-
crettsrs subsc~~ucnt :rmit io:icyI ttxtrsl’cr :tt :L lonc~ GSIJ t~onccn-
Iration. Our qJwitncttt ~II n-hich the (XII ~ottc,cntt,: ttion W:IS
lowcretl bcl’orc adtlittg c~?-c~lohc,sitnitlc (Fig. ti) tlt~ntottst txlcs that.
nlercaIJ1: ttt :iiitl tht, itihilJitot~ (10 ttot ui~tlcrgo tliiw~1 t~lit~tiiiwl
rcnction.
l’rotectiori by WIT against, itthiljitiott of tr:ttts(‘etxsc I I :tllows
one to lost, othc~ :u~~il~ioiics in ortlcr to tlrterttt ittc wht~thw thq
act in the sanic way :LS t:~clo~lc~r;inlitlc.
‘I’hc :rc:liott or high (X313
concctitr :rtiotis ott th(t itthibitioii 01’ ~Jrotcitt sgill,lic~sis l)y slixljto-
vilacin .I is cxtct ly similar to th:lt of c:\-cloltc,sittritlc, to \vhich it
is structurally rc:latc~l (‘l’ablc I I I). I {Ctttctit tc \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:ttiot t in the cell-l’rw systctn. (~rollm:~~~ (28)
found that Ijrotcitt syttthcsis iti 1-1~1~ ~11s \V:IS itthilJilc~1 IJy c:y~lo-
hesinlitlc, stt,cpto~it:tc :itt , tint1 emclit tc, l~tit wliw Ihc alla wrrc
stis1tc’tid~~l in I’twli tit(~tl iuttt 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 I’IQI~I lllaf , of
strcptorit:ic*in lJy its ittscitsitivil y to GSII. ‘I’ltis tlow itot, of
couwe, c~litnitta tc t txttsl’twse 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 I’OIVII:L~on
rem:tins rtttcc~r1:iiti (32). .\tt ittt*rc: isc iii GSl I c~otrc~c~ti1t~:t1iot~tlso
a t N A T I ON A L I N
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OF
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http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/
8/19/2019 Mechanism of Cycloheximide
10/10
Issue of August 25, 1969 B. S. Baliga, A. W. Proncmlc, and H. N. Muwo
4489
failed to prevent the inhibition caused bg this antibiotic. An
interesting feature o f these inhibitors was that cgclohcsimide
and streptovitacin inhibited GTI’ hydro lysis to the same extent
as amino acid transfer from aminoacyl-tRNA. Emetine also
inhibited GTP hydro lysis fai rly strongly, but sparsomynin was
almost without ef fect on this reaction while causing extensive
inhibition of amino acid incorporation. This demonstrates a
considerable uncoupling of G’l’l’ hydrolysis from peptide bond
formation when this antibiotic is lxesent.
REFERENCES
1. KERIUDQE, I>., J. Gen. Microbial., 19, 497 (1958).
2.
YOUNG, C. W., ROBINSON, I’. F., AND S:UXOR, B., Biochenr.
Phar&col., 12, 855 (1963).
3. COLOMIIO. B.. FEI~CE TTI. I,.. .\NU BAGLIONI. C.. Uiochem.
Biophys. Res. Comm un.,‘ll, 989 (1965).
’ ’
4. COLOMBO, B., FELICETTI, L., AND B.\GLIONI, C., Riochim.
Biophys. Acta, 119, 109 (1966).
5. TRIKATELLIS, A. C., MONTJ,~R, M., AND AXELROD, A. E.,
Biochemistry, 4, 2065 (1965).
6. KORNER, A., Biochem . J., 101,627 (1966).
7. GODCH.~UX. W.. ADAMSON, S. II., IIND HERBERT, E., J. Mol.
Biol., 27; 57 11967).
8. SIEGEL, M. It., AND SISI~E R, H. l)., Nature, 200, 675 (IOG3);
Biochim . Biophys. Acta, 87, 83 (1964).
9. ENKIS, H. L., AND LUBIN, M., Fed. Pro c., 23, 269 (1964).
10. WETTSTEIN, F. O., NOLL, H., )IND PENMAN, S., Biochim.
Biophys. Acta, 87, 525 (1964).
11. FELICETTI. L., COLOM~O, B., ,\NI) B.IGLIONI, C., Xochim.
Biophys: A&, 119, 120 (196G).
12.
LIN. S.
Y..
MOSL’ELLER.
R. D..
AXI) I~.II., Proc. A-at. Acad. Sci. U. S. A., 61, 585 (1964).
SKOGERSON, I,., AND MOLDAVE, K., Arch. Biochem . Biophys.,
126, 497 (1968).
NISHIZUIU, Y., .\NI) LIPMANN,
F., Arch. Bioc hem. Biophys.,
116, 344 (1966).
GROLLMIN, A. P., Proc. Nat. Acad. Sci. U. S. A., 66, 1867
(1966).
GROLLMAN, A. P., J. Biol. Chem., 243,4089 (1968).
TRAKATELLIS, A. C., Proc. Nat. Acad. Sci. U. S. A., 69, 854
(1968).
STANNEHS, C. P., Biochem . Biophys. IL’es. Commun., 24, 758
(1966).
CLARIS, J. M., JR., AND CH.~XG, A. Y., J. Biol. Chem., 240,
4734 (1965).
JAYAR~IMAN, J., .\ND GOLDBERG, I. H., Biochemistry, 7, 418
(1968).
a t N A T I ON A L I N
S T I T
U T E
OF
S C I E N
C E E D
U C A T I ON
& R E
S E A R
C H
, onA
p
r i l 9 ,2
0 1 2
www. j b
c .
or g
D ownl o
a d e d f r om
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/