Partial characterization of the protein labeled in a cell-free system from Bacillus cereus 569

491

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95686

PARTIAL CHARACTERIZATION OF T H E P R O T E I N LABELED IN A C E L L - F R E E SYSTEM FROM B A C I L L U S C E R E U S 569

A L A N G. A T H E R L Y ° AND J. IMSANDE**

Department o] Biology, Western Reserve University, Cleveland, Ohio (U.S.A.) (Received March 7th, 1967)

SUMMARY

A partial characterization was made of the protein formed in a cell-free amino acid incorporation system from Bacillus cereus 569/H which contained only endogenous messenger. More than 50 % of the labeled product is released from the ribosomal complex during synthesis. The molecular weights of the released and bound fractions were found to be approximately 5000 and 30 ooo, respectively. Analysis of the two fractions b y carboxypeptidase digestion showed that approximately 96 % of the chains in each class contain only one labeled amino acid residue, that residue being the carboxyl terminal residue, whereas the remaining 4 % of the chains in each class contain an average of 2o (with a maximum of 40) labeled amino acid residues at the carboxyl end. Thus, it appears that the maiority of nascent chains were either released prior to completion or, if not released, unable to continue sustained growth. Variations in the concentration of K+ or Mgz+ during cell-free synthesis did not affect the rate or extent of abortive release from the ribosomes. Calculations show that at least 75 % of the ribosomes isolated from the intact organism are engaged in amino acid incorporation in the absence of added mRNA.

INTRODUCTION

Numerous attempts have been made to obtain synthesis of specific proteins in cell-free systems. To date, the polypeptide chains of hemoglobin have been formed in a cell-free rabbit reticulocyte system 1 and the coat proteins of several bacterial viruses have been synthesized de novo in a cell-free system from Escherichia coli upon the addition of the corresponding viral RNA ~-5. However, it has not been demonstrated as yet that bacterial cell-free systems containing only endogenous mRNA are capable either of de novo synthesis of complete polypeptide chains, or of * Dr. A t h e r l y ' s c u r r e n t a d d r e s s is: I n s t i t u t e of Molecular Biology, U n i v e r s i t y of Oregon, E u g e n e , Oregon. *° R e q u e s t s for r e p r i n t s should be directed to this a u t h o r .

Biochim. Biophys. Acta, 145 (1967) 491-5Ol

492

A. G. ATHERLY, J. IMSANDE

the completion of nascent chains. Experiments have shown that the growing (nascent) peptide chain remains attached to the ribosomal complex throughout its synthesis in vivo s. Yet in the absence of exogenous messenger, approx. 50 ~o of the amino acid residues incorporated into protein in the cell-~ree system are released from the ribosomal complex 7. These two facts can be reconciled if, in the cell-free system, one of the following possibilities, or various combinations thereof, occur: (I) chains are formed de novo and released; (2) nascent chains are completed but not all are released; (3) some nascent chains are completed and released while other nascent chains are only partially completed (elongated) and therefore not released; (4) some nascent chains are elongated and abortively released while other nascent chains are elongated but not released. This commtmication describes the partial characterization of the product formed in a cell-free system from Bacillus cereus 569/H containing only endogenous messenger. I t was found that the soluble protein released from the ribosomes is a heterogenous class of low molecular weight (5 ooo to IO ooo) polypeptides, the majority of which have accepted only one E14Clamino acid residue during cell-free synthesis. Thus, it appears that nascent chains are released prior to completion. Furthermore, it was shown that small variations in the concentration of K + or Mg 2+ do not affect the rate or extent of abortive release of the soluble product from the ribosomes used in this study. Calculations show that at least 75 % of the ribosomes isolated from the intact organism are engaged in amino acid incorporation in the absence of added mRNA.

MATERIALS AND METHODS

The preparation of ribosomes and other components of the cell-free system has been described previously 7. t R N A from B. cereus 569/H was prepared by phenol extraction s of the 19o ooo ×g supernatant fluid. E. coli B t R N A was obtained from General Biochemicals Inc. Amino acyl t R N A was prepared from a reconstituted ElaClprotein hydrolysate (Schwarz Bioresearch Co.) containing 13 labeled amino acids. NS-formyl tetrahydrofolic acid (General Biochemicals) was converted to Nl°-formyl tetrahydrofolic acid and the latter was used to prepare N-formyl-amino acyl t R N A (refs. 9, IO). Amino acyl t R N A contained all of the usual 20 amino acids. The specific activity was calculated to be I . I × lO4 counts/min per m/~mole tRNA.

Conditions o/incubation

The complete system for amino acid incorporation contained, in I . o m l : IO/~inoles Tris (pH 7.2); I / , m o l e GTP; 5/,moles phosphoenolpyruvate; 8o/,moles KC1; I 4 # m o l e s of Mg2+; IOO/~g pyruvate kinase; 1.7mg E14C3-1abeled N-formylamino acyl t R N A and 5 mg of ribosomes. Reaction mixtures were incubated 20 min at 360 and immediately centrifuged at 44 ooo rev./min (Spinco rotor z~ 50) for one hour. Except in the experiments shown in Figs. I and 2, the supernatant fluid was adjusted to p H 12 with I M NaOH, heated at 5 °0 for 40 min, and dialyzed for 2 h against 0.05 M NaHCO3 (pH 8.1) at 4 °. The dialyzed preparation was incubated with DFP-treated carboxypeptidases A and B (Worthington Biochemical CorporaBiochim. Biophys. Actor, 145 (1967) 491-5Ol

CHARACTERIZATION OF PROTEIN SYNTHESIZED , n w t r o

493

tion), as described in the figure legends. Aliquots were removed from the incubation mixture, combined with an equal volume of IO % trichloroacetic acid, and allowed to stand several hours at o ° before centrifugation. Ninhydrin analysis was performed on the supernatant fluid n and the precipitate was washed with 35 to 4o ml of 5 O,o/ trichloroacetic acid over a Millipore filter. Radioactivity was assayed in a Tricarb liquid scintillation spectrometer as previously describedL

Fluorodinitrobenzene treatment After dialysis, 3oo to 4oo ffl (o.3 to o.5 mg protein) of the 19o ooo ×g supernatant fluid was lyophilized to dryness in a I5-ml screw cap vial and subsequently dissolved in 200 #1 of IO °/o NaHCO 3. 300 ffl of IO mg/ml fluorodinitrobenzene (Sigma Chemical Co.) in 95 % ethanol were added and the vial incubated 3 h at 36°. The product was precipitated with trichloroacetic acid and washed 3 or 4 times with io ml of cold 5 % trichloroacetic acid and twice with 3 ml of peroxide-free ether. The dry pellet was then dissolved in 2 to 3 ml of 6 M HC1, sealed in a tube under vacuum and hydrolyzed at i o 5 ° ~ 2 ° for 16 h. The HC1 was diluted three-fold and extracted 3 times with 3 ml of ether. After removal of the ether, the product was dissolved in 2 ml of 1 % NaHCO 3 and extracted 3 times with 5 to IO ml of ether to remove the dinitrobenzene formed during hydrolysis. Ether was removed b y a current of air and the absorption spectrum determined with a Zeiss spectrophotometer.

RESULTS

Effect o[ Mg 2+ and K+ on amino acid incorporation and chain termination The kinetics of amino acid incorporation into protein in tile B. cereus cell-free system is shown in Fig. I. I t should be noted that about 50 % of the radioactivity incorporated is released from the ribosomal complex (Fig. i, curve b and Figs. 2A

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Fig. i. K i n e t i c s of i n c o r p o r a t i o n a n d t h e r a t e of r e l e a s e of p o l y p e p t i d e s f r o m t h e r i b o s o m a l c o m p l e x d u r i n g s y n t h e s i s . C o n d i t i o n s for i n c u b a t i o n w e r e as d e s c r i b e d i n MATERIALS AND METHODS. B. cereus N - f o r m y l - [ 1 4 C ] a m i n o a c y l - t R N A w a s used. T h e r e a c t i o n w a s s t o p p e d b y a d d i t i o n of N a N 3 to 0.30 M. T o t a l vol. w a s 0.5o ml. C u r v e a, t o t a l r a d i o a c t i v i t y i n c o r p o r a t e d i n t o h o t t r i c h l o r o a c e t i c a c i d i n s o l u b l e m a t e r i a l ( O - Q ) ; c u r v e b, r a d i o a c t i v e p r o t e i n r e l e a s e d f r o m t h e r i b o s o m e s d u r i n g s y n t h e s i s ( x - - - x ).

Biochim. Biophys. Acta, 145 (1967) 4 9 I - 5 O I

A. G. ATHERLY, J. IMSANDE

494

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Fig. 2. E f f e c t of Mg z+ a n d K + c o n c e n t r a t i o n on t h e r e l e a s e of p o l y p e p t i d e s f r o m t h e r i b o s o m a l c o m p l e x d u r i n g s y n t h e s i s . E x c e p t for t h e v a r i a t i o n s i n i oni c c o n c e n t r a t i o n s n o t e d a b o v e , c o n d i t i o n s for i n c u b a t i o n w e r e as d e s c r i b e d i n MATERIALS AND METHODS. T o t a l vol. w a s 0.5 ° ml. T h e r e a c t i o n w a s s t o p p e d b y a d d i t i o n of N a N 3 to 0.3o M. Mg 2+ a d d e d w i t h t h e r i b o s o m e s is inc l u d e d in t h e o r d i n a t e of t h e g r a p h A; h o w e v e r , in g r a p h B t h e c o n c e n t r a t i o n of I(+ s h o w n doe s n o t i n c l u d e t h e s t a n d a r d a m o u n t of NH4+ a d d e d .

and B). The effect of variations in the concentration of Mg 2+ or K+ on the release of soluble radioactive protein from the ribosomes is shown in Fig. 2. It can be seen that maximum incorporation occurs at o.o15 M Mg2÷; however, the percentage of newly synthesized protein released from the ribosomal complex is not influenced by the variation in Mg2+ concentration. Essentially identical results are obtained in the K + concentration range 0.02 to o.14 M (Fig. 2B). Thus small variations in either Mg2+ or K + do not cause abortive release of the nascent chains during synthesis.

Molecular weight determination o/the product/ormed in the cell-/ree system In order to characterize the protein-like product formed in the cell-free system its molecular weight was estimated by: (i) molecular seive column chromatography in 6 M urea, and (2) amino-terminal analysis. Prior to chromatography on Sephadex G-I5O , the 'soluble' [14C]protein (i.e., labeled chains released from the ribosomes during synthesis) was incubated at pH 12 at 5°o for 4 ° rain to cleave all peptidylt R N A or amino acyl-tRNA bonds. To prevent aggregation, Sephadex column chromatography was performed in 6 M urea and o.ooi M EDTA (pH 7.3). A molecular seive column profile of the product obtained b y this treatment is shown in Fig. 3. It can be seen that the greater part (7° to 80 %) of the trichloroacetic acid precipitable radioactive product elutes at a position indicating an average molecular weight of approx. IO ooo, while a smaller fraction (20 to 3° °/o) has an apparent molecular weight greater than IOO ooo. In the absence of urea, more than 9° % of the material appears as high molecular weight proteinL These data suggest that the higher molecular weight fraction might be an aggregate of low molecular weight polypeptide chains. Since about 20 to 50 % of the amino acids incorporated by the cell-free system Biochim. Biophys. Acta, 145 (1967) 491-5Ol

CHARACTERIZATION OF PROTEIN SYNTHESIZED *n v~tro

495

are not released from the ribosome during synthesis, an effort was made to separate this material from the ribosomes and to analyze it in the manner described for the soluble (released) fraction. The bound [14Clpolypeptides were released from the ribosomes (io mg/ml) incubation at p H i i for I h at 37 °. Subsequently the ribosomes and ribosomal sub-units were removed by centrifugation at 19o ooo ×g for 2 h. By this treatment, 80 to 9 ° % of the bound [14Clprotein, and a considerable amount of unlabeled bound protein, was released from the ribosomes. In contrast to the released [14Clpolypeptides which appear to have a molecular weight of less than IO ooo, the bound protein has an apparent molecular weight of 25 ooo to 35 ooo (Fig. 3).

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Fig. 3. Gel-filtration profiles of t h e released and b o u n d [14C]protein o b t a i n e d d u r i n g cell-free synthesis. Conditions for i n c u b a t i o n were described in MATERIALS AND METHODS. 2-ml p o r t i o n s of each of the t r e a t e d s u p e r n a t a n t fluids (see t e x t ) were p a s s e d t h r o u g h a 1.5 cm x 7 ° cm c o l u m n of S e p h a d e x G - i 5 o (Pharmacia) at a flow r a t e of 2 m l / c m ~ per ix. 5-ml fractions were collected, carrier a l b u m i n (500/,g) w a s added to each, and the p r o t e i n w a s precipitated w i t h 5 % trichloroacetic acid. The precipitate was collected on a Millipore filter, washed, and assayed for radioa c t i v i t y in a liquid scintillation s p e c t r o m e t e r . T h e b r a c k e t s indicate the position where blue d e x t r a n (A), a l b u m i n (B), l y s o z y m e (C), and t r y p t o p h a n (D) were eluted ill relation to t h e trichloroacetic acid insoluble radioactivity. The molecular weights of the m a r k e r s are a p p r o x . 2 ooo ooo, 60 ooo, 13 ooo and 204, respectively.

The average molecular weight of all protein (i.e., labeled and unlabeled) released from ribosomes during cell-free synthesis was determined b y amino-terminal analysis. Assuming an 80 % recovery of the DNP-amino acid after hydrolysis 12, a number-average molecular weight of 4800 was established for the released protein by this procedure (Table I). These data, combined with those from molecular seive column chromatography, strongly suggest that tile soluble product of cell-free protein synthesis is a heterogeneous group of polypeptides, the average molecular weight of which is approx. 5000.

Estimation o] the number o/ [14C]amino acids incorporated per chain In an effort to establish the number of p~Clamino acid residues incorporated per nascent polypeptide chain, the rate of liberation of a~C from the carboxyl termini of the soluble protein b y carboxypeptidases A and B was examined. As shown in Fig. 4 A, the carboxypeptidase treatment rapidly liberates the greater part of the radioactivity from the labeled trichloroacetic acid-precipitable proteins. The rate of Biochim. Biophys. Acta, 145 (1967) 491-5Ol

490

A.G. ATFIERLY, J. IMSANDE

TABLE I MOLECULAR WEIGHT OF RELEASED PROTEIN BY AMINO-TERMINAL ANALYSIS Conditions for these determinations are described under Materials and Methods.

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Fig. 4 A. Rate of liberation of radioactivity from released protein by carboxypeptidase A and B. Conditions for preparation of the 14C-soluble protein are described in MATERIALS AND METHODS. Carboxypeptidase digestion was conducted at p H 8.1, 37 °, as described in MATERIALS ANn METHODS. The molar ratios of carboxypeptidase A and B to soluble protein were I :IO and 1:2o, respectively. O - O , carboxypeptidase A and B; × - ×, no carboxypeptidase. 4 B. Rate of liberation of amino acid residues from released protein by carboxypeptidase A and B. Conditions for incubation and analysis are described above and in MATERIALS AND METHODS. O - - O , carboxypeptidase A and B; × - × , no carboxypeptidase.

r e l e a s e of t o t a l (i.e., l a b e l e d a n d n o n - l a b e l e d ) a m i n o a c i d s f r o m t h e p r o t e i n , e x p r e s s e d as l e u c i n e e q u i v a l e n t s , is d e p i c t e d i n Fig. 4 B. S i n c e t h e a m o u n t of p r o t e i n u s e d i n e a c h e x p e r i m e n t h a d b e e n d e t e r m i n e d b y a s t a n d a r d p r o c e d u r e 13 a n d s i n c e t h e a v e r a g e m o l e c u l a r w e i g h t of t h e p o l y p e p t i d e c h a i n s h a d b e e n e s t a b l i s h e d b y free a m i n o t e r m i n a l a n a l y s i s ( a p p r o x . 5000) t h e n u m b e r of p r o t e i n m o l e c u l e s (i.e., t h e n u m b e r of carboxyl-terminal end groups) available for carboxypeptidase digestion can be c a l c u l a t e d . S i n c e t h e r a t e of r e l e a s e of t o t a l l e u c i n e e q u i v a l e n t s is k n o w n (Fig. 4 B ) , it is p o s s i b l e t o c a l c u l a t e t h e l e u c i n e e q u i v a l e n t s r e l e a s e d p e r p o l y p e p t i d e c h a i n p e r h o u r . C o n s e q u e n t l y , t h e r a t e of r e l e a s e of r a d i o a c t i v i t y f r o m t h e l a b e l e d p o l y p e p t i d e c h a i n s (Fig. 4) c a n b e e x p r e s s e d as l e u c i n e e q u i v a l e n t s r e m o v e d p e r c h a i n (Fig. 5). F r o m t h e d a t a p r e s e n t e d i n F i g . 5 i t c a n b e s e e n t h a t a b o u t 58 % of t h e r a d i o a c t i v e amino acids incorporated into the released protein fraction are carboxyl terminal

Biochim. Biophys. Acta, 145 (1967) 491-5Ol

497

CHARACTERIZATION OF PROTEIN SYNTHESIZED , n w t r o

amino acids whereas the remainder of the labeled amino acids extend inward as far as 4 ° residues from the carboxyl terminus. Thus it appears that approx. 96 % of the released nascent chains have accepted only one labeled amino acid, that amino acid representing the carboxyl terminal residue, whereas approx. 4 % of the nascent chains have accepted an average of 20 labeled residues per chain [i.e., the percentage of the label incorporated that occurs as the C-terminal residue (58 %) is equal to the number of chains (96 ) that contain only one residue per chain divided by the sum of the labeled interior residues (4 chains at 20 residues per chain) and C4erminal residues (96 chains at I residue per chain)]. Similar results were obtained for the [~4C]protein that remained bound to the ribosome during amino acid incorporation (Figs. 5 and 6). ~100 t .

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Fig. 5. N u m b e r of labeled a m i n o acid r e s i d u e s i n c o r p o r a t e d p e r n a s c e n t chain. The curves depicted in this figure are a s u m m a r y of t h e d a t a s h o w n in Figs. 4 A, 4 B and 6. Fig. 6. R a t e of liberation of r a d i o a c t i v i t y f r o m b o u n d p r o t e i n b y c a r b o x y p e p t i d a s e A and ]3. P r e p a r a t i o n of t h e [l~C]protein is described in t h e t e x t . Conditions for i n c u b a t i o n and e n z y m e c o n c e n t r a t i o n s are described in Fig. 4 and MATERIALS AND METHODS.

It should be noted that the labeled material that was attacked slowly by carboxypeptidase was not rapidly degraded by leucine aminopeptidase. Therefore these labeled amino acid residues are not located at the amino termini of the chains. The possibility that the decreased rate of digestion by carboxypeptidase at extended times (see Figs. 4 A and B) may result from aggregation of the [14C]protein rather than extended runs of labeled amino acids in the labeled protein was considered. In an effort to investigate this possibility, [l~C]protein that had been subjected to carboxypeptidase digestion for 7 h was chromatographed on a Sephadex G-I5O column. The column elution profile (Fig. 7) shows that, although carboxypeptidase does release labeled amino acids from the low molecular weight species preferentially, some low molecular weight material still contains labeled amino acids. Thus the slow release of radioactivity by carboxypeptidase is not due solely to aggregation. However, the possibility that the slowly digestible, low molecular weight material represents a resistant core has not been ruled out.

Percentage o/ribosomes active in amino acid incorporation TlSSI~RES, SCHLESSINGER AND GROS14 reported that less than IO % of the Biochim. Biophys. Acta, 145 (1967) 491-5Ol

498

a . G . ATHERLY, J. IMSANDE

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isolated E. coli ribosomes are active in amino acid incorporation in the absence of added messenger. Later GILBERT15 calculated that I I to 13 o/~ of the E. coli ribosomes are engaged in polyphenylalanine synthesis in the presence of polyuridylic acid. Assuming that only one [~4Clpolypeptide is obtained from one ribosome, it is possible to calculate the percent of ribosomes engaged in amino acid incorporation from the data obtained b y the use of carboxypeptidase. The parameters required for this calculation are: (I) the rate at which amino acids are liberated by the carboxypeptidase treatment, (2) the specific activity of the tRNA, and (3) the number of ribosomes in the incorporation mixture. In the experiment shown in Fig. 4, 28oo counts/min per mg of ribosomes were released in 4 ° min (i.e., per leucine equivalent) from the trichloroacetic acid precipitable [14Clprotein b y carboxypeptidase. Since the specific activity of the t R N A is I . I . IO~ counts/min per mffmole of amino acid, o.25 mffmoles of labeled amino acids are liberated per mg ribosomes. Furthermore, I mg of ribosomes represents o.33 m#moles of ribosomes. Therefore, assuming that one ribosome makes only one peptide chain, 75 % (i.e., o.25/o.33 ×IOO) of the ribosomes are engaged in amino acid incorporation.

DISCUSSION

The enzymatic transfer of amino acids from amino acyl-tRNA to polypeptide chains has received wide attention. However, the number of enzymes required, as well as their precise roles in this process, remains in question. In the reticulocyte system 16,]7 two enzymes are required for peptide chain elongation: one enzyme promotes the binding of phenylalanine-tRNA to a washed ribosome-polyuridylic acid complex, while the other enzyme catalyzes the formation of peptide bonds. In the E. coli system three or more enzymes m a y be required18,19, yet the binding of Biochim. Biophys. Acta, 145 (1967) 491-5Ol

CHARACTERIZATION OF PROTEIN SYNTHESIZED in vitro

499

amino acyl-tRNA appears to be non-enzymatic ~°. On the other hand, transfer of amino acids from amino acyl-tRNA to nascent polypeptide chains in the B . cereus 569 system is mediated by a highly organized ribosomal structure in the absence of added mRNA or soluble enzymes ~. A further suggestion that all cell-free systems are not alike, and that there may be variations in the universal mechanism for protein synthesis, is provided by the fact that cycloheximide but not chloramphenicol inhibits amino acid incorporation in the rabbit reticulocyte system while the opposite is true for the bacterial cell-free systems. Undoubtedly additional differences are introduced into the postulated mechanism of protein synthesis by studies that employ synthetic homo- or heteropolymers as messenger since these artificial messengers do not contain normal initiation and termination codons. Therefore, we have undertaken a study on the mechanism of protein synthesis using the totally endogenous cell-free system from B . cereus 569. A useful but neglected approach in the investigation of the mechanism of protein synthesis would include a careful analysis of the protein-like products formed in the cell-free system. After an incubation period of 20 min, approx. 50 % of the radioactivity incorporated is detached from the ribosomes (Fig. I). It has been suggested that high concentrations of Mg~+ cause misreading of the message and might therefore be responsible for abortive release. However, as shown in Fig. 2, the extent of polypeptide release during synthesis is not affected by variations in the concentration of Mg 2+ or K +. Thus, the integrity of the mRNA-ribosome-polypeptide chain complex apparently is not dependent upon a precise concentration of these ions. In order to determine the properties of the 1~C products formed during cell-free protein synthesis, it is first necessary to estimate their molecular weight. These 14C products can be divided into two classes: a fraction that is released from the ribosome during synthesis, and a fraction that remains attached to the ribosomal complex. The molecular weights of the two fractions, which are analyzed by molecular seive column chromatography, amino-terminal analysis, and sucrose density gradient centrifugation, differ considerably. The 14C products released from the ribosomal complex during synthesis has a number average molecular weight of approx. 5 ooo while that of the bound fraction is approx. 30 ooo. Since few completed protein chains have a molecular weight as low as 5000, these data suggest that the chains released from the ribosomal complex during cell-free synthesis are not complete proteins, but rather proteins that have been aborted during synthesis. However, the ~4C-protein which remains attached to ribosomes after a short period of synthesis is in the range expected for an average completed peptide chain (i.e., approx. 30 ooo). The question whether de novo protein synthesis occurs during cell-free synthesis was investigated by the use of carboxypeptidase. Carboxypeptidases specifically and systematically attack the carboxyl end of the protein. Since protein synthesis proceeds from the amino-terminal toward the carboxyl-terminal end, liberation of the labeled product by carboxypeptidase would be expected to proceed slowly if the protein were uniformly labeled from one end to the other. On the other hand, if only a few labeled amino acids were added to the earboxyl termini then the radioactivity would become acid soluble very rapidly. It was found that, upon digestion of the ~C-product with the carboxypeptidases, approx. 58 % of the E14C~amino acids incorporated into trichloroacetic acid precipitable protein becomes acid soluble when only one leucine equivalent is removed from the labeled product (Fig. 5). The remaining counts are Biochim. Biophys. Acta, 145 (1967) 4 9 I - 5 O I

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A . G . ATHERLY, J. IMSANDE

liberated only very slowly from the trichloroacetic acid precipitable protein by the carboxypeptidase treatment. Extrapolated values suggest that some peptides may contain a maximum of 4 ° labeled amino acids. On the other hand, the decreased rate of digestion by carboxypeptidase may result from aggregation of the labeled protein, the presence of a slowly digestible core, or from proline which is known to be resistant to carboxypeptidase. Furthermore, the labeled material which is attacked slowly by carboxypeptidase is not rapidly liberated by leucine aminopeptidase. Therefore these counts are not at the amino-termini of the chains. The fact that the majority (96 %) of the nascent chains have accepted only one amino acid before incorporation ceases suggests that: (i) the ribosomal complex is damaged during preparation in such a way that only one peptide bond can be formed, or (2) a component necessary for synthesis is depleted during incorporation, or (3) one of the required enzymes (excluding peptide synthetase) is missing from most of the ribosomal complexes. Amino acid incorporation by ribosomes isolated from cells that were disrupted by passage through a pressure cell or by the lysozyme-hypertonic sucrose method was approximately equal to that obtained with ribosomes prepared by the standard alumina grinding techniqueT, 21. Therefore there is no direct evidence for damage during preparation. If, on the other hand, an unknown but necessary component is depleted (other than N-formyl methionyl-tRNA) 22 or if one of the required enzymes is missing (preliminary results suggest the latter), then this system stands as an excellent assay system for further investigation into the mechanism of protein synthesis. Knowing the specific activity of tRNA and the rate of release of amino acids by carboxypeptidase it is possible to calculate the percent of ribosomes engaged in protein synthesis. Approx. 75 % of the ribosomes were found to incorporate at least one amino acid. This value is based on an assumed molecular weight of 5000 for the released fraction. This value is much higher than that obtained by GILBERT15 who used polyuridylic acid as messenger and TISSII~RES, SCHLESSINGER AND GROS 14, who used endogenous mRNA.

ACKNOWLEDGEMENT

This investigation was supported in part by grant HD-o2168 from the United States Public Health Service.

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CHARACTERIZATION OF PROTEIN SYNTHESIZED i n vitro IO ii 12 13 14 15 16 17 18 19 20 21 22

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