In vitro protein synthesis in yeast

Biochimica et Biophysica Acta, 294 (1973) 517-526 (~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97538

I N VITRO P R O T E I N SYNTHESIS IN YEAST POLYURIDYLIC ACID-DEPENDENT INCORPORATION OF P H E N Y L ALANINE INTO ENDOGENOUS P E P T I D Y L T R A N S F E R RNA BEN A, M. VAN D E R ZEIJST*, K E E S J. M. E N G E L ' " AND H E N R I P. J. BLOEMERS*'"

Van 't H o f f Laboratorium, Sterrenbos 19, Utrecht (The Netherlands) (Received July i8th, 1972) (Revised manuscript received October i6th, 1972)

SUMMARY

In a subceUular system programmed with polyuridylic acid and derived from Saccharomyces carlsbergensis approximately I I phenylalanine residues per h and per ribosome were incorporated into material which was insoluble in hot trichloroacetic acid. The incorporation was optimal at 8 mM MgC1, in the presence of yeast tRNAPh% In the presence of crude yeast tRNA, a maximal level of 0. 5 phenylalanine residue per ribosome was reached. In the latter case, hydrazinolysis of the precipitate obtained in hot trichloroacetic acid indicated that 74 % of the label was incorporated in carboxy-terminal positions of peptides with physical properties different from hot trichloroacetic acid-insoluble oligophenylalanines. In the presence of fusidic acid the maximum level of incorporation was 0.2 residue per ribosome, 9 1 % of the label being located at carboxy-terminal positions. It was concluded that the observed polyuridylic acid-dependent incorporation had taken place into preexisting nascent polypeptide chains. No conclusions are presented on the unknown mechanism of inhibition observed in the presence of crude tRNA. Normal or "classic" oligophenylalanine formation was optimal at 15-18 mM MgC12 and virtually absent at 8 mM MgC12.

INTRODUCTION

In their classic paper Nirenberg and Matthaei 1 reported the ability of polyuridylic acid to direct the synthesis of polyphenylalanine in a subcellular system from Escherichia coli. They observed that unlike mixed polypeptides both the radioactive reaction product and synthetic polyphenylalanine were extremely insoluble in 16 different solvents. In virtually all reports since that time the poly (U)-dependent phenylalanine incorporation has been interpreted as polyphenylalanine synthesis. In this paper we present experiments which make it necessary to reconsider this general interpretation. * To whom reprint requests should be directed. Present address: Instituut voor virologie, Faculteit Diergeneeskunde Rijksuniversiteit Utrecht, Yalelaan, Utrecht {The Netherlands). ** Present address: Chemische Fabriek Naarden, Naarden (The Netherlands). *** Present address: Department of Biochemistry, University of Nijmegen, Nijmegen (The Netherlands).

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B . A . M . VAN DER ZEIJST dtal.

A number of subcellular systems from yeast programmed with polyuridylic acid have been described in the literature 2-9. In these systems peptide synthesis proceeds at a low rate and stops after a short time. Maximal incorporations were found which varied between io (ref. 4) and o.oo2 (ref. 6) phenylalanine residues per ribosome. Initially, using crude yeast tRNA, we found a maximal incorporation of o. 5 phenylalanine residue per ribosome. In later experiments, using yeast tRNA phe, a maximal incorporation of I I phenylalanine residues per h per ribosome was reached. Seemingly, as phenylalanine oligomers containing less than 4 residues are soluble in hot trichloroacetic acid 1°, no more than 12.5 °"o of tile ribosomes had been active when an average incorporation of 0. 5 phenylalanine residue per ribosome was found. In a search for the exact percentage of active ribosomes we measured the amount of label located in carboxy-terminal positions. Surprisingly, we found 74 °/o of tile label, insoluble in hot trichloroacetic acid, in carboxy-terminal positions. When phenylalanine incorporation was restricted by fusidic acid, using either crude tRNA or tRNA Phe, 91 ° o was found in a carboxy-terminal position. Obviously, the products formed in the cell-free system could not be polyphenylalanine. In this paper evidence is presented that at low Mg2+ concentrations (less than IO mM) poly(U) is capable of supporting phenylalanine incorporation into preexisting nascent polypeptides only, while at higher Mg2+ concentrations both the normal or "classic" polyphenylalanine synthesis and this novel type of incorporation take place. Recently, Castles et al. n studying ribosomes from rat muscle, and Sturani et al. ~ with a system from Neurospora crassa suggested similar mechanisms of phenylalanine incorporation. Part of the results of this paper have been presented in a review published elsewhere 13.

MATERIALS AND METHODS

Preparation o/ribosomes Saecharomyces carlsbergensis (strain 74 N.C.Y.C. England) was cultured and protoplasts were prepared as described previously 14. Protoplasts were incubated at a concentration of about 1. 5 • lO8 protoplasts per ml for 30 min at 30 °C in a medimn containing per ml 12o mg iuannitol, IO mg casamino acids, 25/,moles sodium-potassium phosphate (pH 6.2) and 3 ° mg glucose. The protoplasts were lysed in ice-cold Buffer A (0.05 M Tris-HC1 (pH 7.6), 5 mM magnesium acetate, IO mM KC1, io mM 2-mercaptoethanol and 0. 5 mM spermidine) at a concentration of 1.2 • lO9 protoplasts per ml. After centrifugation of the lysate at IO ooo x g for io rain, the supernatant was centrifuged at lO5 ooo x g for 12o rain. From the crude ribosomal pellet a suspension of 30 mg/ml of ribosomes in Buffer A supplemented with 33 O/o/ (v/v) of glycerol was prepared. For all calculations it was assumed that yeast ribosomes have a tool. wt of 4 " los and that a suspension of I m g ribosomes per ml has an A260nm of II.O. The ribosomes could be stored for 6 months without loss of activity at -- 15 °C. Analysis o/poIysomes on sucrose gradients Samples (o.15 ml) of the crude lysate were layered onto 5-ml isokinetic sucrose gradients for the Spineo-Beckman SW 5oL rotor and were analyzed as described 15.

PEPTIDYL-tRNA AND POLY(U)-DEPEI~BENTPEPTIDE SYNTHESIS

519

The temperature was 5 °C, the top concentration 15 % (w/w), and the centrifugation time 25 rain. The parameters for the construction of the gradients were taken from ref. 16.

Isolation o] polysomes Polysomes were separated by a centrifugation in a B-XV zonal rotor as describedlL Trisomes, tetrasomes and pentasomes were collected by centrifugation at 5 °C for 16 h ill the Spinco-Beckman 3o-rotor.

Measurement o] in vitro peptide synthesis Unless otherwise stated, the subcellular system consisted of a mixture which contained per ml 5o#moles Tris-HC1 (pH 7.6), o.15/,mole Na,GTP, 0. 5 #mole Na2ATP, 6.25#moles trisodium phosphoenol pyruvate, 25/*g pyruvate kinase, 0.3/zmole spermidine, 8/,moles MgCI,, 5 °/,moles KC1, 6.6 nmoles [14C]phenylalanine (475 Ci/mole), 500/zg poly(U), 20 Ilmoles of crude tRNA or 2 nmoles tRNA phe and 25O-lOOO pmoles ribosomes. The incubation was performed at 25 °C. For the determination of the phenylalanine incorporation, IO-IOO-/A samples were removed at appropriate times and transferred to Whatman 3 MM filter disks. The disks were washed once with hot and two times with cold trichloroacetic acid, with ethanol and ether, dried and counted at 60 % efficiency in a Nuclear Chicago Mark II liquid scintillation counter.

Chromatographic analysis of the reaction products o/ the celI-]ree system A cell-free system was incubated (0.2 ml) and the ribosomes were separated from the bulk of the radioactive label by centrifugation through 15 % (w/w) sucrose in Buffer A (0.2 ml) b y centrifugation for 2 h ill the Spinco-Beckman 4o-rotor. The sediment was resuspended in 0.05 ml I M NH4OH and incubated for 30 rain at 37 °C in order to hydrolyze peptidyl-tRNA. The extract was spotted on Whatman No. I chromatography paper together with 25 jug of each mono- to pentaphenylalanine. Chromatography was perfol-med in n-butanol-ethyl acetate (2 : I, by vol.) saturated with 2 M NH4OH. The chromatogram was cut into strips of I cm, which were counted at 60 % efficiency.

Solubility o[ the reaction products o[ the eelI-/ree system A sample (0. 4 ml) from the incubated subcellular system was brought to 0.2 M NaOH and incubated for 3 ° min at 37 °C in order to hydrolyze peptidyl-tRNA. Subsequently, the mixture was neutralized with HC1 and I mg per o.6 ml polyphenylalanine was added (average chain length 48 monomers according to the manufacturer). 2o-/zl samples from the mixture were added to IOO #1 of the solvents to be tested, shaken for I h and centrifuged. IOO/zl of the resulting supernatant was transferred to Whatman 3 MM filter disks, dried and counted.

Acid hydrolysis o/ the reaction product o/ the cell-]tee system A o.4-ml sample from the incubated subceUular system was added to 0. 4 ml 12 M HC1, sealed in vacuo and treated for 8 h at IOO °C. Under these conditions polyphenylalanine is not hydrolysed completely1. The reaction mixture was dried in vacuo over solid NaOH. The dried sample was chromatographed in n-butanol-acetic acid-water (4 : I : I, by vol.) on Whatman 3 MM chromatography paper.

520

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et al.

H ydrazinolysis

Hydrazinolysis of the reaction product was performed according to Akabori et al. TM with several modifications 19,e°. I M NaOH was added to o.6-ml samples, to a

final concentration of 0.2 M, and the samples were incubated at 37 °C for 3 ° rain in order to hydrolyze peptidyl-tRNA. The labelled peptide chains were isolated by trichloroacetic acid precipitation 21. To the dried samples 0. 5 nil hydrazine containing I M hydrazine sulphate was added. The samples were incubated in vacuo for 16 h at 60 °C, dried over H2SO4 and extracted with 0.6 ml water. A sample of the extract was counted directly, another after extracting with 0. 5 vol. of heptanal in order to eliminate hydrazides. The percentage carboxy-terminal radioactivity found was corrected for the conversion of free phenylalanine into its hydrazides as explained in Table I. Controls were made to test the methodology of the hydrazinolysis for the detection of carboxy-terminal phenylalanine in oligophenylalanine and polyphenyV alanine. Firstly, unlabelled polyphenylalanine of an average chain length of 4 8 monomers was subjected to the procedure. This compound did not dissolve in hydrazinc and remained undegraded since no ninhydrin-positive material was found in the position of reference phenylalanine hydrazide in an electropherogram (electrophoresis at pH 3.5, 4 ° V/cm for 6o min; the hydrazide was prepared from diphenylalanine according to ref. 18). Secondly, penta- and tetraphenylalanine subjected to tile hydrazynolysis method travelled for the larger part like the untreated compound in the chromatography system for oligophenylalanines described above. Only a small amount of phenylalanine could be detected. These observations indicate that when a carboxy-terminal phenylalanine is preceded by other phenylalanines in polypeptide chains the hydrazinolysis is less reliable and gives an underestimate of the amount of carboxy-terminal phenylalanine. Materials

All chemicals not mentioned were the purest preparations commercially available. The other reagents were from the following sources: phosphoenol pyruvate, pyruvate kinase and the oligophenylalanines: Sigma Chemical Co., St. Louis, Mo., U.S.A. GTP and ATP: PL-Biochemicals, Milwaukee, Wisc., U.S.A. Poly(U) and polyphenylalanine: Miles, Elkhart, Ind., U.S.A. 14C-labelled amino acids: the Radiochemical Centre, Amersham, England. Hydrazine: K and K Laboratories, New York, U.S.A. Brewers yeast tRNA, sodium salt, lot 22o67 (acceptor activity 52 pmoles phenylalanine per A26onm unit) and tRNA phe (acceptor activity 11o8 pmoles phenylalanine per A260nm unit): Boehringer, Mannheim, Germany. The sodium salt of fusidic acid was a gift from Dr W. O. Godtfredsen of Leo Pharmaceutical Products, Copenhagen, Denmark.

RESULTS AND DISCUSSION

Characterization o / t h e ribosomes

It is relevant to realize that ribosomal preparations routinely used in this and other studies are heterogeneous. When, before lysis, protoplasts are incubated in rich medium, most ribosomes are found in polysomes. This is shown in Fig. IA. In this

PEPTIDYL-tRNA

AND P O L Y ( U ) - D E P E N D E N T PEPTIDE SYNTHESIS

521

experiment, cycloheximide was added before lysis. However, when protoplasts were lysed in the absence of cycloheximide as described for the preparation of ribosomes, smaller polysomes and more 8o-S ribosomes were found (Fig. IB). In this case most of these 8o-S ribosomes dissociate in sucrose gradients containing 0.5 M KC1 (not shown). Apparently, they represent free ribosomes formed by "run off" during lysis.

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Fig. i. P o l y s o m e c o n t e n t of y e a s t protoplasts; t h e occurrence of r u n off d u r i n g lysis. P r o t o p l a s t s were i n c u b a t e d a n d s a m p l e s were t a k e n for t h e a n a l y s i s of p o l y s o m e s as described in Materials a n d M e t h o d s . T h e control (lO -3 M c y c l o h e x i m i d e a d d e d to t h e i n c u b a t i o n m i x t u r e before lysis) is s h o w n in P a n e l A. T h e effect of o m i t t i n g c y c l o h e x i m i d e before lysis is given in P a n e l B.

Activity o/the ribosomes in peptide synthesis Fig. 2 illustrates the poly(U)-dependent incorporation of phenylalanine into hot trichloroacetic acid-insoluble material; I I phenylalanine residues per ribosome are obtained after an incubation of i h. For reasons not well understood, the activity in the presence of crude yeast tRNA is remarkably low. Yet, poly(U) gave a 2o-fold stimulation of the phenylalanine incorporation in the presence of crude tRNA, whereas the incorporation of a mixture of lysine and isoleucine was not enhanced by poly(U) (Fig. 3). Therefore, the phenylalanine incorporation is not the result of protection of endogenous messengers against nucleases by added poly(U).

Analysis o] the products ]ormed in vitro When the reaction product formed after incubation of the system during I h in tile presence of crude tRNA was subjected to hydrazinolysis, it appeared that 74 % of the [14C]phenylalanine in the hot trichloroacetic acid-insoluble material had been

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Fig. 2. P o l y ( U ) - d e p e n d e n t p h e n y l a l a n i n e i n c o r p o r a t i o n i n t h e pre s e nc e of t R N A Phi. The i nc uba tions were: complete system ( 0 - 0 ) ; c o m p l e t e s y s t e m + l m M fus i di c a c i d ( A - A ) ; p o l y ( U ) omitted (I-R). Fig. 3- L o w i n c o r p o r a t i o n in t h e p o l y ( U ) - d e p e n d e n t s y s t e n l in t h e pre s e nc e of c r u d e t R N A . C o n l p l e t e s y s t e m ( • - • ) ; c o n l p l e t e s y s t e m + i mM fusidic a c i d ( I k - • ) ; p o l y (U) o m i t t e d ( I t - I t ) . L a c k of s t i m u l a t i o n of i n c o r p o r a t i o n of isoleucine a n d l y s i n e b y p o l y ( U ) . [14C]Phenylalanine w a s o m i t t e d a n d i n s t e a d [14C~lysine a n d [14C~isoleucine (1. 5 • i o 5 M, 3Ol Ci/mole) w e re a d d e d to~ g e t h e r w i t h 18 u n l a b e l l e d a m i n o a c i d s (1.5 • IO -s M). The i n c u b a t i o n w a s p e r f o r m e d w i t h ( O @) and without ( ~ - ~ ) poly(U).

incorporated in a carboxy-terminal position (Table I). The total amount incorporated was 0. 5 phenylalanine residue per ribosome (Fig. 3). Consequently, 37 % of the ribosomes had participated in the incorporation of an average of no more than 1.35 phenylalanine residue. In an other experiment, fusidic acid was used, which as an inhibitor of the translocation step restricts the incorporation to one or two amino acids per ribosome ~2. In the presence of fusidic acid and of either crude t R N A (Fig. 3)

TABLE I HYDRAZINOLYSIS

OF T H E P R O D U C T S OF A

CELL-FREE INCUBATION

The h y d r a z i n o l y s i s w a s p e r f o r m e d as d e s c r i b e d in M a t e r i a l s a n d Methods. A f t e r h y d r a z i n o l y s i s a b o u t 80 % of t h e 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 (as m e a s u r e d b y t h e W h a t m a n fi l t e r p a p e r m e t h o d ) w a s reco vered . The r e c o v e r y of t h e control, i n c u b a t e d w i t h u n l a b e l l e d p h e n y l a l a n i n e to w h i c h l a b e l l e d p h e n y l a l a n i n e w a s a d d e d a f t e r t r i e h l o r o a c e t i c acid p r e c i p i t a t i o n b u t before h y d r a z i n o l y s i s , was o v e r 95 %. T h e p e r c e n t a g e 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 a c a r b o x y - t e r m i n a l pos i t i on, was c a l c u l a t e d for t h e n o r m a l i n c u b a t i o n as follows: 38.3/61.3 × 1 . 3 2 / I . I I × i o o % = 74 %. I n t h i s w a y for t h e i n c o r p o r a t i o n in t h e presence of f u s i d i c acid a v a l u e of 9 1 % w a s found.

Incubation

cpm phenylalanine recovered after hydrazinolysis

cpm phenylalanine recovered in the water phase after extraction with heptanal

Normal + f u s i d i c acid Control

61. 3 - lO 3 24. 5 103 1.32 • IO"

38.3 • lO 3 18.8 " 103 1 . I I • lO 6

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PEPTIDYL-tRNA

AND POLY(U)-DEPENDENT

PEPTIDE

SYNTHESIS

523

or t R N A Pbe (Fig. 2) the maximal incorporation was o.2 phenylalanine residue per ribosome, 9 1 % of the label being located in carboxy-terminal positions (Table I). This experiment indicated t h a t in the presence of fusidic acid 18 % of the ribosomes incorporated an average of I . I phenylalanine residues. If the product of the uninhibited reaction had been polyphenylalanine, the presence of fusidic acid would have led to the formation of the trichloroacetic acid-soluble x° di- or triphenylalanine only. Therefore, we conclude t h a t phenylalanine had been incorporated into preexisting polypeptides; nascent polypeptides are the only plausible candidates for this acceptor function. I n agreement with this conclusion, it was found t h a t unlike polyphenylalanine 1 the reaction product formed in the presence of either fusidic acid or crude t R N A was completely hydrolyzed in 6 M HC1 in 8 h at ioo °C. Also the significant solubility of the reaction product in a n u m b e r of solvents (Table II) is different from t h a t of polyphenylalanine. TABLE II T H E S O L U B I L I T y OF T H E REACTION PRODUCT FORMED D U R I N G T H E P O L Y ( U ) - D E P E N D E N T A L A N I N E INCORPORATION IN T H E P R E S E N C E OF CRUDE t R N A

PHENYL-

For experimental details see Materials and Methods. The samples extracted contained 27oo cpm. Solvent

Extracted amount qf radioactivity (cpm)

Solubility of polyphenylalanine according to ref. I

33 % HBr in glacial acetic acid Trichloroacetic acid Water Benzene N,N-Dimethylformamide Ethanol Glacial acetic acid Phenol Acetone Ethyl acetate Pyridine Formic acid

1141 ioo 145°

soluble insoluble* insoluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble insoluble

IO

157° 200 900 2180 63° ioo 13o0 650

* In ref. I a product was defined as insoluble when less than 0.002 g of the product was soluble in IOO ml of solvent at 24 °C. Finally, as a control experiment, it was shown (Fig. 4) t h a t polysomes freed from 8o-S ribosomes b y zonal centrifugation did incorporate phenylalanine. This incorporation, unlike t h a t of a mixture of lysine and isoleucine, was stimulated b y poly(U). For the sake of brevity we have referred to the poly(U)-dependent phenylalanine incorporation into preexisting nascent polypeptides as the "novel t y p e of incorporation" and to the formation of poly- or oligophenylalanine as the "classic" incorporation. Obviously, the three methods used to reveal the occurrence of the novel t y p e of incorporation (hydrazinolysis, acid hydrolysis and determination of solubility of the reaction product) fail to discriminate between polyphenylalanine and mixed polypeptides with long carboxy-terminal polyphenylalanine sequences, t h a t supposedly are formed in the presence of t R N A Phe and the absence of fusidic

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Fig. 4. Stimulation by poly(U) of phenylalaniue incorporation mediated by polysomes. Polysomes were incubated in the cell-free s y s t e m : complete s y s t e m ( 0 - 0 ) ; poly(U) o m i t t e d (m-m). Lack of s t i m u l a t i o n of isoleucine and lysine b y poly(U). Conditions were as given in the legend of Fig. 3- The incubation was performed w i t h ((2) 0 ) and w i t h o u t ([2] [2]) poly(U). In this inc u b a t i o n in addition to the n o r m a l ingredients of in vitro peptide synthesis crude t r a n s f e r factors (ref. 15) at a concentration of o.2 mg/inl were present. Crude t R N A was used. Fig. 5. Polymerization of phenylalanine into endogenous peptidyl chains and into oligophenylalanine at various Mg 2+ concentrations. Cell-free s y s t e m s were incubated for 4 ° min and the reaction p r o d u c t was analyzed by c h r o m a t o g r a p h y . The incorporation into material s t a y i n g at the origin is plotted in Panel A ( O - O , complete system; A - A , complete s y s t e m + t mM fusidic acid). Panel B shows the a m o u n t of phenylalanine incorporated into material with an R F value equal to or greater t h a n t h a t of diphenylalanine. In this e x p e r i m e n t t R N A P'" was used.

acid. This might well be one of the reasons wily tile novel type of incorporation has been overlooked so far with a few exceptions 11,12.

Classic polyphenylalaninc ~vnthesis Further experiments were aimed at investigating whether classic pplyphenylalanine synthesis could occur at the same time as the novel type of incorporation and whether perhaps one type or the other would predominate under different conditions. Therefore, ribosomes were isolated after incubation in the cell-free system and the reaction product was chromatographed. In the chromatographic system used naturally occurring peptides stay at the origin, while short oligophenylalanine molecules up to five residues have characteristic RF values. A disadvantage is that long polyphenylalanines also stay at the origin. Consequently, label at the origin can represent a mixture of both types of incorporation, but any radioactive label with an Rv value equal to or greater than that of diphenylalanine has to be attributed to classic phenylalanine polymerization. The effect of the Mg z+ concentration on distribution of label

P E P T I D Y L - t R N A AND POLY(U)-DEPENDENT PEPTIDE SYNTHESIS

525

in the chromatogram is shown in Fig. 5. The classic type of incorporation functions optimally at 15 mM Mg~+ and not at all at 8 mM Mg*+, the concentration selected for our previous experiments. The formation of radioactive material at the origin of the chromatogram (Fig. 5A), representing a mixture of the two types of incorporation, is bimodal with respect to the Mg2+ concentration. The second optimum is absent when the experiment is carried out in the presence of fusidic acid which prevents the formation of long polyphenytalanine chains (Fig. 5A). Conclusions

From these experiments the following picture arises: I. At an Mg2+ optimum of 15 mM short oligophenylalanines (di- to penta-) are formed. At a slightly higher Mg2+ optimum oligophenylalanine sequences with more than 5 residues are formed. This synthesis represents true oligophenylalanine synthesis, since it is absent in the presence of fusidic acid. Presumably, it is mediated by free ribosomes and poly(U). 2. At 8 mM Mg~+ no true oligophenylalanine synthesis takes place, since in this case no di- to pentaphenylalanine synthesis could be detected. On the other hand, at 8 mM Mg2+ there is poly(U)-dependent incorporation of phenylalanine into material which remains at the origin during chromatography (Fig. 5). Its formation is not prevented, when the incorporation of amino acids is restricted to one or possibly two amino acids per ribosome by the action of fusidic acid 22. This incorporation represents the addition of carboxy-terminal phenylalanines to endogenous nascent chains. It is mediated by the interaction between ribosomes carrying endogenous peptidyl-tRNA and poly(U). These conclusions are in agreement with those of Castles et al. 11 in a system derived from skeletal muscle of normal and diabetic rats. Moreover, they supplement the suggestion made by Sturani et al. ~2 "that the presence of the peptidyl-tRNA bound to the 6o-S subunit induces an increased phenylalanine polymerization at low Mg~+ concentration". It is not known whether or not prokaryotic systems can support the novel type of incorporation. However, it should be noted that bacterial ribosomal preparations prepared as described by Nirenberg and Matthaei ~ probably contain free ribosomes only. Consequently, in this case one can find true polyphenylalanine formation only.

ACKNOWLEDGEMENTS

This investigation was partly supported by the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.). The skilful technical assistance of Miss M. H. Roos is gratefully acknowledged. REFERENCES

I Nirenberg, M. W. and Matthaei, J. H. (1961) Proc. Natl. Acad. Sci. U.S. 47, 1588-16o2 2 Bretthauer, R. K., Marcus, L., Chaloupka, J., Halvorson, H. O. and Bock, R. M. (1963) Biochemistry 2, lO79-1o84 3 de Kloet, S. I~. (1965) Proc. K . z~kad. Wet., Ser. B 68, 266-283 4 Heredia, C. F. and Halvorson, H. O. (I966) Biochemistry 5, 946-952

526 5 6 7 8 9 io ii 12 i3 14 15 16 17 18 19 20 21 22

B.A. M, VAN DER ZEIJST ~'t al.

Ayuso, M. S. and Heredia, C. F. (1967) Biochim. Biophys. Acta 145, 199-2Ol Richter, D., Halneister, H., Petersen, H. G. and Klink, F. (1968) Biochemistry 7, 3753-376t Hutchinson, H. T., Hartwell, L. H. and McLaughlin, C. S. (1969) J. Bacteriol. 99, 8o7-814 Tiboni, O., Parisi, B., Perani, A. and Ciferri, O. (197 o) J. Mol. Biol. 47, 467-476 Richter, D. and Lipmann, F. (197 o) Biochemistry 9, 5o65-5o7o Pestka, S., Scolnick, 1~. M. and Heck, B. H. (1969) Anal. Biochem. 28, 376-384 Castles, J. J., Rolleston, F. S. and Wool, I. G. (1971) J. Biol. Chem. 246, 1799-18o 5 Sturani, E., Alberghina, F. A. M. and Casacci, F. (1971) Biochim. Biophys. Acta 254, 296-303 Amesz, W. J. C., Bloemers, H. P. J., Engel, C. J. M., van der Mast, C. A., van der Saag, P. T. M. and van der Zeijst, B. A. M. (1972) J. Electroanal. Chem. 37, 393--4°6 Hartlief, R. and Noningsberger, V. V. (1968) Biochim. Biophys. Acta 166, 512-531 van tier Zeijst, B. A. M., Kool, A. J. and Bloemers, H. P. J. (1972) Eur. J. Biochim. 3o, 15-25 McCarty, K. S., Stafford, D. and Brown, O. (1968) Anal. Biochem. 24, 314-329 van der Zeijst, B. A. M. and Bult, H. (1972) Eur. J. Biochem. 28, 463-474 Akabori, S., Ohno, K. and Narita, K. (1952) Bull. Chem. Soc. Jap. 25, 214-218 Bradbury, J. l-I. (1958) Biochem. J. 68, 482-486 Kusama, K. (1957) J. Biochem. Tokyo 44, 375-381 Siekevitz, P. (1952) J. Biol. Chem. 195, 549-565 Bodley, J. XV., Zieve, F. J. and Lin, L. (197 o) J. Biol. Chem. 245, 5662-5667