THE SELECTIVE [NHEIBYSION
OF PROTEIN ASSEMBLY BY GOfiGEROTlN

Thesis for the Degree of M. S.
MICHEGAN STATE Uzz‘HVERSlTY
SHERWOOD REID CASJENS
19767

 

 
  
 
 

   
 
  

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ABSTRACT
THE SELECTIVE INHIBITION OF PROTEIN

ASSEMBLY BY GOUGEROTIN

By Sherwood Reid Casjens

The mechanism by which gougerotin inhibits protein synthesis
has been investigated. Gougerotin has been found to specifically
inhibit the transfer of amino acids from aminoacyl-SRNA into poly-
peptide. Gougerotin was found to inhibit the incorporation of
amino acids more strongly than the release of finished globin
chains. The breakdown of polysomes, which normally occurred with
protein synthesis in the cell-free system, was inhibited by the
antibiotic. The action of gougerotin was not reversed by GT? or
supernatant enzyme in the concentrations tested. The action of
puromycin and gougerotin were compared. Gougerotin did not cause
release of polypeptides from the ribosomes as did puromycin. In
fact gougerotin inhibited the puromycin dependent release of peptides
from ribosomes. Thus the site of action of gougerotin appeared
to be primarily the inhibition of peptide synthetase. Several
mechanisms for the action of gougerotin in the inhibition of

protein synthesis are discussed.

THE SELECTIVE INHIBITION OF PROTEIN

ASSEMBLY BY GOUGEROTIN

By
Sherwood Reid Casjens

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
MASTER OF SCIENCE

Department of Biochemistry

1967

AC KICO'JLEDG-ZENTS

I would like to thank Dr. Allan J. Norris for his advice and
assistance during this work and also for the financial assistance
received from the National Institutes of Health through him. I would
also like to thank Ron Slabaugh and A. Bruce MacDonald for some
stimulating discussions and the latter for use of some unpublished data.

SoReCe

ii

DEDICATION

To my parents

iii

TABLE OF CONTENTS

quODUCflON 0 O O O O O O . . O . I I 0 O O 0 .

mstICAL . . O . . O O . O O O . O O . O . O .

I'IETHODS AND I'meIAI-IS e e e e e e o e o 0

Compounds . . . ..... . . . . . . . . . .

Biological.Materials . . . . . . . . . . .

Preparation of Rabbit Reticuloqyte

Page
. 1
. 2
. 6
. 6

7

Ribosomes 7
Preparation of Supernatant Enzyme
Fraction . . . . . . . . . . . . . . . . 8
IN . .
Preparation of C-Peptidyl - Ribo-
Sanes ......OO....0.0. . 8
Cell Free Assays . . . . . . . . . . . . . . 10
14
Assay for C-Valine Incorporation
intoProtein............. .10
Determination of GTP or Puromycin
Dependent Release of Polypeptides
fromRibosomes............ .10
Assay.for Incorporation of 3H-
Puromycin into Trichloroacetic
Acid Insoluble Product . ...... . . . ll
ANALYTICAL PROCEDURES . . . . . . . . . . . . . . 12
RESULTS .IIOIOOOOOOOOOOOOQOOOblu
Effect of Gougerotin on Amino Acid Incorp-
oration into Protein . . . . . . . . . . . . . 14
Effect of Gougerotin on Polysome Break-
do‘m .0........I......OOO.19
Effect of Supernatant Enzyme Fraction and
GTP on the Inhibition of Protein Synthesis
by GougerOtj-rl o o o > o e e o e o o e e o e e o o 19

iv

Effect of Gougerotin on GTP Dependent

Release of Globin Chains . . . . .

Comparison of the Effects of Gougerotin
and Puromycin . . . . . . . . . . . . .

Effect of Gougerotin on Puromycin
Dependent Release of Polypeptides
from Ribosomes . . . . . . . . . .

Studies with 3H-Puromycin . . . .

DISCUSSION

BIBLIOGRAPHY

Page

..... 21+

0 0 21+

..35

TABLE

III

LIST OF TABLES

Page

Effect of Supernatant Enzyme and GTP on the
Inhibition of Amino Acid Incorporation by
Gougerotin..................23

Dissociation of Nascent Polypeptide from
Ribosomes by Puromycin and Gougerotin . . . . 28

Effect of Gougerotin on Puromycin Dependent
Release of Polypeptides from Ribosomes . . . . 31

FIGURE

LIST OF FIGURES
Page
Structures of Gougerotin, Puromycin, and Amino

Acyl-SR-NA0000cocoon-000000005

The Effect of Gougerotin on Amino Acid Incorp-
oration into Protein in a Cell Free System . . 16

The Inhibition of Amino Acid Incorporation by
Various Concentrations of Gougerotin . . . . 18

The Effect of Gougerotin on Polysomal Break-
down During Protein Synthesis. . . . . . . . . 21

The Effect of Gougerotin on GTP Dependent Release
ofGlobinChains................26

Incorporation of 3H-Methoxyz-Puromycin into
Trichloroacetic Acid Insoluble Material . . . . 33

Schematic Representation of Protein Biosyn-
theSis. . O O O O . . 0 O O O O I . O O O O . O 39

INTRODUCTION

Aminoacyl nucleoside antibiotics (1) such as puromycin (2-5),
chloramphenicol (6-8), gougerotin (9), and a number of others,
as well as a number of other types of antibiotics such as cyclo-
heximide (10,11) and streptomycin (12,13) have been found to be
specific inhibitors of protein synthesis. In the past the study
of the mechanism of action of these compounds has been of interest
in the study of protein biosynthesis as well as in the study of
antimicrobial agents, since some antibiotics have been found to
inhibit specific steps in the protein biosynthetic pathway, and
hence can be used to help clarify these steps. Thus the present
study was undertaken with the primary goal of the determination of
the mode of action of the aminoacyl nucleoside antibiotic gougerotin
in the inhibition of protein synthesis and the secondary goal of

making gougerotin a useful tool in the study of protein biosynthesis.

HISTORICAL

Gougerotin was first isolated from Streptomyces gougeroti and
found to have antibiotic action by Kanzaki gt_al;_(1h). Its structure
was first investigated by Iwasaki (15), who proposed a structure which
was later shown by'Fox‘gt‘al£ (16) to be incorrect. Fox has proposed
the structure for gougerotin shown in figure 1. Gougerotin is an
aminoacyl nucleoside antibiotic in that it contains peptidyl, carbohy-
drate and pyrimidine moieties.

Clark and Gunther (9) showed that gougerotin was an inhibitor of
protein synthesis 2p;yitgg.with.the use of a polyuridylic acid directed
synthesizing system from Escherichia coli. they found that gougerotin
inhibited protein synthesis at the stage of amino acid transfer from
aminoacyl-SRNA to polypeptide. They suggested that gougerotin's action
may be similar to that of puromycin since they both inhibit amino acid
transfer into protein in the cell-free system.

Sinohara and Sky-Peck (17) used a cell-free amino acid incorporating
system from rate liver microsomes to obtain similar results to those of
Clark and Gunther, in that gougerotin had no effect on amino acid
activation and appeared to inhibit protein synthesis at the transfer
reaction. However they proposed that since gougerotin did not seem to
fit the specificities of puromycin analogs found by Nathans gtflgl;
(18), it probably inhibited the transfer reaction in a different manner
than puromycin.

Since the time the bulk of the work described in this thesis was

completed, one simultaneous study (19) and two subsequent studies have

3
been published (20,21) which deal with the site of action of gougerotin.

These reports will be considered in the discussion.

Figure l. The structures of gougerotin (16), puromycin and
and the 3' terminal nucleotide of aminoacyl-SRKA. All are
similar in that they contain a nitrogen base, a carbohy-
drate and an amino acid type moiety. Gougerotin, however,
differs from puromycin and aminoacyl-SHEA in that it contains
a pyrimidine rather than a purine base, a hexose rather than
a pentose carbohydrate, and a dipeptide instead of a single

amino acid.

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METHODS AND MATERIALS

COMPOUNDS

Gougerotin was a gift of Dr. J. M. Clark Jr., Biochemistry
Division, University of Illinois, Urbana, Illinois and Dr. A.
Miyake of Takeda Chemical Industries, Ltd., Osaka, Japan. Puromycin-
methoxy (3H) dihydrochloride was purchased from New England Nuclear
Corp., Boston, Mass. Unlabeled puromycin dihydrochloride and unlabeled
amino acids were purchased from Nutritional Biochemicals Corp., Cleveland,
Ohio. Uniformly labeled L-(luC)-valine was a product of Schwartz Bio-
Research Inc., Orangburg, New York. Nucleoside triphosphates were
obtained from P—L Laboratories, Milwaukee, Wisconsin. Reduced glutathione
was purchased from Mann Research Laboratories, Inc., New Ybrk, New
York. The scintillators.and thixotropic gel powder were aquired from
Packard Instrument Company, Inc., Downers Grove, Illinois. Nembutal was
obtained from Abbott Laboratories, North Chicago, Illinois. Dioxane,
naphthalene, and phenylhydrazine hydrochloride were purchased from
Distillation Products Industries, Rochester, New York. Heparin sodium
and analytical grade toluene were from Fisher Scientific Company,
Chicago, Illinois. All other materials were purchased from Sigma

Chemical Company, St. Louis, Missouri, in the highest purity available.

BIOLOGICAL MATERIALS
I. Preparation of Rabbit Reticulocyte Ribosomes.

Male New Zealand rabbits weighing between six and eight pounds were
made reticulocytic by four daily injections of 0.175 ml of 2.5% neutralized
phenylhydrazine per pound of body weight. The injections were made sub—
cutaneously. Each day's supply of phenlyhydrazine was frozen in an
individual container. On the fifth day no injection was given, and on
the sixth day after the initial injection the rabbit received a solution
containing 2000 I. U. of heparin and 75 mg of Nembutal by intravenous
injection. The blood was collected immediately by heart puncture. The
blood was the cooled to 4° and centrifuged in a Sorvall cetrifuge for
20 minutes at 2000 x gravity. All further operations were done at 4°,

The pelleted cells were resuspended in a solution containing 0.0075 .11
EgClZ, 0.13 N.Na01, and 0.005 M KCl, with a volume equal to that of the
supernatant plasma. The suspension was filtered through glass wool and
centrifuged again for 20 minutes at 2000 x g. The supernatant liquid was
removed by aspiration, and the cells were lysed by addition of n times the
packed cell volume of a 0.025 §_MgC12 solution and stirred very gently for
10 minutes. One packed cell volume of 1.5 fl_sucrose containing 0.15
Q’KCl'was then added and the suspension was centrifuged at 10,000 x g

for 10 minutes to remove the cell debris present. The supernatant

liquid was centrifuged for 90 minutes at 78,000 x g. The high speed
supernatant fluid so obtained was saved for the preparation of the super-
natant enzyme fraction. The ribosomal pellets were resuspended were
resuspended gently in a volume of Medium B (0.25 N sucrose, 0.017 g
KHCOB, and 0.002 E'MgC12) which was two thirds that of the previous super-

natant fluid. The solution was cetrifuged again for 90 minutes at 78,000

8
x g centrifugations were carried out in a Spinco Model L-2 Ultra-
centrifuge (22).
II. Preparation of the Supernatant Enzyme Fraction.

The supernatant enzyme fraction was prepared from the first 78,000
x g supernatant by addition of Tris-RC1 buffer (pH 7.5) to a final
concentration of 0.1 M, Then powdered ammonium sulfate was added to
40% saturation at No. The precipitate which formed was removed by
centrifugation and discarded. Ammonium sulfate was added to 70% of
saturation, and the resultant precipitate was removed by centrifugation
and taken up in a small volume of a solution containing 0.1.MDTris-

HCl buffer (pH 7.5) and 0.001.M'glutathione. Ammonium sulfate was

again added to 70% of saturation and the precipitate was removed as before
and dissolved in a mixture containing 0.02 M Tris-HCl buffer (pH 7.5),
0.001 M EDTA, 0.001 M glutathione, and 0.001 M MgClZ, and dialyzed
overnight against 100 volumes of the same solution. The dialyzed
preparation was then adjusted to 0.02 M.glutathione and stored at

-l8oor --l96o until it was used. The preparation, so obtained, is called
the "supernatant enzyme fraction" (23).

III. Preparation of 14C-Peptidyl-Ribosomes.

The preincubation of ribosomes with 1Ll'C-uvaline to produce 1’40--
labeled ribosomes was carried out in a medium containing l.mM ATP, 2.5
mfi_phosphoenol pyruvate, lO'ng/ml pyruvate kinase, 0.05 M_Tris-HCl buffer
(pH 7.5), Air 13;; MgClZ, 0.05 M KCl, 0.02 M glutathione, 0.05 111:1 in each
of the 20 amino acids except valine, 5 mg/ml ribosomes, h mg/ml
supernatant enzyme fraction, and 0.05 mfl'in 1uC-valine (specific
activity 10 C/M). The solution was incubated for 10 minutes at 37°,

and the reaction was stopped by the addition of 10-12 volumes of

9
Medium B (described above) containing a 100 fold excess in unlabeled
valine. The ribosomes were isolated by one centrifugation at
78,000 ng for 90 minutes. The ribosomal pellets were resuspended
in a small volume of 0.25 M sucrose and centrifuged at 1000 x g to

remove debris. The supernatant solution contained the 1u'C-peptidyl-

ribosomes.

11".“

10
CELL FREE ASSAIS
I. Assay for 1uC-Valine Incorporation into Protein.

The cell-free hemoglobin synthesizing system used to detect radio-
active valine incorporation contained 0.25 mM GTP. 1.0 mM ATP, 5 mM phos-
phoenol pyruvate, 401ag pyruvate kinase, 20 mM glutathione, 50 mM KCl,

4 mM MgClZ, 50 mM Tris-RC1 buffer (pH 7.5), 2 or 3 mg ribosomes as
indicated, 4 mg of supernatant enzyme fraction, 0.05 mM of each of the 20
amino acids except valine, and 0.05 mM in L-(luC)-valine (specific activity
3 C/M), in a total volume of 1.0 ml. The phosphoenol pyruvate was pre-
pared from the barium salt, with HCl, removal of barium with K250” treat-
ment and neutralization with KOH. The glutathione was brought to pH 6.0 and
nucleoside triphosphate solutions were brought to pH 7.0 with KOH.

II. Determination of GTP Dependent Release of Polypeptides from

Ribosomes.

Studies of the effect of gougerotin on the GTP dependent release
of polypeptides from ribosomes were performed by incubation of the prelabeled
ribosomes in a solution containing 50 mM KCl, 4 mM MgClZ, 0.2 mM GTP,
and gougerotin as indicated in a total volume of 1.0 ml. No incorpora-
tion of amino acids occurs under these conditions, although finished at
and Q chains are released from the ribosomes. The amount of GTP dependent
release was calculated by subtracting the amount of protein non-specifi-
cally released in a similar assay to which no GTP had been added. The
value of the nonspecific release was usually about 10% of the total.
radioactivity in the assay. Following incubation for 40 minutes at

37° the solutions were transferred to 4 ml cellulose centrifuge tubes

11
and centrifuged at 105,000 x g for 60 minutes. Each supernatant was then
analyzed for radioactive protein (23). If the puromycin dependent release
of polypeptides was to be measured, the procedure was the same except
GTP was omitted and puromycin was added in the quantities indicated.

III. Assay for Incorporation of 3

H-Puromycin into Peptidyl-Puromycin.

The incubation mixture for the formation of peptidyl-puromycin
included Tris-HCl buffer (pH 7.5), glutathione, Cl, and MgClZin the
concentrations indicated for the puromycin dependent release system
(above). Ribosomes and 3H-puromycin were present in the amounts indica-
ted. The total volume of the assay was 50 microliters. The reaction was
stopped by pipetting a 5 microliter aliquot into 10% (w/v) trichloro-
acetic acid. The resulting precipitate was analyzed for the incorpora-
tion of 3H-puromycin into polypeptides as described in analytical

procedures.

12
ANALYTICAL PROCEDURES

The concentrations of ribosomes were determined by the spectro-
photometric method of TS'O and Vinog’rad (24).

The analytical ultracentrifuge run was made in a Spinco Model E
Analytical Ultracentrifuge at 42,040 r.p.m.

Incorporation of radioactive amino acids into protein was measured
by the following procedure. Fifteen mg of bovine serum albumin was
added to each of the one ml assays followed by precipitation with 5%
(w/v) trichloroacetic acid. After allowing 30 minutes for complete
precipitation, the precipitate was collected by centrifugation in a
clinical centrifuge. This precipitate was washed by resuSpension in 5%
trichloroacetic acid and centrifugation. The resulting pellet was
dissolved in 0.5 ml of 1 M_NaOH to hydrolyze any aminoacyl-SRNA present,
and reprecipitated with 5% trichloroacetic acid and the precipitate
washed as before with 5% trichloroacetic acid. The pellet was then re-
suspended in acetone which had been made 0.1 NDin HCl. The precipitate
was centrifuged and suspended in a 2:3 (v/v) mixture of the acid acetone
and diethyl ether. After centrifugation the pellet was suspended in di-
ethyl ether, centrifuged and air dried. The resulting powder was trans-
ferred very carefully to a glass counting vial (the last bit was trans-
ferred with the help of 0.5 ml of 1.0 E.NaOH and several drops of dioxane
were added to the powder in the counting vial to aid dissolving). When
all of the powder had dissolved in the NaOH solution, 15 ml of thixatrOpic

counting fluid was added, and the vial was shaken vigorously. The

13

counting mixture was prepared by combining 7 g‘of 2,5-diphenyloxazole,

150 mg of 1,4-bis-2-(5-phenyloxazole)-benzene, 50 g of naphthalene,

and 36 g of thixotropic gel powder dissolved in 200 ml of toluene,

30 ml of ethanol, and 800 ml of p-dioxane. The samples were then

counted in a model 3003 Packard Liquid Scintillation Counter.
Incorporation of tritiated puromycin into peptidyl-puromycin

was measured by a procedure similar to that given above for measuring

amino acid incorporation except 10% trichloroacetic acid was used

in place of the 5% tricloroacetic acid and the NaOH treatment and

subsequent wash of the precipitate were omitted.

RESULTS

In the present studies the results of Clark and Gunther (9) and
Sinohara and Sky-Peck were comfirmed and extended in that gougerotin
was found to inhibit amino acid incorporation in the rabbit reticulocyte
celquree system in a manner similar to that shown by the above workers
using the Eg'ggli and rat liver systems. The time course of amino
acid incorporation into trichloroacetic acid precipitable protein
in the cell-free system is shown in figure 2. It can be seen that
gougerotin causes a decrease in the total incorporation as well as a
decrease in the rate of amino acid incorporation. Increasing the
concentration of gougerotin resulted in further lowering of the rate
of amino acid incorporation and total incorporation. At all concentrations
tested the reaction was essentially complete by'ho minutes.

To characterize the effect of gougerotin in more detail, the
inhibition of the amino acid incorporation system was studied as a func-
tion of antibiotic concentration. Figure 3 shows the results of the study.
Gougerotin was added to the complete system in the indicated concen-
trations and incubated for 40 minutes at 37° (at which time the reactions
were completed). The concentration required to reach maximum inhibition
was approximately ten times that reported for puromycin (3).

Gougerotin also showed an effect on the breakdown of polysomal
structure during protein synthesis. According to current models (25-27),
protein synthesis is accompanied by an orderly breakdown process as the

ribosomes reach the end of the messenger RNA molecule and become detached

Figure 2. The effect of gougerotin on amino acid incorporation
into protein in a cell free system. The complete system (0.15)
contained 3 mg of ribosomes, 1U"C-valine ( specific activity
4.0 C/H) and other components as described in flETHODS (assay

I
for incorporation of l4’C--valine into proteins). To the
complete system gougerotin was added to a concentration of
0.1 3.2.3. (o...) and 1.0 m}: (o- 0). Following incubation at 37°C
for the times indicated the reaction was stepped by the addition
of u ml of 5% trichloroacetic acid, and the total radioactive

protein present was determined as described in HALYTICAL

PROCEDURES.

 

 

 

 

 

 

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TIME (MINUTES)

Figure 3. The inhibition of amino acid incorporation by
various concentrations of gougerotin. Antibiotic was added
to the complete system (described in METHODS - assay for 1&0-
valine incorporation into protein) in the indicated concen-
trations and incubated at 37°C for 40 minutes. The total
radioactive protein was determined as described in AHALYTICAL

PROCEDURES.

 

 

 

 

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19
from the polysome structure. Since it has been shown that only a limited
amount of reattachment of single ribosomes to the messenger REA occurs
in the reticulocyte cell free system (28-31),the net effect is that
the proportion of polysomes decreases with a simultaneous increase in
the amount of single ribosomes as protein is synthesized.

The addition of gougerotin to a cell free system which would normally
be actively synthesizing protein caused inhibition of polysome breakdown
as well as inhibition of amino acid incorporation (figure H). Concurrent
studies by A.J. Morris (32) using sucrose density gradient centrifugation
yielded similar results, and the use of increasing concentrations of
gougerotin caused increasingly stronger inhibition of polysome breakdown
by gougerotin.

The evidence available seems to implicate the utilization of GTP
in ribosome movement along the messenger RNA; thus inhibition of the
GTP utilization system was a possible site for the action of gougerotin.
However the data shown in table I indicate that increased GTP content
of the assay had no significant effect on the amount of inhibition of
amino acid incorporation that was observed in the cell free system. Thus
it may'be stated that gougerotin is not competing with GTP for a Specific
ribosomal site. Similarly, the degree of inhibition was not markedly
affected by variations in the levels of supernatant enzyme used in the
assay. These results find some support in studies of 1LPG-GTP binding
to ribosomes and GTP hydrolysis by ribosomes by A.B. MacDonald (35).

In both cases no effect by gougerotin was found.

Finished globin chains are released from the polysomes by a GTP

dependent process which can be distinguished from amino acid incorpora-

Figure 4. The effect of gougerotin on polysomal breakdown
during protein synthesis. Ribosomes (3 mg) were incubated
in the complete system for amino acid incorporation for

20 minutes at 37°C and then chilled and transferred into

a 1.2 cm.prismatic cell (upper trace). An identical assay
which was 1.0 mfl_in gougerotin was placed in the material
plane cell (lower trace). The photograph was taken approx-
imately 6 minutes after attaining u2,o¢+o rev./minute in a
Spinco Model E Analytical Ultracentrifuge (at 5°C). Sedi-
mentation was from left to right. Total radioactivity
present in protein was determined in identical assays
containing 0.05 mM 1“Cavaline (specific activity 3.0).

The assay with no gougerotin contained 660? counts/minute,
and the assay with 1.0 m§.gougerotin contained 805 counts/

minute of radioactive protein.

 

 

 

 

 

 

 

rI‘OP BOTTOM

Table I. Effect of supernatant enzyme preparation and GT
1%

on the inhibition of amino acid incorporation of C-valine

(specific activity 3.0) into polypeptides was measured as

described in METHODS. The assays in which GTP was varied

contained o mg of supernatant enzyme fraction, and the

assays in whi‘h the supernatant ens; 0 fraction was varied

contained 0.25 microuoles of GT . All assays contained

gougerotin as indicated.

 

TABLE I

 

14
C-VALIN E INCORPORATION

 

GTP ADDED GOUGEROTIN
(Amoles) ADDED counts/ min % inhibition
(Amoles)
0. 25 none 757 6 0%
0.25 0.10 2206 71%
O . lO 0. 10 2983 61%,)
0.50 0.10 2832 63%
1.00 0.10 2552 66%
SUPERNATANT
ENZYME ADDED
(mg)

6. 0 none 7576 0%
10 . 0 none 7 601 0%

6.0 0.10 2206 71%
10. 0 0. 10 2580 66%

 

24
tion into protein (23). Thus is seemed possible that gougerotin blocked
polysome breakdown, and hence amino acid incorporation, at the release
step. However data presented in figure 5 indicated that this step is not
the primary site of action since the polypeptide release is significantly
less suceptible than the amino acid incorporating system to the anti-
biotic (see also figure 3) both in maximum inhibition found (about 60% and
80-90% respectively) and in the concentration of gougerotin required
to reach maximum inhibition (about 1 mfl_and 0.3 mg respectively).

The mechanism of action of puromycin in the inhibition of protein
synthesis has been reasonably well characterized. Yarmolinsky and De La
Haba (2) have suggested that it acts as an analog of the amino acid acceptor
end of the tranfer RNA molecule, since their structures are very similar.
Puromycin has since been shown to act in this manner and to release unfin-
ished peptides (3,36) in the form of a peptide with puromycin bound to its
C-terminal by a peptide bond (5,37,38). Thus puromycin acts like an
aminoacyl-sRNA, however after the peptidyl-puromycin bond is formed it
is released from the ribosomal particle since the remainder of the sRNA
is not present with its binding sites for ribosomes. Allen and Zamecnik
(5) have suggested, however, that this release of peptidyl-puromycin
may not be entirely quantitative.

Clark and Gunther (9) found that gougerotin inhibited protein synthe-
sis and suggested its effect might be similar to that of puromycin.
Therefore the action of the two antibiotics was compared. Puromycin or
gougerotin was added to a cell-free release system, and the appearance of
11;C-polypeptides was measured. Table II presents data which show that
puromycin and gougerotin do not act in the same manner. Gougerotin

showed no release of polypeptide from the ribosomes as did puromycin.

Figure 5. The effect of gougerotin on GTP dependent release
of globin chains. Gougerotin was added to the complete
releasing system described in FETHODS (determination of

GTP dependent release of polypeptides from ribosomes).
Prelabeled ribosomes (3 mg) containing lOfl13 counts/minute
were incubated for 40 minutes at 37°C , and the released
globin chains were determined as described in RETHODS. The
values in the figure are average values of two identical

experiments.

 

 

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Table II. Dissociation of nascent polypeptide from ribosomes
(2 mg containing 3720 counts/minute total radioactive poly-
peptides) by puromycin and gougerotin in a medium containing
agelz, K01, glutathione and Tris-H01 buffer in the concen-
trations described in.METHODS, and puromycin and gougerotin
as indicated.. Ribosomes were removed by centrifugation and
the soluble labeled polypeptides determined as indicated in

ANALYTICAL PROCEDURES.

HI

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Gougerotin
Puromycin

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In fact gougerotin appeared to depress the basal amount of release to a
value less than that seen when no antibiotic was added to the assay.
Further indication that the two antibiotics act by different mechanisms
can be seen in the fact that gougerotin inhibits polysome breakdown
(see figure 4), whereas puromycin has been reported to actually accelerate
polysome breakdown (3,11,30).

Since the formation of the puromycin-polypeptide bond probably
is the only step taking place under the conditions of the release assay,
it has been considered as a possible system for the study of the formation
of a model peptide bond (37,39). Thus the effect of gougerotin onthe
reaction between puromycin and peptidyl-SRNA may meaningful in terms of
the actual peptide bond forming step in normal protein synthesis. Results
of the studies of the effect of gougerotin on the puromycin dependent
release are presented in table III. It is evident that gougerotin has a
very marked effect on this reaction, and hence the site of action appears
to be inhibition of peptide synthetase. In concurrent studies A. J.
Morris defined the effects of gougerotin concentration upon the puromycin
dependent release of labeled polypeptides from the ribosome in more
detail using the same analytical procedure. It was found that the data
best fit a Lineweaver-Burk plot (40) which showed gougerotin to be a
competitive inhibitor of puromycin dependent release reaction (32).
Attempts to measure the initial rate of 3H-methoxy-puromycin incorpora-
tion, and hence to do more extensive kinetic studies, were not successful
since the reaction rate was too fast to be measured by available

techniques (see figure 6). It is evident, however, that gougerotin does

Table III. The effect of gougerotin on puromycin induced
release of polypeptides from prelabeled ribosomes was
determined by adding gougerotin in the indicated amounts,

to the puromycin dependent release system described in
HETHODS. The experiments with 0.025 micromoles of puromycin
were done with 2 mg of ribosomes containing 3208 counts/min.
and were incubated for 20 minutes at 37°C before centrifu-
gation. The assays with 0.50 micromoles of puromycin
contained 3 mg of prelabeled ribosomes containing #001
counts/minute and were incubated for 40 minutes at 37°C
before centrifugation. Each of the counts/minute values
was obtained by subtracting the amount of radioactivity in
a no antibiotic blank from the amount of released radio—

activity found in the assay cantaining puromycin.

TABLE III

 

 

.. “q .4... -....~.

11:.
LnromYCin Added Gougerotin Added C-rolypeptide
(amoles) @moles) Released

counts/min % inhibition

 

0.025 none 600 0%
0.07.5 0.05 589 2%
O, 0?. '3 O. 25 11-14 31.73
0. 0.7.5 1. 00 29]. 52%
0.50 none 1721 0%

0.5-O 0.05 1.376 2073
0.50 0.10 117% 32%

0.50 1.00 779 55%

 

Figure 6. Incorporation of H—methoxy—puromycin with time.
Incorporation of puromycin into trichloroacetic acid precip-
itable material was measured as described in HETHCDS.

Tritiated puromycin (specific activity 1000) was present in

1'

a concentration of l x 10 and there were 0.0 mg of

;g
ribosomes in the complete system (e-e). To the complete

system gougerotin was added to a final concentration of

3

A
-

p

.I‘
'1

l x 10- (0-0). he total volume of the assay was 50 micro-
liters. At the indicated times 5 microliter aliquots were
pipetted into two ml of 10% trichloroacetic acid, 15 mg of

bovine serum albumin was added and the incorporated H-

puromycin determined as described in AXALITICAL PROCEDURES.

'* ’~ ', a ‘ N
b; ‘urtL‘LJ - l

-. .. 7"" .‘ . , - . ‘ A
“JJLL UL“ 1;... kn JI-

-- .r.
‘1'"; 'v

4\

 

14—-

 

 

10

 

 

 

 

 

 

 

 

 

 

 

 

 

TIM 3

(MI N JJ.‘ _. S )

3“
indeed inhibit the formation of peptidyl-puromycin, as approximately
one half as much tritium labeled puromycin was incorporated into
trichloroacetic acid insoluble material when l x lO-zfl’gougerotin

was present as was incorporated in the absence of gougerotin.

DISCUSSION

The study of the mechanism of action of antibiotics which specifically
inhibit protein synthesis has been very valuable in the study of the
various steps in protein synthesis. This study on the mode of action
of gougerotin indicates that it may also be of considerable value
in studies of this type.

Since GTP dependent release did not appear to be the primary
site of action of gougerotin (release was inhibited up to 50-60% at
high antibiotic concentration, however at lower concentrations the incorp-
oration reaction was inhibited 70-801 and release was only inhibited
10-20%), and the inhibition was not reversed by the concentrations of
GTP and soluble enzymes tested, the step at which gougerotin acts primarily
is at or before the peptide bond forming reaction. It has been shown pre-
viously that gougerotin does not effect binding of aminoacyl-SRNA to
the ribosome-messenger RNA complex in an E‘Hggli system (19) and that
gougerotin does not inhibit the activation of amino acids in the rat
liver system (17). These observations along with the findings that
gougerotin inhibits the reaction of puromycin with peptidyl-sRNA on
ribosomes (a system where it is thought that only the peptide bond
forming step takes place) lead to the conclusion that gougerotin is
a.specific inhibitor of the peptide bond forming enzyme.

There are still several possibilities for the detailed mechanism
of action of gougerotin. The first of these is pertinent if one assumes
that gougerotin and puromycin do not displace each other once they have

bound. Then it can be proposed that gougerotin and puromycin compete

36
for the same site of the peptide synthetase molecule.

However recent reports (20,21) indicate that the above mechanism
may not be entirely correct. Using §&_ggli_ribosomes with polylysyl—
sRNA Goldberg and Mitsugi (20) measured the release of polylysine by puro-
mycin. They found that gougerotin did reduce the rate of puromycin
dependent release, but Lineweaver-Burk plots of their data indicated
that the inhibition was not purely competitive, but was of a mixed type
(41), that is both the maximum velocity and the affinity of the enzyme
for the substrate (puromycin) are altered. Coutsogeorgopoulos (21)
used an _E_I_._ 221.1; system with polyuridylic acid and measured the effect of
various inhibitors on the initial rate of phenylalanine incorporation.

His data also show mixed type inhibition for gougerotin on the reaction.
He also found that two other antibiotics which, like gougerotin

have cytosine as the nitrogen base, showed kinetics very similar to.
that of gougerotin.

Thus at least two more hypothetical models for the action of gougerotin
are possible. These are based on the fact that gougerotin shows mixed
competitive and non-competitive inhibition of peptide bond formation.

This is a two substrate reaction, and since the concentration of one

of the reactants was held constant throughout (bound peptidyl-sRNA).the
equations describing single substrate reactions are useful (#2). Thus

in view of the above mentioned kinetic studies it is possible that gougero-
tin interacts with the peptide synthetase molecule at some site unrela-

ted spacially to the catalytic site, and after binding the conformation

of the protein is changed in such a manner as to alter the maximum velocity

and the binding constant of the enzyme.

37

The third hypothetical model for the action of gougerotin
is as follows. Figure 7 shows a possible diagramatic representation of
peptide bond formation. A possible mechanism for gouerotin could then
be that it is an analog of one of the C's in the ACC nucleotide sequence
on the 3' end of the aminoaCyl-SRRA, and so it could possibly cause
partial hindrance to puromycin binding as well as being close enough to
the catalytic site to have some effect on the velocity of the catalysis.
Coutsogeorgopolos' data (21), which do not appear to support this hypo-
thesis, might be explained by the fact that gougerotin would be competing
with bound aminoacyl-SRNA and that he was actually measuring total amino-
acyl-533A concentration. de did, in fact, find that puromycin did not
show competitive kinetics with aminoacyl-SHEA as might be expected from
its accepted mode of action. These hypotheses should be viewed with ex-
treme caution, however, since they are based on kinetic studies of very
complex systems, and definitive conclusions should await further studies.

Also noted in these studies was the fact that puromycin de-
pendent release of polypeptides from ribosomes did not require the
addition of GTP. Similarly no GTP requirement was found for the inhibi-
tion of the puromycin dependent release by gougerotin. Studies on
the role of GTP in peptide bond synthesis in the reticulocyte system
have, to date, shown no strict stoichiometry'between GTP utilization and
peptide bonds formed (3%), however studies in an §&_gglisystem have
shown a relationship of one peptide bond formed for each GTP hydrolyzed
(43,Qb), The lack of a GTP requirement for the puromycin reaction
reported here may indicate that GTP does not participate in the peptide

bond forming step directly.

"

Fi~ure 7. ApOSSiole sclenatic representation of protein

‘I

ynthesis as adapted from Schweet (33) and-CoutSOgeorg-

C
He
0
(:1

000103 (21 . The upper circle represents the 005 subunit

’1
}.Jo
:‘f‘
(D
m
H:

or amioacyl-SRYA and peptidyl-SRXA,
designated "A" and "P" respectively, and the peptide
synthetase ("3"). he lower semicircle represents the #05
subunit with the messenger REA bound to it. To attempt has
been made to draw the ribosome, binding sites, and sRNA's
to scale. Protein synthesis can be broken down into he
following steps: activation of the amino acid by reaction
with a specific sRflA and concurrent conversion of one ATP
to an AIP (not shown on the diagram), binding of the amino-
acyl-SRNA to the ribosome messenger RNA complex, formation
of a peptide bond by peptide synthetase resulting in a
peptidyl-SRNA in the "A" position, and shifting of this
peptidyl-SRNA to the "P" position with the possible util-
ization of GTP. Special initiation and release mechanisms

are needed, but are not necessary for this discussion.

 

 

 

 

 

 

 

 

moAboC \

40 ‘
Thus gougerotin, as a specific inhibitor of the biosynthesis
of proteins, may be of considerable value for the study of Specific
stages in protein synthesis such as the attachment of aminoacyl-
sRNA to ribosomes and the release reaction, since it specifically
inhibits the peptide bond forming reaction as it occurs on the

ribosomal template.

10.

ll.

12.

13.

1h,

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