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 repeatedly confirmed in mammalian cells (2-F). Using cell-free

Sites of cycloheximide action on protein synthesis were s;!-stems, several authors (%ll) have obtained data suggesting
examined using a cell-free system prepared from rat liver. that the charging of transfer RNA with amino acids is not influ-
If all amino acids or aminoacyl transfer RNA were present enced by the inhibitor, which thus must act at some subsequent
at the start of incubation, the system appeared to incor- stage in protein synthesis. Recent studies suggest that cyclo-
porate W-leucine mainly by elongation of peptide chains. hcsimitlc may affect protein synthesis at more than one l)oint.
Under these conditions, high dose levels of cycloheximide Lin, Mosteller, and Hardesty (12) obtained evidence from es-
were necessary in order to inhibit incorporation extensively. perimellts on reticulocytes that the antibiotic inhibits the in-
The inhibition could be prevented by raising the glutathione tiation of new peptide chains aud the elongation of nascent pep-
content of the reaction mixture, and particularly by prelimi- tides on ribosomes by different mechanisms.
nary incubation of a mixture of transferase I and II with In the studies reported below, we have attempted to localize
high concentrations of glutathione before adding these 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
enzymes to the system. Other sulfhydryl compounds were
also effective in protecting against cycloheximide. It has consisted of liver polyaomes, activating and transferring enz~mcs,
been concluded that the inhibitory action of cycloheximide and cofactors. Since the enzyme fractions were treated to rc-
move frcxe a11d tRK.&bound alnino acids, the system is conse-
on peptide chain elongation involves inactivation of trans-
quentl>. dependent on an exogwous supply of amino acids. If
ferase II, an enzyme known to have a sulfhydryl requirement.
incubated n-ith all 20 amino acids, it will incorporate mainly 1,~
High concentrations of glutathione were also found to pre-
chain elongation. If, however, it is incubated without amino
vent the inhibition of cell-free protein synthesis caused by
acids, the polysomes disaggregate and amino acid incorporation
streptovitacin A, a derivative of cycloheximide, but not
is mininlal; if at this point amino acids arc added to the incubn-
inhibition caused by emetine or sparsomycin.
tion medium, the polysomes can be reaggrcgatcd and labeled
If the protein-synthesizing system was first incubated
amino acids are once more incorporated into l)eptide chain<.
without amino acids or aminoacyl-tRNA, polysomes present
The nature of this action of delayed amino a&d supl)lementation
at the start of incubation underwent disaggregation. On
is not fully understood, but may be dependent on chain initiation.
addition of amino acids at this point, the polysomes became
This system has been used by us to study the actions of cycle-
reaggregated and incorporation of 14C-leucine was stimu-
hesimidc and some other antibiotics on elongation of peptitle
lated, probably by a process involving chain initiation. The
chains and on the phenomenon of polysome aggregation in re-
response of polysome aggregation and coincident 14C-leucine
sponse to delayed amino acid supplementation. With this SJ-S-
uptake could be inhibited by low doses of cycloheximide.
tern, KC have now shown that chain elongation and pol\,some
Furthermore, this inhibitory action of cycloheximide could
aggregation are inhibited by different concentrations of cycle-
not be prevented by raising the glutathione content of the
hesimidc. The action of the antibiotic on chain elongation
medium. This suggests that the action of cycloheximide on
aplxws to be directed against aminoacyltransferase II and can
polysome aggregation differs from its effect on peptide chain
be prewnted b,v high concentrations of sulfh\-dry1 compounda,
elongation.
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. *\ pre-
liminary report of the action of t,his antibiotic 011 chain elonpa-
tion has :~p~~~rd (14).
(‘~~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
Rengejzts and I/e&n-The amino ackk uwtl n-crc obt;tincd
* This investigation \~as srqlported 1)~ Public Ilralth Service from Sutritional l<iochemicals. Othrr chemicals \VCW rwgcllt
Gratlt (A-08893-03. grade. Rat lircr tRS.1 stril)lw(\ of amino acsid< n-:ts ~~~ucl~awtl

4480

This is an Open Access article under the CC BY license.

Issue of August 25, 1969 B. X. Baliga, A. W. Prommk, and H. N. Munro 4431

from General Biochemicals. Cyclohesimide was provided by p*g of :Irniiio:LcSltranxfcrnsc protein, 500 g of polysomc twotcin,
ZUdrirh, and puromycin dihydrochloride was obtained from and 20,000 cpm of 14C-aminoacyl-tRN-1 in a final volurrrc~ of 1
Nutritional Biochemicals. Streptovitacin A was a ljroduct of ml. Inrubations were carried out at 37”, and radioactivity in-
Ultjohn, and cmctinc was provided by the S. B. Penick Company, corporated into protein was measured as described above.
Scn- York, Sew York. Sparsomycin was kindly supplied as Hydrolysis of GTl’ by the protein-synthesizing system wts
a gift by Dr. I. H. Goldberg of the Harvard Medical School. The determined by measuring radioactive inorganic phosphorw re-
uniformly labeled 14C-L-leucine (specific act’ivity, 250 mCi per leased from (y-32P-GTl’ as described by Cor~wny and Lipmann
mmole) and a mixture of %-amino acids (1 &i per mg of uni- (I 8). The reaction mixture was similar to that used for 14C-
formly lab&xl amino acid mixture) were purchased from sew arninoacyl transfer to lxptides, except that, only ‘ZC-aminoacyl-
England Suclear. International Chemical and n’uclcar Corpora- tRN=\ was present and the GTP used WIS labeled with 821’ in the
tioll, Irvine, California, provided (+*P-GTP (specific radioac- terminal phosphate (11,000 cpm per ml of rrartion rnixtuw).
tivity, 22 mCi per pmole), which n-as further purified by chroma- The reaction mixture was incubated at 3T” for 30 mill a11tl the
tography on DEhE-cellulose. Most of the media used were reaction was terminated by adding equal amounts of 0.2 RI silico-
matlc ul, in TK(1\1 buffer (0.05 M Tris-HCl, pH 7.6, 0.025 M K(‘1, tungstic acid in 0.02 N I-180, and 1 ml of 0.001 M pot,assium t)hos-
and 0.005 nr ;\IgCIZ) as described by Wcttstein, Staehclin, and l,hatc (pH 6.8) as carrier. The extent of GTP hydrolysis was
so11 (15). measured by extraction of the released inorganic l)hosl)h:rte as
Preparation 0jPolysomes and Cell Sap ~nzyl,ies-Pol~aomes (C- the phosphomolybdate complex into isobutyl alcohol; the extract
ribosomes) were obtained from the livers of fasting 150-g rats by was counted for radioactivity using t,hc Suclcar-Chic,:rgo gas
the procedure described by Baliga, l’ronczuk, and Munro (13). flow COUllk~.
To obtain activating and transferring cnzymcs, cell sap was pre- Polysome Prqliles-IIlcubatioll mist,urcs for redimcntation
l):nwl :IJKI denuded, first, of free amino acids by dialysi.s, and analysis of polysome profiles were first diluted with 0.7 \.olume
second, of tRr\‘-1 by the lxotamiire sulfate treatme as described of 0.01 11 Tris-HCl buffer (pH i.6), thcll layered over :I lilrear
1,. IMiga et nl. (13). l’rotein n-as precipitated from this frac- gradient of 10 lo 40 7; sucrose in ‘I’KJI buffer. The gradient xas
tioll b&n-een 30 a11d iO7; saturation with (SH,)$O, to yield a crntrifugcd at 38,000 rl)rn in the Sly-50 rotor of the Spinco model
mixture of activating and transferring enzymes with Illinirnal L2 ultracentrifuge for $0 min. The absorl)tion 1)rofilr at 260 rnp
tontent of free or tRNX-attached amino acids; t,he l~w~~:tration was recorded automatically \\-ith a flow cell device in :I Gilford
was uwd for the system incorporating free amino acids illto 1x1,- nlodcl 2000 spectrol)hotollleter. Radioactivity on thtx gradient
tides. For incorl)oration of amino acids from ami~wacyl-tRr\‘A was nwasured on fractions of 12 droljs; the lxotein \va> I)rrcipi-
int 0 pal>-.qome?, lillft~ac~tioiiated alllii,o:ic3-ltlaIlsfer:l.~e~ free of tated with carrier albumin and the hot t rirhloracetic avitl-illsol-
activating enzymes n-we prepared I’rolrr the cell salI :rftcxr lrent- ublc lnwipitate was collected on :I Millil)orc filter for rolnrting
nwnt with Sephatlcs G-25. The nrtivating rnzymw \vcrc re- as drsrribed above.
moved by the traditional nlrthod of lwc*il)itation at 1111 5, :IJ~ a. k’stimation of Protein-The protein content of the c11zyllles,
mixture of transfcrascs I and II was ~~relwwl from the su~)cr~~a- pol,wornes, and gradient fracl ions was dctcwnincd by thv nwthod
tarot fraction as dcscribed bJ- Gasior :uld Molda\-r (I 6). U.ith of Lowry et al. (19) using bovine serum albunlin :lb the ,~t;~lrtl:wd.
cwvh batch of reaction misturc, the optimal amount< of trans-
fernsc fraction alld (when required) of trnllsfcrase and arti\-sting
enzyrue fxaction wcrc established. IYJkct of Cycloheximide Concentration on .lkno 14&i J worpo-
I+-eparation oj “C-arrlinoacyl~tR~~~ I -For the prelwation of ration-The action of cyclohesimide was ,\tuditJd in a cell-1’rw txo-
radionctire aiilino:rc~l~tRr\TX, the lxocedure dcwribed by teia-synthesizing system consisting of liyc:r pal>-somes, act i\-:rting
l\Ioltl:lre (I T) \r:w wvtl. Rat, liver cell sap WM ndjwtcd to l,II and transferring enzymes together with cofdctors, alltl W-
5 to bring dowi :I precipitate containing tRX.1 ai~d amino arid- leucine. The system had been prepared depleted of frw anlino
acti\-sting ellzymw. This precipitate ~1s thrn ret&sol\-rd and acids (see under “Materials and Methods”) and wxs thus tlc~xwd-
allon-cd to rract n-ith the ‘“C-amino acid nlisture in the tnwcnce cnt on csogenous amino arids. To onr set of tubes, a caonll)lete
of 0.01 JI -iTI-‘, 0.01 hI lIgCls, 0.01 hf GSII, and 0.1 &I l’ris-IICl mixture of amino acids was added at thr st,art of incubation; to
(pI1 7.6). The aminoacyl-tRiSA was then isolated according to the other set,, no amino arids wrrc addctl other than 14(‘~loucine.
the method of Moldave (17). The final product had :I specific Fig. ICCshows that, whwl amino acids w-cre present, inc~wasing
act i\-it). of 350,000 cpm lwr mg of RXA. levels of r\-cloheximidc cawed l)xgrc,wire inhibition of ‘4C-
In&&ion-The incubation mixture for incorporation of free leucinc uptake into l)rotein. -1dditiolr of 0.1 pg per ml hat1 a
amino witis into protein contained 50 mhl Tris-HCI buffer ($I slight action on 14C-lcurinc
_ incorporation; in order to :irliiere
7.6), 1 ml1 GTP, X0 rnhZ NHICl, 2 nor 12’1’1’, 5 nix AIg(‘l?, 4 rnl\f 75%) inhibition, it was necessary to add 1 mg of inhibitor tw ml
GSII, 0.5 FCi of unil’ormlg labeled 14C-L-leurine, 500 pug of mixed of incubation medium, a level coml~:w:rble to t,hnt u-cltl by
artivating and lransfcrring enzyme lnvtein, and 500 ,ug of l)oly- Rcttstein, Noll, and I’cnman (10) in their cell-frw sy-tcnl to
som(x lxotein in 1 ml of final volume. ‘I’hrst amount:: of enzymes retard ribosome movement along the mcsseager stra11cl. The
and 1)oly,wmw have bern shown to bc ol)timal for l)romotilg much smaller residual incorporation of ‘Vleucine obtained in
incorporation of “X~l(~ucine into protein (13). Incubntioils were the absence of added amino acids (Fig. 16) xas little affwt cd by
carried out at, 3i” , and radioacti&g incorporated into 1)rotein cycloheximide at any concentration.
wxs measured wit,h ;I Nuclear-Chicago gas flo~v counter on the It has previously been shown by us (13) with such a -\-stem
rexiduc left after treatment with hot trichloracetic acid (13). that, after 20 min of incubation wilhout amino acids, incorlwra-
The incubation mixture for transfer of 14C-nminoar~l-tR~~~ tion of 14C-leucine tenses altogether but can be restarted by
to l~olyaon~~s contained 50 m&I Tris-HCI buffer (1~1~ 7.6), either a complete mixture of amino acids. Fig. lc shows the effect of
4 or 20 111~ GSH, 0.2 mnf GTP, 5 rnlf 1\2&12, 80 mAI XI-T,Cl, 100 two levels of cycloherimide on this response to delayed amino
 4482 Cycloheximide Action on Protein Synthesis Vol. 244, Wo. 16

Supplementation

fAA

MINUTES
FIG. 1. Effect of various concentrations of cycloheximide on (Cycle.). a, with all amino acids (AA) present throughout incu-
amino acid incorporation. Liver polysomes were incubated with bation; b, without addition of other amino acids to the medium; c,
‘4C-leucine in an amino acid-dependent protein-synthesizing when the medium was supplemented with all amino acids and with
system for 40 min and incorporation into peptide was measured cycloheximide after 20-min incubation. The data are the mean
in the presence of different concentrations of cycloheximide results from two to four experiments.

acid 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, sen-
sitivity to cycloheximide is much greater when the amino acids
are added 20 min after the start of incubation than when the
_..... ,....._._._._.. 22’ ,ncc/bofjon -AA
amino acids are present throughout incubation (Fig. la).
20’ Jd -AA Ej’ect of Cycloheximide on Polysome Aggregation-When the
f2’ 11 fAA 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-
+0.1+/000Jlg Cyd0.
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 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 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
6% LINEAR SUCROSE GRADIENT
> 40% picture for 14C-leucine uptake after amino acid addition coincides
closely with the aggregation and then disaggregation of the
FIG. 2. Inhibition of polysome resynthesis by cycloheximide
(Cycle.). Liver polysomes were incubated for 20 min in the amino
polysomes. Since it has been established that high concentra-
acid-dependent protein-synthesizing system containing 4 mM tions of cycloheximide can stabilize polysomes in vitro (lo), we
GSH without added amino acids (AA), and then the complete examined the effect of various dose levels of cycloheximide on the
mixture of 20 amino acids was added either alone (-) or in the
presence of 0.1 to 1000 rg of cycloheximide (---). Incubation
subsequent disaggregation of polysomes that had been reaggre-
was continued for another 2 min. A control sample was incubated gated by amino acid addition. The polysome system was first
for a similar total period of 22 min without added amino acids incubated for 20 min without exogenous amino acids. Amino
(-----). All three samples were separated simultaneously on acids were then added, followed 2 min later by various levels of
sucrose gradients and the polysome profiles were measured by
ultraviolet absorption. These experiments were replicated four
cycloheximide, and incubation was terminated at 3 min after
times. addition of the inhibitor. Fig. 3b shows that the polysome
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
compounds on inhibitory action of cycloheximide on protein
ZOO’lncvbation /-AA) 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. 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 buffer 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
-I the table. The data shown are the average of three experiments.

FIG. 3. Liver polysomes were incubated in the amino acic l-

dependent protein-synthesizing system for 20 min without amino
acids (AA) and then a mixture of all amino acids was added and
Components

-
incubated
previously
I Additions for incubation
-
GTP,
incubation was continued. a, control samples (changes in poly- Tube POlY- y’
IllCOP
poration
some profile after amino acid supplementation). After 2 min -SH somes,
MC- -SH
s mide
(-) and 5 min (- - -) following amino acid addition, a portion compounds amino- compounds
(1 m&T,
of the mixture was layered onto a sucrose gradient and the poly- XYl- tube)
tRNA
some profiles were obtained. A sample incubated for a total _
period of 22 min without amino acids (-----) was also run. b, lmmles pmles cfim
effect of cycloheximide on polysome profiles after amino acid 1 Non e None Non’ + 4 GSH - 5500
supplementation. After 2 min of incubation as above in the
presence of amino acids, cycloheximide in amounts of 1 pg (-----), 2 Non e None Non’ + 4 GSH + 2600
3 + - - 4 GSH - 5350
100 pg (- - -), or 1000 fig (-) was added to the incubation mix- +
ture and after an additional 3 min of incubation the polysome 4 + - + + 4 GSH - 1100
profiles were examined. 5 + - + + 20 GSH - 2300
6 + 4 GSH + + - - 1900
7 + 20 GSH + + - - 2200
aggregate was well preserved in the presence of 1 mg per ml of - -
8 + 4 GSH + - 5570
cycloheximide, but at lower concentrations breakdown was 9 20 GSH - - -
+ + 5900
considerable. In the samples incubated without addition of 10 + 4 GSH - 1800
+ +
cycloheximide (Fig. 3a), the ratio of polysomes to monosomes 11 + 20 GSH - + - + 3500
was 6.7 at 2 min after addition of amino acids, and 3.4 at 5 min 12 + 10 DTT= - + - + 3800
after addition, which is compatible with disaggregation seen in 13 + 20 Mer- - + - + 4100
the figure. For samples receiving the inhibitor 2 min after the capto-
amino acids (Fig. 3b), the ratio of polysomes to monosomes at ethanol
5 min of incubation is 4.1 for 1 pg of cycloheximide, 5.0 for 100 -
(1DTT, dithiothreitol.
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 be enhanced if the fraction containing the transferases was in-
ml) is much lower than the level necessary to stabilize the poly- cubated beforehand with cycloheximide (tube 3 versus 4). It
some aggregates so formed (1 mg per ml). has been suggested that cycloheximide inhibits transferase II
E$ect of Cycloheximide on Transfer of Amino Acids from tRNA (II), and this enzyme is known to be sulfhydryl-dependent (20).
to Polysomes-It has been reported that cycloheximide has no Consequently, it occurred to us that cycloheximide may retard
effect on amino acid activation and binding to tRNh (8). In peptide elongation by inactivating the sulfhydryl groups of this
order to simplify the protein-synthesizing system, we therefore enzyme and that high concentrations of sulfhydryl compounds
prepared aminoacyl-tRNA charged with a mixture of 14C-amino might compete effectively with the inhibitor and prevent its
acids and added it to an incorporation system consisting of liver action. Accordingly, the GSH content of the medium during
polysomes, partly purified transferases, GTP, and GSH. Such a incubation was raised from 4 pmoles to 20 pmoles. Table I
system incorporated the labeled amino acids efficiently into pro- shows partial protection against inhibitory action of the previ-
tein. Table I shows the mean results obtained from three such ously added cycloheximide (tube 4 versus 5). The action of cy-
studies with this system. Addition of large amounts of cyclo- cloheximide was also diminished by adding 4 pmoles of GSH
heximide at the beginning of incubation caused a 50% inhibition during preliminary incubation (tube 4 versus 6), but no better
of this transfer of label (tube 1 versus 2). It will be noted that, protective effect was obtained if 20 pmoles were added during
in order to achieve this degree of inhibition, a concentration of prior incubation along with cycloheximide than if the GSH was
1 mg of cycloheximide per ml was necessary. This lack of sen- added later during incubation (tube 5 versus 7). A series of
sitivity is comparable to that found above for W-leucine incor- incubations was therefore carried out in which the transferase
poration in the presence of abundant free amino acids in the fraction was incubated with various sulfhydryl compounds before
system (Fig. la). The inhibitory action of the antibiotic could addition of cycloheximide (tubes 9 to 13). Increasing the glu-
 4454 Cycloheximide Action on Pwtein Synthesis Vol. 244, Ko. 16

tathionc lcwl to 20 ~nwlcs did not affect incorl)oration by the 5000 I

I
@cm in thr abwwe of the inhibitor (tube 8 VBTSUS 9), but prior b
incubation \vith thi-: level of glutathionc greatly reduced the z ,ZOmM GSH control
B2 4000-
inhibitory action of cyclohesimidc added subscquentl?- (tube
IO oersus 11). Even more effective protection against cgclohrs-
imidc was obtained by adding dithiothreitol 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. This shows that transfer of 14C’-
amino acids in the absence of cyclohesimide was not significantly 4 mM GSH + cyclohexlmlde
influenced by different levels of the sulfhgdryl compounds.
q ,oLn -
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 IO 20 30 40 50 60
groups, K:W maximally effective at a level of 10 pmoles per tube, MINUTES
whereas a similar dcgrec of protection against inhibition by the FIG. 5. The effect of glutathione level dllring preliminary incu-
other two con~pounds required 20 Fmoles. bation on the subsequent action of cycloheximide during
Since Suttcr and Moldavc (20) have shown that prior incu- subsequent incubation. Transfer of 14C-akino acids from amino-
bation of transfcrase II with glutathione accelerates particularly acyl-tRNA to peptides was examined during differcut times of incu-
bation in the presence or absence of cycloheximide. The system
the initial rata of 14C-aminoacyl transfer t,o ribosomal peptide,
consisted of liver polysomes, GTP, MgZ+, “C-arninoac~l-tl~~A,
we examined t,hc effect of GSH level on both the initial trallsfcr transferase enzymes, and buffer. The transferase fraction was
rate and the final plateau attained in the presence of c\-clohcxi- previouslv incubated at 37” for 5 min with either 4 or 20 mM glu-
rnitl(a. Fig. 6 shows that, in absence of the illhibitor, previous tathione in 0.5 ml of buffer. The other constitllellts of the reac-
tion were then added to a final volume of 1 ml and cycloheximide
incuhtiolr or the transferasc fraction with 20 pmoles of GSII
(1 mg) was added to some of the tubes. Tncubatioll was continued
rcsullcd iti :L marginally grcatcr initial incorporation rate and at 37” for varying periods of time. The figllre shows incorporation
also a slightly higher final plateau than were obtained in the withollt cyrloheximide losing enzyme previously illrllbated at 20
lwesenc’e 01’ 4 pnloles of GSII. When cyclohesimide \VR~ added rnM GSH (A-A) and 4 rn%f GSH (O-O), and illcorporat ion in
presence of cycloheximide using enzyme previollsly incllbatrdwith
to the reaction mixture follolT-ing prior incubation JI-ith 4 pmoles
20 rnM GSH (O-O) and 4 rn>q GSH (O-0). The data are the
of GSH, it had an extensive inhibitory action both on the initial average of two kxperiments.
ratca of rwction alltl 011 the fillal ljlateau attained. Preliminary
incubation with 20 pmoles of GSH protected against cyclohesi-

TABLE II
Effect of preliminary incubalion of washed polysomes with s~tlfhhyclryl
compoluztls 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 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 Additions for
previously incubated incubation I
0
I I I I 1
5 IO 15 20 GTP,
q;- Incorpo-
,umoles of -SH Compound Cyclo- CYCb ration
Poly- Gluta- heximide amino-
Xyl-
Cluta- hexi-
mide
Fro. 4. The effect of cycloheximide on aminoacyl transferases somes thione tRNA: thione
previously incltbated wit,h -SII compounds. The effect of cyclo- trans-
feraw
heximide on transfer of ‘“C-amino acids from aminoacy-tIlNA
to peptides was examined using a system consisting of liver poly- j.moles cpm
somes, lJC-aminoacyl-tl<NA, a mixture of transferases I and II,
None None - zioo
GTP, lIg2f, and buffer. The transferase fraction in this system
was iucubated at 37” for 5 min with various amomits of ghltathione None None + 2400
+ - - 5350
(-----), mercaptoethanol (- - -), or dithiothreitol (---) in a total
volume of 0.5 ml of buffer. The other constitllents of the reac- + - 2000
tion were then added to give a final volume of 1 ml and incubation + - - 5GOO
was contin1led for 30 min at 37”. Cycloheximide (1 mg) XT-as + - - 2350
added to half of the samples at the end of preliminary incubation. + 20 - 5700
The figllre represent,s the mean values obtained in three experi- + 20 + 2900
ments.
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- pmoles. Thus, llrior treatment with a high level of GSII has a
ity that, the inhibitory action of cycloheximide is directed only protective action against the inhibitor.
against the initial rate of aminoacyl transfer, which is particu- EJ’ect oj” Cycloheximide on Release oJ Peptides by Puromycin-
larly subject to GSII stimulation according to Hutter and Nol- Puromycin releases incomplete peptidc chains from ribosomes by
dave (20). substituting for aminoacyl-tRNh at the acceptor site on the
In addition, experiments were performed in which the poly- ribosome and subsequently reacting to form a peptide bond
Homes instead of the transferases were previously incubated with (22-24). It has been shown that cyclohcximidc retards this re-
cyclohesimidc or GSH. Table II shows that prior incubation of leasing action of puromycin (II). hccordingly, the capacity
the polysomes wit’h cycloheximide potentiatcd the action of the of GSH to reverse this effect of cycloheximide was evaluated.
inhibitor very little, and preliminary incubation of the polysomes Polysomes were incubated for 5 min with “C-sminoac~l-tRn’X,
with 20 pmoles of GSH did not confer any additional protective GSH, GTP, and the transfcrase fraction. The polysomes were
effect against cyclohcximidc over and above that obtained by then harrcsted on a sucrose gradient, and the incorporation of
adding the Same amount of GSH during incubation. It is to be W activitr into peptides was examined at various points on the
noted that the polysomcs used in these studies were washed with gradient (Fig. 7a). Most, of the incorporated radio:Lctivit,y was
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 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 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 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
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 pmoles of GSH was much less inhibited by
c\-clohesimide than was enz?-me previously incubated with 4

” ,L!
/+tJ i : : : : : : i ’ :
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 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 trans-
ferase 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
2 4 6 8 IO
MINUTES Mmolcs per tube, and puromycin (200 ,~g) or cycloheximide (1 mg)
or both were added to some samples. The gradients shown
FIG. 6. The effect of preliminary incubation of nminoacyltrans- represent the following conditions of incubation: a, incubation
ferases lvith cycloheximide and glutathione on initial rate of for 5 min lvithollt inhibitors or preliminary incubation; b, similar
amino acid transfer. The system used transferred ‘K-amino to preceding conditions with addition of puromycin after 3 min of
acids from aminoacyl-tRNA to peptides and consisted of a mixed incubation; c, incubation with cycloheximide for first 3 min of
transferase fraction, liver polysomes, GTP, Mgz+, ‘*C-aminoacyl- incubation, followed by an additional 2 min with puromycin; cl,
tRNA, and bluffer. The transferase fraction was incubated at 37” preliminary incubation of transferases for 5 min with 4 pmoles
for 10 min with 4 or 20pmoles of GSH in 0.5 ml of buffer. It was of GSH, followed by 5-min incubation alone, then incubation Tvith
then adjusted by dilution to provide the same amount of enzyme cycloheximide for 5 min, followed by 5 min with puromycin; e,
and 4 pmoles of GSH in all samples. The other constituents of preliminary incubation of trnnsferases with 20 pmoles of GSH,
the reaction \vere then added to give a final volume of 2 ml. Cy- t,hen 5-min incubation alone, then incubation with cycloheximide
cloheximide (1 mg) was added at this point to some of the tubes. for 5 min, followed by 5 mill with puromycin; f, preliminary incu-
Samples were taken at different time intervals for assay of in- bation of transferases m-ith 20 pmoles of GSH, followed by IO-min
corporation into peptidcs. The dat,a are the average of two ex- incubation, and finally 5 min with puromycin. The experiment
periments. was replicated t\vice.
4486 Cycloheximide Action on Protein Synthesis Vol. 244, No. 16

found on the polysome aggregates, with little release into the prevents release of peptide chains by puromycin, an action of
supernatant fraction. When puromycin was added during the cycloheximide that can be prevented by previous incubation of
final 2 min of incubation, the activity in the polysomes was much the transferase enzyme fraction with large amounts of GSH.
reduced and the supernat’ant fraction of the gradient contained According to Skogerson and Moldave (25), puromycin reacts
most of the radioactivity (Fig. 7b). When cycloheximide was more extensively with peptidyl-tRNA4 when it is at the peptidyl
added 3 min before the puromycin, more act,ivity remained on site on the ribosomes; its presence at that site is dependent on
the polysorne aggregates and very little was released (Fig. 7~). transferase II and GTP, thus explaining inhibition of the puro-
Disaggregation of polysomes to monosomes as a result of puro- mycin reaction by cycloheximide.
mycin action was also reduced by addition of cycloheximide Egect of Cycloheximide and Other Inhibitors on GTP Hydrolysis
($ Fig. 7, b and c). To examine the effect of glutathione on and Amino Acid Incorporation in Presence of DiJerenf Concen-
this action of cycloheximide, the transferase fraction was pre- trations of GSH-From the preceding evidence, the inhibitory
viously incubated in the presence of either 4 or 20 pmoles of GSH action of cycloheximide on chain elongation appears to be di-
for 5 min; the “(:-arnirloac?-l-tRNA and cofactors were then rected against transferase II, which is the only sulfhydryl-de-
added, and incubation was continued for 15 min with addition of pendent enzyme involved in aminoacyl transfer (20). Nishizuka
cyclohexirnide and then puromycin as indicated in Fig. 7, d, e, and Lipmann (26) have found that the GTP hydrolysis reaction
and f. Fig. 7d confirms that, cycloheximide reduces total in- required for chain elongation in a bacterial protein-synthesizing
corporation and prevents release of peptide chains by puromycin system also involves a sulfhydryl-dependent enzyme. This en-
when the transfemse has been previously incubated at the lower zyme (translocase) may be identical with transferase II (25).
GSH level (cj. Fig. 7b). However, when the enzyme was first We (14) have recently shown that cycloheximide inhibits GTP
incubated with 20 pmoles of GSH (Fig. 7e), incorporation of the hydrolysis in our mammalian system. The effective concentra-
Y-amino acids was very extensive in spite of the presence of the tions of cycloheximide are similar to those required for inhibition
cyclohcximide, thus confirming protection against inhibition by of W-aminoacyl transfer to peptide chains in the system, and
previous treatment of the transferase fraction with GSH. Fur- the action of cycloheximide can be similarly prevented by gluta-
thermore, the puromycin almost completely released the W- thione (14). This confirms the interrelationship between cy-
peptides into the supernatant fraction. It will also be noted that cloheximide and GSH by means of a separate reaction. In the
the polysome profile under these conditions showed considerable present series of studies, we have used the hydrol>-sis of GTP to
degradation, notably the presence of a large monosome peak. follow the action of other inhibitors of protein synthesis in order
These actions of puromycin were then similar to its effects in the to supplement the information obtained from amino acid in
absence of cycloheximide (Fig. 7f). It will be seen from compari- corporation.
son of Fig. 7.f with Fig. 7b that the presence of high concentra- First, the condit.ions for hydrolysis of (y-3?P-GTl’ xvere
tions of GSH actually potentiates the releasing action of puro- examined using the protein-synthesizing system for amino acid
mycin. transfer from aminoacyl-tRNA to peptide. Fig. 8 shows the
These experiments confirm that cycloheximide retards protein amounts of 32P released as inorganic pho+hate in this cell-free
synthesis without releasing the peptide chains (10). It also protein-synthesizing system when all reagents are present and
also in the absence of aminoacyl-tRNX or ribosomes. A small

‘,0°:
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
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
Minus AA’- tRNA 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
Minus Ribosomes
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
Minutes
when the transferase fraction was previously incubated with
Fro. 8. Ribosome and aminoacyl-tRNA requirements for
GTPase action. Hydrolysis of GTP was examined in a cell-free 20 pmolcs of the sulfhydryl reagent. Streptovitacin -1, a hy-
system n-hich transferred amino acids (AA) from aminoacyl- drosylated derivative of cyclohesimide, Teas also added at a
tRNA to peptides. It contained rat liver polysomes, the trans- concentration of 1 mg per ml, since this is known to inhibit l)ro-
ferase enzyme fraction. ,..(Y-~~P)-GTP. aminoacvl-tRNA. and other tein synthesis effectively in cell-free systems (4). Table HI
components as described under “Materials and Methods.” The
time course is shown for the complete system and in the absence of shows that it behaved like cycloheximide. Grollman (27) has
added aminoacyl-tRNA or polysomal ribosomes. shown that emetine inhibits the amino acid transfer reaction in
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 aggregation caused by delayed addition of amino acids to a me-
may occur because of its structural similarity to cycloheximide. dium lacking free amino acids could be inhibited by much lower
We therefore added 100 pg of emetine per ml to our transfer sys- concentrations of cycloheximide than those necessary to achieve
tem, a concentration shown by Grollman to be within the effec- inhibition of chain elongation. Since this suggests a separate
tive range, and confirmed that both amino acid incorporation action of the inhibitor, we tested the capacity of sulfhydryl
and a2P release were extensively inhibited. However, unlike compounds to protect against this effect of cycloheximide. Fig.
cycloheximide, neither reaction could be protected against in- 9 shows polysome reaggregation in response to addition of amino
hibition when the glutathione concentration was raised. Groll- acids when the GSH concentration in the medium was raised
man (28) has recently found that emetine differs from cyclo- from 4 mM to 20 mM. The usual reduction in monosomes and
heximide in its action on HeLa cells. On suspending the cells accumulation of polysomes was obtained, and the presence of
in fresh medium, the inhibitory action of cycloheximide could be 0.1 pg of cycloheximide was again sufficient to prevent reaggre-
reversed, whereas that of emetine could not; however, in Groll- gation (compare Fig. 2). Thus, GSH does not influence this
man’s system the action of streptovitacin A was also found to be action of cycloheximide.
irreversible. Finally, the action of sparsomycin was studied in In the same experiments, YXeucine was present in the me-
our system at a concentration of 2 pg per ml, which is known to dium, and incorporation of radioactivity into protein was ex-
inhibit protein synthesis in mammalian cell-free systems (29). amined after 40 min of incubation. When no other amino acids
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,
inhibition of 32P release by sparsomycin was slight. This result,
which implies an unexpected dissociation between the two re- ----_-. 22’ incubation - AA
quirements of peptide bond formation on ribosomes, has been - { 2$ incybation 4:
confirmed on several occasions and may indicate uncoupling by
20’ incubation - AA
sparsomycin of GTP hydrolysis from other events in protein +2’ incubation +AA and
synthesis. +O.Ipg cycloheximide
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 constituent,s were then added and incuba-
tion was continued for 30 min.

I Prior
incubation
I
_-
,STP,
Incubation
MC-
Amino-
acyl-
Tube tRNA
IPolY-
hzyme (;hlta- somes, Antibiotic incor-
: 1thione a mino- porated’l
t “R’i
.-
I moles @m/lube
1 + 4 + - 1000 4170 ,
2 + 20 +
- 1200 4350
3 + 4 + Cycloheximide 200 1140
J
4 + 20 + Cycloheximide 800 3900 LINEAR SUCROSE GRADIENT
5 + 4 + Streptovitacin 210 830 16% = 40%
6 + 20 + Streptovitadin 790 4000 FIG. 9. Prevention by cycloheximide of polysome reaggregation
7 + 4 + Emetine 530 560 on addition of amino acids (AA) in a GSH-rich medium. Liver
8 + 20 + Emetine 530 580 polysomes were incubated for 20 min in an amino acid-dependent
9 + 4 + Sparsomycin 830 660 protein-synthesizing system similar to that of Fig. 2 but contain-
ing 20 mM GSH without added amino acids, and then a complete
10 + 20 + Sparsomycin 980 700
mixture of 20 amino acids w&s added either alone (-) or in the
--
presence of 0.1 rg of cycloheximide (-- -). Incubation was
a The value for 32P released in tube 1 is equivalent to 4600
continued for an additional 2 min. A control sample was incu-
pmoles of GTP hydrolyzed, and the value for 14C incorporated in bated for a similar total period of 22 min without added amino
tube 1 is equivalent to 480 pmoles of amino acid incorporated. acids (-----). All three samples were separated simultaneously
Conway and Lipmann (18) also report a considerable discrepancy on sucrose gradients and the profiles were measured by ultraviolet
between GTP hydrolysis and aminoacyl incorporation. absorption. The experiment was replicated twice.
44% Cyclohc:cimide Action on Protein S7y~lhesis
mw :~tltlctl to the tirc~tlium, 22’70 rpm lwr tr11Jc~were ittcorIJoratct1.
\\?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~,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).
‘I’hcsc studies Iwovitle furthw c\-itlcttcc that the actioti of cy-
clohcsimitlc on rc:rggwg:ttiott :iiid on 1wlJtitl~ t~lotlgxtioti is tlue to
diffrrcttt trtcch:tttisttts.
chain ittitiation atid oti (*lt:titt c~loirg:tlioti. \ilieti the\- ittcrtbated
intact rcticulocytcs with S:iF xnd cyclolwsintitlc, both con-
pomtds itthibitcd ch:titt initiation, but c~~c~1oIic~sittiitlc:ilso aftfccted
elong:~tiori of nt~swtt1 Iwl)titIc~ c~haitts;. Orir wsults 1)rovidc di-
rect 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 ribo-
comes. 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 re-
aggrcgatc~ 1)~ a 1Jrowss that ~Jrwttttlabl\- xquircs ittitiation of
pq)tide chains. ‘I’hcb c*ottc*c~tili~:ttioti of c:\-~lollc~sittlitlt! iteedcd
for effcctivc itihilJitiotr of chairi c~lottg:ilioii iii this syslcni is 10”
times greater Ihan 1h:tt tic&d lo ~wvc~it r0tnplct.r: 1Jolysome
reconstitution on adding amino acidh. ‘l’his is the rcvwe of Ihe
finding of’ Stanners (30), w110 estrmittcd t,ht: ac*tiott of cyclohcsi-
mide 011 hamster ctnbryonic cells iti t issuc: c~ultrrix~ and found
that low doses inhibited movcmcttt of ribosontw along the mes-
senger, 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. ‘l’hcse 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 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: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) I’ailcd to obtain
an inhil)itory c~ffcc~l.of cyc~lohcsitttitlc on I~urottt~~in-tlc~~)(~tt(Ic~~t
rcleasc ol 1x1)1 itlw 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~otrtyc~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
Vol. 244, No. 16
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 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: Iwl’oi~~ I’otxtitig 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, 51inlttl:iitl c,ffcct,
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 \vhctt 1)wviousl~
itrcubatctl with \atious sulf’I~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:n‘s that, I)urifiic~tl ~1x11sCw:tst~I I is
readily itt:tc:t iwt.ed Wough its suII’hytIry1 g~~rt~w :rtrtl that it twt
be regctiwttcd 1Jy sulfhydi~yl tlonot~s. Ill th? (‘:I’( ol’ Ihc t:1wde
trnnsferasc IJrc:I):lt’at,iotis usctl I)y us, whi& iitt~ludc l)olh cti-
zymcs I and II, it is ~~robablc that transfctxw 1I is less uitst:ible
(20). ‘I’his is srtIJIwt?,c~tl by the tlata itt E’ig. 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). ‘I’his 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).
‘I’his 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 iii-
crettsrs subsc~~ucnt :rmitio: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~ntottsttxlcs 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 tlrtertttittc 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 cxtctly similar to th:lt of c:\-cloltc,sittritlc, to \vhich it
is structurally rc:latc~l (‘l’ablc 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-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 emclittc, l~tit wliw Ihc alla wrrc
stis1tc’tid~~l in I’twli 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 I’IQI~I lllaf, of
strcptorit:ic*in lJy its ittscitsitivily to GSII. ‘I’ltis tlow itot, of
couwe, c~litnittatc 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~ion
rem:tins rtttcc~r1:iiti (32). .\tt ittt*rc:isc iii GSl I c~otrc~c~ti1t~:t1iot~ :tlso
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 14. MUNRO, 13. N., BAI,IG.\, B. S., .\AX PI~ONC~UK, A. W., Nature,
219, 944 (1968).
interesting feature of these inhibitors was that cgclohcsimide
15. WE’r’rs’rEIiv, F. O., S.~.\EHELII\, T., AND NOLL, H., Nature,
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inhibited GTP hydrolysis fairly strongly, but sparsomynin was 17. MOLD.ZVE, K., in S. P. COLOXICK AND N. 0. KAPLAN (Editors),
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inhibition of amino acid incorporation. This demonstrates a 18. CONI~~Y, W. T., .\ND LIPMINN, F., 1’1-0~. Nat. Acad. Sci.
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