Mechanism of Cycloheximide

8/19/2019 Mechanism of Cycloheximide

1/10

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


2/10

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

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3/10

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


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4/10

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-


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5/10

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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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


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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);

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SKOGERSON, I,., AND MOLDAVE, K., Arch. Biochem . Biophys.,

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NISHIZUIU, Y., .\NI) LIPMANN,

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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

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STANNEHS, C. P., Biochem . Biophys. IL’es. Commun., 24, 758

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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).


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Mechanism of Cycloheximide

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