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216 BIOCHIMICA ET BIOPHYSICA ACTA BBA 96973 METABOLISM OF PUROMYCIN BY YEAST CELLS* ULRICH MELCHER ~" Department o[ Biochemistry and MSU/AEC Plant Research Laboratory, Michigan State Univer- sity, East Lansing, Mich. 48823 (U.SA.) (Received March 8th, 1971) SUMMARY Yeast cells, supplied with high concentrations of puromycin, formed acyl amino- acyl puromycins and aminoacyl puromycins. These compounds were identified by their behaviour in chromatography and electrophoresis, by the incorporation of radioactive amino acids, acetate, and formate, and by reaction with I-dimethyl- amino naphthalene-5-sulfonyl chloride. Methionyl puromycin, contrary to expecta- tion, was not a major product. Although formation of puromycin derivatives closely resembled inhibition of protein synthesis as to its dependence on puromycin concen- tration, chloramphenicol, cycloheximide, and anisomycin did not affect the formation of these compounds. INTRODUCTION High concentrations of puromycin have been used in vivo to obtain puromycin derivatives of the initial amino acid polymerized during protein synthesis in prokary- otes 1, chloroplasts 2, and hen's oviduct minces 3, What is known about the mechanism of puromycin action iustifies this approach. Puromycin reacts with ribosomal pep- tidyl tRNA to produce peptidyl puromycin 4. After a translocation step, the ribosomes may dissociate from the mRNA 5,6 or they may reinitiate synthesis of short chains 7,8. After dissociation, the ribosomes are available for reinitiation since, in the presence of puromycin, more N-terminal than C-terminal peptides are synthesized 9. Thus, at high enough concentrations of puromycin, only derivatives of the initial amino acid should be formed. I report here an attempt to apply this technique to yeast cells. Yeast was cho- sen since a variety of proteins are synthesized and since the N-terminal amino acids of total cellular proteins are known 1°. MATERIALS One pound cakes of Red Star brand pressed baker's yeast were stored at 4 °, never for more than a week, prior to use. Puromycin dihydrochloride (Nutritional Bio- * Taken from a dissertation submitted in partial fulfillment of the requirements for a Ph. D. Degree to the Department of 13iochemistry, Michigan State University. "* Present address: Institute for Molecular Biology, University of Aarhus, 8ooo Aarhus, Denmark. Abbreviation: DNS, i-dimethylamino naphthalene-5-sulfonyl. Biochim. Biophys. $cta, 246 (1971) 216-224

Metabolism of puromycin by yeast cells

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216 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96973

METABOLISM OF PUROMYCIN BY YEAST CELLS*

U L R I C H M E L C H E R ~"

Department o[ Biochemistry and MSU/AEC Plant Research Laboratory, Michigan State Univer- sity, East Lansing, Mich. 48823 (U.SA.)

(Received March 8th, 1971)

SUMMARY

Yeast cells, supplied with high concentrations of puromycin, formed acyl amino- acyl puromycins and aminoacyl puromycins. These compounds were identified by their behaviour in chromatography and electrophoresis, by the incorporation of radioactive amino acids, acetate, and formate, and by reaction with I-dimethyl- amino naphthalene-5-sulfonyl chloride. Methionyl puromycin, contrary to expecta- tion, was not a major product. Although formation of puromycin derivatives closely resembled inhibition of protein synthesis as to its dependence on puromycin concen- tration, chloramphenicol, cycloheximide, and anisomycin did not affect the formation of these compounds.

INTRODUCTION

High concentrations of puromycin have been used in vivo to obtain puromycin derivatives of the initial amino acid polymerized during protein synthesis in prokary- otes 1, chloroplasts 2, and hen's oviduct minces 3, What is known about the mechanism of puromycin action iustifies this approach. Puromycin reacts with ribosomal pep- t idyl tRNA to produce peptidyl puromycin 4. After a translocation step, the ribosomes may dissociate from the mRNA 5,6 or they may reinitiate synthesis of short chains 7,8. After dissociation, the ribosomes are available for reinitiation since, in the presence of puromycin, more N-terminal than C-terminal peptides are synthesized 9. Thus, at high enough concentrations of puromycin, only derivatives of the initial amino acid should be formed.

I report here an at tempt to apply this technique to yeast cells. Yeast was cho- sen since a variety of proteins are synthesized and since the N-terminal amino acids of total cellular proteins are known 1°.

MATERIALS

One pound cakes of Red Star brand pressed baker's yeast were stored at 4 ° , never for more than a week, prior to use. Puromycin dihydrochloride (Nutritional Bio-

* Taken from a dissertat ion submi t ted in partial fulfillment of the requirements for a Ph. D. Degree to the Depar tmen t of 13iochemistry, Michigan State University.

"* Present address: Ins t i tu te for Molecular Biology, Universi ty of Aarhus, 8ooo Aarhus, Denmark.

Abbreviat ion: DNS, i -d imethylamino naphthalene-5-sulfonyl.

Biochim. Biophys. $cta, 246 (1971) 216-224

PUROMYCIN METABOLISM 217

chemical Co.) was dissolved in sterile water and neutralized with NaOH. Cyclohexi- mide (Nutritional Biochemicals Co.) and chloramphenicol (California Biochemical Co.) were dissolved in water. Anisomycin, a gift from Dr. N. Belcher (Pfizer and Co.) was dissolved in sterile water with the addition of HC1. N-Formyl methionyl Emethoxy- 8H]puromycin and E14Clglycyl glycyl puromycin, synthesized by the method of LEDER AND BURSZTYN 11, were a gift from Dr. W. H. Evins. Methanol, ethyl acetate, and pyridine were redistilled prior to use.

METHODS

Incubation o/yeast cells and isolation o/ puromycin derivatives Yeast (2.0 g) was pleincubated 5 rain at room temperature in IO ml 0.25 M

glucose, o.I M sodium citrate, pH 4.8, containing 4o~ug/ml chloramphenicol. 15/,I yeast suspension was added to 35/~1 incubation solution (puromycin, radioactive precursors, etc.). After incubation at 37 °, samples were chilled, diluted to 2 ml, made alkaline with NaOH and extracted 4 times with 2-3 ml ethyl acetate 11. The combina- tion of alkaline pH and ethyl acetate was sufficient to release most of the cell con- tents.

The concentrated ethyl acetate phases were chromatographed on thin layers of silica gel HF~54 using chloroform-95 % ethanol-aq.ammonia (4 ° : 58 : 2, by vol.) as solvent. Puromycin derivatives were eluted with ethyl acetate-methanol-water (I : I : I, by vol.). The gel eluates were electrophoresed in I.O M acetic acid adjusted to pH 1.8 with 9 ° % formic acid I on paper at 350 V for 2.5 h. The position of radio- active components was determined with a Packard radiochromatogram scanner. Radioactive bands were cut out for radioactivity determination or for elution and further analysis.

Steam distillation The distillation flask of the steam distillation apparatus contained the sample,

I ml I M H~P04, and 9 ml water. The alkaline trap flask contained IO ml o.oi M NaOH. During steam distillation, the volume in both flasks was maintained by intermittent heating. After 12o ml water were collected from the condenser, the contents of the alkaline trap flask were dried and counted. The mean value for per- cent recovery of known amounts of sodium E14C]acetate was 94.8 with a mean devia- tion of 7 .1%.

Reaction with DNS-CI and separation o[ DNS amino acids Puromycin derivatives in 20/,1 o.I M NaHCO 3 and 30/,1 acetone were reacted

with 2-3 #1 20 mg/ml I-dimethylaminonaphthalene-5-sulfo~l chloride (DNS-C1) TM.

Puromycin derivatives were hydrolyzed either before or after reaction with DNS-C1 in I ml 6 M HC1 for lO-18 h at lO5 °. DNS amino acids were separated by two-dimen- sional thin-layer chromatography on silica gel H using benzene-pyridine-acetic acid ( 8 0 : 2 0 : 2 , by vol.) TM and chloroform-95 % ethanol-aq.ammonia ( 4 0 : 5 8 : 2 , by vol.) as solvents.

Biochim. Biophys. Acta, 246 (1971) 216-224

218 u. MELCHER

RESULTS

Isolation o] puromycin derivatives Puromycin derivatives (resulting from incubation of yeast cells with puromy-

cin and radioactive precursors) were separated from other labeled products by a three-step procedure. After incubation, the chilled, diluted reaction mixture was made alkaline and extracted with ethyl acetate. At alkaline pH, puromycin and its derivatives are extracted into ethyl acetate zl, while the radioactive precursors used were only poorly extracted. The extracts were chromatographed on thin layers of silica gel HF254. Puromycin and its derivatives had RF values between 0.65 and 0.75, while most interfering radioactive substances, including amino acids, had RF values lower than 0.5. Eluates from the thin-layer chromatoglams were electrophoresed on paper at pH 1.8 (ref. I). The radiochromatogram scan of an electropherogram of material labelled with a mixture of [14Clamino acids is shown in Fig. I. Aminoacyl puromycins were separated from N-blocked puromycin derivatives and to a lesser extent from puromycin. All three were well separated from the only major remain- ing radioactive contaminant, which barely moved from the origin. Little, if any, radioactivity was found in the amino acid region. Thus, the combination of ethyl acetate extraction, thin-layer chromatography, and papei electrophoresis separated puromycin derivatives from other radioactive materials. This procedure was used in all experiments described here.

4OO I

.~ 300 A 4 ~ 2 i

2oo!

IO0

O ( - ) t) 6 4 2 0 2(+)

CM FROM ORIGIN

Fig. I. Electrophoresis of amino acids, puromycin, and puromycin derivatives at pH 1.8. Yeast cells were incubated with i.o/zC 14C-labeled amino acid mixture (I mC]mg) and 75 mM puromy- cin for 6 h. The electrophoretogram was scanned for radioactivity at o. 5 cm/min, 30 sec time constant, and 5 mm slit width. Standard compounds: I, N-formyl methionyl puromycin; 2, methionine, glutamic acid; 3, glycyl glycyl puromycin; 4, puromycin.

Identi/ication o/acyl aminoacyl puromycins As shown in Fig. I, the above procedure yielded two labeled electrophoretic

bands. The first coelectrophoresed with N-formyl methionyl puromycin and the sec- ond with a dipeptidyl puromycin. This behavior suggested that the bands contained acyl aminoacyl puromycins and aminoacyl puromycins, respectively. The suggestion was confirmed by the following observations.

The compounds coeleetrophoresing with N-formyl methionyl puromycin con- tained amino acid iesidues. When yeast cells were supplied with a mixture of uni- formly 14C-labeled amino acids (all protein amino acids except methionine, cysteine, glutamine, asparagine, and tryptophan) during incubation with puromycin, label appeared in the product (Fig. i and Table I, line I). Such label could be recovered in DNS amino acids after acid hydrolysis and reaction with DNS-C1 (Table II, column I).

Biochim. Biophys. Acta, 246 (1971) 216-224

PUROMYCIN METABOLISM 219

TABLE I

C O U N T S / M I N I N C O R P O R A T E D I N T O Y E A S T A C Y L A M I N O A C Y L P U R O M Y C I N S F R O M D I F F E R E N T R A D I O ~

A C T I V ~ P R E C U R S O R S

Amounts and specific radioactivities of precursors were: [laC]amino acid mixture (Volk), I.O/,C (i mC/mg); sodium [14C]formate, 5.o/~C (4.o mC/mmole); sodium [2-14CJacetate, o,5/~C (o.59 mC/mmole); L-EMe-SH]methionine, 5.o#C (i.omC/2.2 rag). Concentrations of puromyciu and NaC1 were 5 ° mM, except with amino acid mixture, where they were 75 mM. Incubation was for 3 h.

Treatment Counts/rain

Amino Formate A celate Methionine acids

Puromycin lO58 658 274 300 NaCI 56 2 6 3 ° NaC1, 0.34 mM cycloheximide 49 - - - - - - Puromycin, boiled yeast* 72 61 34 80

* Yeast suspension was placed 5 min in a boiling water bath prior to incubation.

TABLE I1

D I S T R I B U T I O N O F R A D I O A C T I V I T Y A M O N G D N S A M I N O A C I D S F R O M [ 1 4 C ] A M I N O A C I D L A B E L E D

P U R O M Y C I N D E R I V A T I V E S

Puromycin derivatives were obtained by incubating yeast cells with 2.o/2C E14C~amino acid mixture (I mC/mg) and 75 mM puromycin for 3 h. DNS amino acids were obtained after hydro- lysis of the derivatives and reaction with DNS-CI as described in methods. Background counts/ min have been subtracted. Standard counting error = 4-5 counts/rain

D N S amino acid Counts/rain in D N S amino acids/rom:

A cyl aminoacyl A minoacyl puromycin puromycin

Isoleucine 32.2 64.o Leucine 36. i 75.7 Valine 31.3 36.3 Proline 1.4 24.4 Phenylalanine 24 . 8 Alanine 6. 3 114. I Lysine, tyrosine 20. 7 27.0 Glycine, tryptophan 21.5 lO2.8 Serine, threonine i7.I 19.5 Glutamic Acid 42.3 15. 5 Aspartic Acid 22.o - -

T h e a m i n o ac id res idues of these c o m p o u n d s h a d b locked ~ -amino groups .

A c y l a m i n o a c y l p u r o m y c i n s , l abe led w i t h a [14Clamino acid m i x t u r e , were i so la ted .

One ha l f was r e a c t e d w i t h DNS-C1 a n d t h e n h y d r o l y z e d , whi le t h e o t h e r was h y d r o -

l yzed before r eac t i on w i t h DNS-C1. B o t h samples were t h e n a n a l y z e d for r a d i o a c 0 v e D N S a m i n o acids. Of t h e D N S a m i n o ac id r a d i o a c t i v i t y o b t a i n e d a f t e r hyd ro lys i s

a n d d a n s y l a t i o n , o n l y 3 % cou ld be found in D N S a m i n o acids if d a n s y l a t i o n pre- c eded hydro lys i s . There fo re , mos t , if n o t all , of t h e a m i n o ac id res idues h a d b locked

a m i n o groups . T h e b lock ing g r o u p was an acy l res idue. R a d i o a c t i v i t y f r o m [J4C~formate a n d

[saC~acetate was i n c o r p o r a t e d in to t h e e l e c t r o p h o l e t i c b a n d (Table I, l ine I) . S o m e

Biochim. Biophys. Acta, 246 (197 I) 216-224

2 2 0 u . MELCHER

of the radioactivity was present in N-acyl residues. This could be determined by steam distillation ~4 since the radioactive acids liberated after hydrolysis are volatile. Table I I I shows that [~4Clacetate-derived radioactivity was not steam distillable before hydrolysis, but was almost completely distillable after hydrolysis. Radio- activity from [14Clformate was 40-5 ° % steam distillable with or without prior hydrolysis. N-Formyl groups are sufficiently acid labile 15,1e that they were readily hydrolyzed during steam distillation.

T A B L E I I I

STEAM DISTILLATION OF RADIOACTIVITY IN ACYL AMINOACYL PUROMYCINS

Amounts and specific radioactivities of precursors used to label acyl amino acyl puromycin during a 3-h incubat ion with 5 ° mM puromycin were as in Table I. Eluates from electrophoretograms were divided into three portions. One was counted directly; another was s team distilled; a third was concentrated and hydrolyzed wi th 6 N H3PO 4 at lO5 ° for 12 h before s team distillation. Background counts/rain have been subt rac ted

[I4C]A cetate [. a*C] Formate

Counts~rain % Counts~rain %

Total 2o6 ioo 5oo ioo Steam distilled io 5 2o6 41 Steam distilled after hydrolysis 2Ol 97 231 46

The carboxyl group of the amino acid residues in this band must also have been blocked, since a free carboxyl would have hindered extraction into ethyl ace- tate. The carboxyl was most probably in peptide linkage to puromycin. The com- pounds were not formed when puromycin was omitted (Table I, lines I and 2). Incorporation of [14Clamino acids did not occui when protein synthesis was inhibited with cycloheximide, rather than puromycin (Table I, lines I and 3). Further, the band cochromatographed on silica gel and coelectrophoresed at pH 1.8 and 5.4 with N-formyl methionyl puromycin. Therefore, the most probable structure for com- pounds in the fkst band was: N-acyl aminoacyl puromycin.

Identification o] aminoacyl puromycins Similar arguments identify the second band as aminoacyl puromycin. The

compounds in this band contained amino acid residues. Fig. I and Table IV, line I show that the compounds were labeled by incubating yeast cells and puromycin with a [14C]amino acid mixture. Table II, column 2, shows that such radioactivity was recovered in DNS amino acids after hydrolysis and reaction with DNS-C1.

T A B L E IV C O U N T S / M I N INCORPORATED INTO AMINOACYL PUROMYCIN FROM D I F F E R E N T RADIOACTIVE PRE-

CURSORS*

Treatment A mino Formate Acetate Methionine acids

Puromycin 718 65 29 18 i NaC1 32 3 8 58 NaC1, 0.34 mM cycloheximide 82 - - - - Puromycin, boiled yeast lO 4 27 17 83

* Compare Table I.

Biochim. Biophys. Acta, 246 (1971) 216-224

PUROMYCIN METABOLISM 221

The amino acid residues had free amino groups. First, radioactivity from E14C]acetate or E14CJformate was not incorporated into these compounds (Table IV, columns 2 and 3). Second, of the DNS amino acid radioactivity obtained after hydrol- ysis and dansylation, 87 % could be found in DNS amino acids if dansylation pre- ceded hydrolysis.

Since the compounds did not behave like amino acids in ethyl acetate extrac- tion or thin-layer chromatography, their carboxyl must have been blocked. It was probably linked to puromycin. Filst, incorporation of E14C]amino acids was depen- dent on the addition of puromycin and was not obtained when protein synthesis was inhibited with cycloheximide, rather than puromycin (Table IV, lines I, 2 and 3). Second, the band cochromatographed and coelectropboresed with a dipeptidyl puro- mycin. The most probable structure for these compounds was therefore: aminoacyl puromycin.

Formation o/ puromycin derivatives by yeast cells Since radioactive precursors labeled puromycin derivatives, the appearance of

radioactivity in the products was used as a measure of their formation. The results show that the rate of incorporation of [14C]amino acids into both compounds (Fig. 2A) and the incorporation of [14Clformate into acyl aminoacyl puormycins (Fig 2B) decreased only slightly during three hours of incubation. E14C]Acetate incorporation (Fig. 2B) declined more rapidly, perhaps due to more rapid metabolism.

3 0 0

2oo

0 2. .'o ,. ' ,;o MINUTES OF INCUBATION

8 0 0

I ZOO

0

/Y I ! I I

0 BO 180

MINUTE8 OF INCUBATION

Fig. 2. Kinetics of incorporation of radioactive precursors into puromycin derivatives. A. Acyl aminoacyl puromycin ( 0 ) and aminoacyl puromycin (O) were obtained by incubation of yeast with o. 5/ ,C reconsti tuted 14C-labeled protein hydrolyzate (z mC/mg) and 50 mM puromycin. B. Acyl aminoacyl puromycin was obtained by incubation of yeast with 2.0/,C sodiumLl~C]formate (4.0 mC/mmole) ( 0 ) or 0. 5 t,C sodium [2-14C~acetate (5o/~C/7.i mg) (O) and 5 ° mM puromycin.

The above results demonstrate that puromycin was converted to acyl amino- acyl puromycins and aminoacyl puromycins during incubation with yeast cells. Since this reaction can be abolished by boiling yeast cells prior to incubation (Tables I and IV, lines I and 4), the conversion must be catalyzed by live yeast cells.

Relation to protein synthesis initiation If these products of puromycin metabolism are derivatives of the initial amino

acid used in protein synthesis, then they must be formyl methionyl puromycin and/or

Biochim. Biophys. ~lena, 246 ( i97i) 216--224

222 U. MELCHER

methionyl puromycin. Formyl methionine is the initiator in mitochondria 17 while methionine is the initiator in cytoplasm 18,19.

IaH]Methionine was incorporated into acyl aminoacyl puromycin (Table I, column 4) and into aminoacyl puromycin (Table IV, column 4), But, as was shown above (Table II), methionine was not the only amino acid incorporated. Further, since a higher input of E3Hlmethionine than of IlaClamino acid radioactivity had to be used to obtain a similar incorporation, the methionine derivatives were not the major products of puromycin metabolism. Therefore, puromycin metabolism ob- served here was unrelated to initiation of protein synthesis as currently understood.

Relation to protein synthesis If puromycin metabolism is at all related to protein synthesis, then the follow-

ing conditions should be met. First, the puromycin concentration dependence of inhibition of protein synthesis must be the same as that for formation of the deriva- tives. Second, formation of these compounds must be sensitive to inhibitors of ribo- some catalyzed peptide bond folmation.

Fig. 3 shows that synthesis of both acyl aminoacyl puromycin and aminoacyl puromycin (as measured by ElaC~amino acid incorporation) was maximal at 50 mM puromycin. Fig. 4 shows the puromycin concentration dependence of protein syn- thesis inhibition. Since the concentration dependences were similar, the compounds may have been formed by the action of puromycin on protein synthesis.

300

~ 200

~ iO0

P, 0

/ ' ' ; ' 'o 20 4 60 8

mM PUROMYCIN

IOO

N 4o

2o

/ , O0 20 410

mM PUROMYCIN

O J D ~ O

~o ~0

Fig. 3. P u r o m y c i n concen t ra t ion dependence of incorpora t ion of E14C]amino acids into p u r o m y - c in der ivat ives . Compare Fig. 2A. All i ncuba t ions were for 3 h wi th 75 m M NaC1.

Fig. 4- Inh ib i t ion by p u r o m y c i n of El~C]amino acid incorpora t ion in to macromolecu les by yea s t cells. All i ncuba t ions were for 3 h wi th 75 mlV[ NaC1. R esu l t s are t he average of two expe r imen t s wi th 0. 5/~C and one w i t h 2.0 pC [14Clamino acid m i x t u r e (I mC/mg) . Macromolecules were pre- c ip i ta ted f rom a q u e o u s phases af ter e thy l ace ta te ex t r ac t ion by add i t ion of 2.0 ml 3 ° % (w/w) t r ichloroacet ic acid con ta in ing o. i % (w/w) casein hyd ro lyza t e and collected on m e m b r a n e filters.

Synthesis of these compounds was, however, not sensitive to inhibitors of pep- tide bond formation. Chloramphenicol inhibits peptide bond formation by bacterial type ribosomes ~°, but is ineffective on the same reaction of eukaryotic ribosomes 21. At a concentration of I mg/ml, chloramphenicol did not affect incorporation of any of the precursors tested.

Biochim. Biophys, Acta, 246 (1971) 216-224

PUROMYCIN METABOLISM 223

Cycloheximide inhibits the translocation of peptidyl tRNA in eukaryotes 22,2s. It may, thus, inhibit peptide bond formation indirectly. Anisomycin inhibits the fragment reaction between acyl aminoacyl hexanucleotide and puromycin catalyzed by yeast ribosomes 21. Neither of these inhibitors at concentrations that inhibited protein synthesis by more than 9 ° % (0.34 mM cycloheximide and I mM anisomy- cin) was able to reduce formation of puromycin derivatives (as measured by labeling with ~l~Clformate, acetate, or amino acids) by more than IO %.

DISCUSSION

The present results demonstrate that yeast cells convert puromycin into acyl aminoacyl puromycins and aminoacyl puromycins. The formation of other com- pounds, such as acyl puromycins (lacking the amino acid residue), and N-blocked aminoacyl puromycins with blocking groups othei than acetate or formate, was, however, not ruled out.

Although the dose response curves for formation of these compounds and for inhibition of protein synthesis were similar, the lack of sensitivity to other inhibitors argues that these compounds were not formed as a consequence of puromycin ac- tion on protein synthesis the inability to detect the expected methionyl puromycin formation could be due to several causes. First, tile quantity of methionyl puromycin formed through protein synthesis may have been small compared with that of puro- mycin derivatives not formed by ribosomes. Second, ribosomal puromycin deriva- tives may have been large peptidyl puromycins and consequently not extracted with ethyl acetate. Third, a rapid enzymatic hydrolysis of methionyl puromycins when compared to othei aminoacyl puromycins is possible.

Other actions of puromycin have been reported 24,~5, but it is unclear how these actions could give rise to the observed compounds. The data reported here thus pro- vide evidence of a novel reaction of puromycin in eukaryotic cells.

The results suggest that caution must be exercised when applying the method of BACHMEYER AND KREIL 1 to eukaryotes. Specifically, the non-ribosomal formation of acyl aminoacyl puromycins in the presence of high concentrations of puromycin, observed here with yeast cells, may be responsible for the formation of N-acetyl glycyl puromycin by hen's oviduct minces reported by NARITA et al. 3.

ACKNOWLEDGEMENTS

The author thanks Dr. N. Belcher for the gift of anisomycin, Dr. W. H. Evins for the gift of puromycin standards, and Dr. J. E. Varner for patient guidance and encouragement. This work was supported by the U.S. Atomic Energy Commission under contract No. AT (II-I) 1338.

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I H. BACHMEYER AND G. KREIL, Biochim. Biophys. dora, 169 (1968) 95. 2 H. ]3ACHMEYER, Biochim. Biophys. dcta, 209 (I97 o) 584.

Biochim. Biophys. Acta, 246 (x97 I) 216-224

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4 D. NATHANS, Proc. Natl. Aead. Sci. U.S., 51 (1964) 585. 5 A. R. WILLIAMSON AND R. SCHWEET, Nature, 202 (1964) 435. 6 B. COLOMBO, L. FELICETTI AND C. BAGLIONI, Biochem. Biophys. Res. Commun., 18 (1965) 389. 7 A. R. WILLIAMSON AND R. SCHWEET, J . Mol. Biol., I I (1965) 358, 8 J. D. SMITH, R. i~. TRAUT, C'. M. BLACKBURN AND R. E. MONRO, J. Mol. Biol., 13 (1965) 617. 9 D. NATHANS, J. Mol. Biol., 13 (1965) 521.

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Biochim. Biophys. Aeta, 246 (1971) 216-224