Anfinsen,'0 and the cystines were then reduced and converted to analogues of

Transcription

1 ON THE STRUCTURE AND FUNCTION OF BOVINE TRYPSINOGEN AND TRYPSIN* BY KENNETH A. WALSH, DOROTHY L. KAUFFMAN, K. S. V. SAMPATH KUMAR, AND HANS NEURATH UNIVERSITY OF WASHINGTON, SEATTLE Communicated December 30, 1963 The detailed primary structure of bovine trypsinogen has been the subject of independent but concurrent studies in this laboratory1-4 and in the laboratories of Sorm, Keil et al. in Prague5-7 for several years. Although the determination of the structure is not yet complete, recent progress in this laboratory4 8 has made possible the formulation of a working hypothesis for a structure which includes all of the 229 amino acid residues. Examination of this preliminary structure suggests certain tentative conclusions concerning the relationship between the structure and the function of the protein. Trypsinogen consists of a linear sequence of 229 amino acid residues with six disulfide cross-linkages. Enzymatic degradations of DIP-trypsin9 or of S-sulfotrypsinogen by trypsin' 3, 6, 7 chymotrypsin,3 or pepsin5 have given rise to many peptides which have been isolated and their sequences determined. However, it has not been possible to recover all of the amino acids of the parent protein in soluble peptides from any single digest. Hence, it has been necessary to degrade the molecule in several ways to ensure that all of the residues will be isolated from one or another of such degraded preparations. Toward this end, the narrow specificity of trypsin (used as an enzyme) was "redirected" toward new points of cleavage of trypsinogen (used as a substrate) by the following modification of trypsinogen: the c-amino groups of lysine were trifluoroacetylated by the method of Goldberger and Anfinsen,'0 and the cystines were then reduced and converted to analogues of lysine by alkylation with 2-bromo-ethylamine by an adaptation of the technique of Lindley."1 Trypsin cleaved this product at the carboxyl side of the aminoethylcysteinyl residues, rather than at the lysine residues, and gave rise to peptides with C-terminal AEC residues. In many cases these peptides contained acylated lysine residues and overlapped peptides from a conventional tryptic digest (Table 1). The feasibility of this approach to trypsinogen was facilitated by the occurrence of six disulfides and by the low content of arginine (two residues) which provides points of cleavage in both conventional and redirected digestion. Hofmann8 recently applied the cyanogen bromide reagent of Gross and Witkop'2 to the cleavage of trypsinogen at the two methionines. Subsequent reduction and alkylation gave rise to three fragments which were separated by Sephadex gel filtration and selective precipitation techniques.8 The sum of the compositions of the three isolated fragments accounted for all of the amino acids in trypsinogen, indicating that the fragments represent three different but contiguous pieces of the molecule (Table 2). The fragments, A, B, and C, have been identified by end-group analysis with the N-terminal, middle, and C-terminal regions, respectively, of the molecule. Comparison of the compositions of the purified fragments with the compositions of peptides of proven structure has made possible the prediction of the occurrence of certain peptides in certain fragments. These predictions have been 301

2 302 BIOCHEMISTRY: WALSH ET AL. PROC. N. A. S. TABLE 1 PEPTIDES ISOLATED FROM A "REDIRECTED" TRYPTIC DIGEST OF MODIFIED TRYPSINOGEN* RT-1 (Val,Asp4,Lys) (Lu,Val,Gly2,Tyr) (Thr,AEC) RT-2 Ala.(Gly,Tyr,Leu,GluG1y2,Lys) (Asp,Ser) AEC RT-3 (Ala,Gly,Tyr,Lu,Glu,Gly2,Lys,Asp,SerAEC,.Glu,Gly2,Asp,SerGly,Pro,Val2,AEC) RT-4 Gln.Val.(Ser,Lu,Asn,Ser,Gly,Tyr) RT-5 (Gly,Ala,Asn,Thr,Pro,Val,Tyr) RT-6 (Glu,Gly2,Asp,Ser,Gly,Pro,ValAEC) RT-8 Leu.Lys.Ala.(Pro,Ileu,Leu,SerAsp,Ser2,AEC) RT-9 (Lu2,Ser,Gly,Try) (Gly,Asn,Thr,Lys,Ser2,Gly,Thr,Ser,Tyr,Pro,Asp,Val,Lu) (Lys,AEC) RT-10 Ala.(GluLys,Asp,Lys,Pro,Gly,Val,Tyr) RT-12 (LysSer,Ala,Tyr,Pro,GlyGlu,Lu,Thr,Ser,Asn,Met,Phe).AEC RT-13 Val.Ala.Ser.Ileu(Ser,Pro,Leu,Thr,Ser)AEC RT-14 Ala.Ser.Ala.(Gly,Thr,Gln)AEC RT-15 (Met,Phe,AEC) * Tryptic cleavages were "redirected" from lysines (by trifluoroacetylation) to cysteines (by conversion to the S-p3-aminoethylcysteinyl derivatives). The peptides were purified by paper electrophoresis and chromatography. almost entirely verified by partial enzymatic hydrolysis of the fragments and identification of the isolated peptides (Table 3). Preliminary data on the digestion of S-sulfotrypsinogen by carboxypeptidase A have identified the C-terminal sequence of the molecule (Table 4). Hofmann8 has indicated that fragment C must be C-terminal because it alone did not contain homoserine. This conclusion is confirmed here by comparison in Table 4 of the C-terminal residues of fragment C with those of S-sulfotrypsinogen, the data being consistent with the C-terminal sequence: -Gln.(Thr,A]a).Ileu.Ser.Asn.OH. The only peptides consistent with this structure are ct-ad (Table 3) and C-Ha,'3 which are therefore assumed to occupy the C-terminal position in fragment C and S-sulfotrypsinogen, respectively. It is not clear why the previous carboxypeptidase digestion by Pechere, Dixon, Maybury. and Neurath14 did not reveal these C-terminal residues. As discussed by Hofmann,8 the presence of N-terminal phenylalanine in fragment C and the occurrence of a Met. Phe sequence in peptide C-5a3 indicate that. TABLE 2 COMPARISON OF THE COMPOSITIONS OF FRAGMENTS OBTAINED BY CYANOGEN BROMIDE CLEAVAGE8 AND THE HYPOTHETICAL STRUCTURES BETWEEN THE METHIONINE RESIDUES IN FIGURE 1,-Fragment A- - -Fragment B --Fragment C--. Fig. 1 Fig. 1 Fig. 1 Isolated (1-92) Isolated (93-16,6) Isolated ( ) Aspartic acid Threonine* Serine* Glutamic acid Proline Glycine Alanine Valinet Isoleucinet Leucine Tyrosine Phenylalanine 2 2 Trace Methionine derivatives Trace 0 Lysine Histidine Trace 0 Arginine Cysteine or derivative Tryptophan * Hydrolysis data extrapolated to zero time. t Hydrolysis data after 90 hr.

3 VOL. 51, 1964 BIOCHEMISTRY: WALSH ET AL. 303 this peptide joins fragment B and fragment C. The only other methionine-containing peptide, C-12a,'5 must overlap fragments A and B. Carboxypeptidase digestions of fragments A and B (Table 4) support these conclusions. The location of the six disulfide bridges in trypsinogen is currently under investigation in this laboratory. The close juxtaposition of two histidines in one of the cystinyl peptides isolated from a peptic digest of the zymogen prompts its presentation in this report. It has the following structure: Asn.Ser.Gly.Tyr.His.Phe.Cys.Gly.Gly.Ser.Leu S S Val.Val.Ser.Ala.Ala.His.Cys.Tyr.Lys.Ser.Gly.Ileu.Gln. Discussion.-Conventional approaches to the determination of the amino acid sequence in proteins were not adequate for the long and heavily cross-linked polypeptide chain of bovine trypsinogen. The success of the present work has depended on the application of two additional methods for determining overlaps and for positioning peptides derived by enzymatic hydrolysis. One of these is the redirection of the action of trypsin from peptide bonds adjacent to lysine residues to those adjacent to half-cystine residues, by masking the eamino groups of lysine and converting the cystine residues into lysine analogues after reductive cleavage. The other method involves the reaction of the two methionine residues of trypsinogen with cyanogen bromide, which causes fragmentation of the polypeptide chain into three segments of comparable length after reduction of the disulfide bonds. The results of these studies are summarized in the partial structure presented in Figure 1, which accounts for all of the 229 amino acid residues found in trypsinogen but does not indicate the locations of the six disulfide bonds. This structure should be regarded as a working hypothesis, since certain sequences still require verifica- Val.Asp.Asp.Asp.Asp.Lys.Ileu.Val.Gly.Gly.Tyr.Thr.Cys.Gly.Ala.Asn.Thr.Val.Pro.Tyr.Gln.Val. 40 Ser.Leu.Asn.Ser.Gly.Tyr:His.Phe:Cys;Gly.Gly.Ser.Leu[Val.Val.Ser.Ala.Ala.His.Cys.Tyr.Lys.Ser. Gly.Ileu.Gln.Val.Arg.Leu;Gly;Gln;Asp;(Asn,Ileu,Asn).Val.Val.Glx.Gly.Asx.Glx.Gln.Phe] [Ileu. 80 Asn.Ser.Gln.Try] Ileu.Ser.Ala.Ser.Lys.Ser;(Pro2,His,Ser,Tyr,Asn2,Thr,Val,Ileu,Leu) :Asn.Asn.Asp. 100 Ileu.Met.Leu :Ileu.Lys :Leu.Lys.Ser.Ala.Ala.Ser.Leu.Asn.Ser.Arg;Val.Ala.Ser.Ileu.(Ser,Pro,Leu, 120 Thr,Ser).Cys;Ala.Ser.Ala.(Gly,Thr,Gln).Cys;Leu;Ileu.Ser.Gly.Try.Gly.Asn.Thr.Lys.Ser.Ser.Gly. 140 Thr.Ser.Tyr.Pro.Asp.Val.Leu.Lys :Cys.Leu.Lys.Ala.(Pro;Ileu).Leu.Ser.Asp.Ser.Ser.Cys.Lys :Ser. 160 Ala.Tyr.(Pro;Gly).Gln.Ileu.Thr.Ser.Asn.Met.Phe.Cys.Ala.Gly.Tyr.Leu.Glu.Gly.Gly.Lys.Asn. Ser.Cys.Gln.Gly.Gly.Asp.Ser.Gly.Pro.Val:(Val) :Cys.Ser.Gly.Lys :Leu;Gln.(Gly;Val;Ser;Ileu; Try);Gly.(Ser;Gly;Cys).Ala.Gln.Lys.Asn.Lys.Pro.Gly.Val.Tyr.Thr.Lys.Val.Cys.Asn.Tyr:Val. 220 Ser.Try:Ileu.Lys:Gln.Thr;Ala;Ileu.Ser.Asn FIG. 1.-Tentative structure of bovine trypsinogen. Residues and sequences deduced in this laboratory are designated in the usual manner, with the following additions: Colons indicate sequences which are probable but require further verification. Semicolons indicate sequences proved elsewhere.5-7 Square brackets enclose two peptides whose relative positions are not yet established. The numbering of residues may be in error by five residues if the relative positions of the two unplaced peptides is reversed. The asterisk indicates a valyl residue whose occurrence at position 188 is not yet firmly established. 20

4 304 BIOCHEMISTRY: WALSH ET AL. PROC. N. A. S. TABLE 3 PEPTIDES ISOLATED FROM ENZYMATIC DIGESTS OF EACH OF THE THREE FRAGMENTS RESULTING FROM CLEAVAGE OF TRYPSINOGEN WITH CYANOGEN BROMIDE FRAGMENT A FRAGMENT B Chymotryptic digest: Tryptic digest: ac-a (ValAsp4,LysLuValGly2,Tyr) bt-a2 (Lu2,Ser,Gly,Try) ac-d2 (Asn,Ser,GlyTyr) bt-a4 (Ala,Pro,Lu2,Ser,Asp,Ser2,AEC) ac-fa (Thr,AEC,Gly,Ala,Asn,Thr,Pro,Val, bt-c, (Ser,Ala,Tyr,Pro,Gly,Gln,Lu,Thr,Ser, Tyr) HSer) ac-gb (His,Phe) bt-ba (Val,MAa,Ser,Lu,Ser,Pro,Lu,Thr,Ser, Peptic digest: AEC) ap-ea (Val, Asp4,Lys,Lu,Val,Gly,Gly,Tyr) bt-ca (Ala,Gly,Ala,Ser,Thr,Gln,AEC) (Thr,AEC,Gly,Ala,Asn,Thr,Val,Pro,Tyr) bt-da (Gly,Asn,Thr,Lys) (Gln,Val,Ser,Leu) bt-dc (Lu2,Lys) ap-4j Val. (Val,Ser,Ala2,His,AEC,Tyr,Lys, bt-e (Lu,Lys) Ser,GlyIleu,Gln) bt-f (Cys,Lu,Lys) ap-5j Ala.Ala (His,AEC,Tyr,Lys,Ser,Gly,Ileu. bt-g1 (AEC) Gin) bt-g2 (Lys) ap-6b Val (Asp4,Lys,Ileu,Val,Gly2) FRAGMENT C ap-7d Val (Arg,Leu),(Gly,Glu,Asp),(Asn,Ileu, Tryptic digest: Asn) ct-aa Asp (Ser,AEC) ap-7h Asn (Ser,Gly,Tyr) (His,Phe,AEC).Gly. ct-ac (Gln,Gly2,Asp,Ser,Gly,Pro,Vall.5, Gly.Ser.Leu AEC) ap-7j lleu (Ser2,AlaLys).Ser ct-ad Gln.(Thr,Ala,Lu,Ser,Asn) ap-9c Val.Val.Glx.Gly.Asx.Glx.Gln.Phe ct-ae (Ala,Gly,Tyr,Leu,Glu,Gly2,Lys) ap-9ga Ileu.Asn.Ser.Gln ct-af Asn.(Tyr,Val).(Ser,Try) Nagarse digest: ct-ba (Asn,Lys,Pro,Gly,Val,Tyr) an-ab (ValAsp4,LysLuValGly2,Tyr) ct-ga Thr.Lys an-dlb (Asn,Thr,Pro,Val,Tyr) an-d3a (Tyr,Asn) ct-h Val.AEC Peptic digest: cp-a (Tyr,Val,Ser,Try) tion as indicated. One small peptide, the pentapeptide tentatively assigned to position 66-70, could be in position The present work dispels the mystery of the identity of the C-terminal residue in trypsinogen, a problem that has puzzled observers for a decade or longer.14' 16 In retrospect, it seems surprising that the C-terminal asparagine has remained undetected since it is hydrolyzed rather readily by carboxypeptidase A in S-sulfotrypsinogen. In this connection it is worthy of note that four proteolytic enzymes or their precursors in the bovine pancreas, carboxypeptidase A, chymotrypsinogens A and B, and trypsinogen have asparagine in this position.'7 The tentative structure of trypsinogen, although not entirely complete, nevertheless reveals certain features which relate to the two biological properties of paramount interest, namely, the process of activation and the mechanism of action of the product of activation, trypsin. Several years ago a model was proposed for the process of activation, suggesting that the two amino acid residues which were implicated as part of the active site, namely a specific serine and a histidine residue, were well separated from each other along the polypeptide chain, but were only brought into juxtaposition through a process of tertiary folding18 accompanying the activation. The present structure of the zymogen, depicted in Figure 1, indeed corroborates the first part of this prediction, since all three histidines occur in the amino-terminal region of the molecule, whereas the reactive serine (residue 184) is in the carboxyl-terminal region. An analogous situation has recently been described for chymotrypsinogen A.23 It is of interest to note that both trypsin and chymotrypsin have the identical N-terminal

5 VOL. 51, 1964 BIOCHEMISTRY: WALSH ET AL. 305 tripeptide sequence of Ileu.Val.Gly, which in each case appears as a direct result of the cleavage of one peptide bond during activation of the parent zymogen.'7 It is not yet clear from the present structure why the primary chemical events involved in zymogen activation must be so specific. The lysyl-isoleucine bond that is cleaved (residues 6-7) precedes in the linear sequence the first half-cystine residue, and thus cleavage of this bond cannot possibly remove any structural impediments occasioned by the location of disulfide bridges. The most noteworthy structural element removed by tryptic hydrolysis is the highly charged tetra-aspartyl sequence, but it is not obvious why cleavage of a bond beyond residue 7 could not possibly also induce zymogen activation. An answer to this problem must await, in the first place, the positioning of all six disulfide bridges in the molecule, and more importantly, the elucidation of the tertiary structure of the molecule at a resolution which permits side-chain interactions to be predicted from the topography of the molecule. The many similarities between trypsin and chymotrypsin in certain physicochemical properties, in sites and rates of pancreatic secretion, and in amino acid composition are well known (cf. ref. 19). The extension of this concept of similarity to the level of catalytic reaction mechanism is evident from their common susceptibility to organo-phosphorus inhibitors and from the demonstration of analogous acyl-enzyme intermediates in their catalytic function.20 In striking contrast to these analogies is the lack of resemblance in primary structure.21 Till now, only one common tetrapeptide sequence has been evident, i.e., -Gly-Asp-Ser-Gly-, which contains in each case the serine residue of the active site.1' 22 A histidine residue has been repeatedly implicated in mechanisms of the action of both enzymes on the basis of many lines of kinetic and chemical evidence, and hence the position of the histidines in the respective primary structures of trypsin and chymotrypsin becomes a matter of considerable interest. Chymotrypsin contains two histidine residues and both have been recently found in a single cystinyl peptide having the structure :23-26 His.Phe.Cys.Gly.Gly.Ser.Leu S S Ala.His.Cys. The present discovery of the identical sequence in the same disulfide-bridged structure in trypsinogen draws attention to a further element of similarity between trypsin and chymotrypsin, and raises the question of a possible functional significance of this unique structure. It has been suggested, on the basis of the identity of the Gly.Asp.Ser.Gly. sequence, that the active site of trypsin, chymotrypsin, and elastase might have evolved from a common precursor." 27 The present finding of an even larger area of identity, containing residues which have been implicated in the mechanisms of both enzymes for many years, lends strong support to the hypothesis that structural similarities between these enzymes are found only in certain areas of functional importance. That is, the unique chemical structure may itself be an essential component of the active center and may play a vital role in the hydrolytic mechanism. It would, presumably, be expected that the same

6 306 BIOCHEMISTRY: WALSH ET AL. PROC. N. A. S. TABLE 4 LIBERATION OF AMiNo ACIDS BY CARBOXYPEPTIDASE A FROM DERIVATIVES OF TRYPSINOGEN S-sulfo- Fragments of Cleavage by CNBr trypsinogen A B C Weight ratio of substrate: enzyme 20:1 45:1 10:1 45:1 Time of digestion at ph 9, 250C (hr) Major amino acids liberated (fractional yield) Asparagine 0.28 Asparagine + glutamine Glutamine Serine Isoleucine Alanine Threonine Valine 0.3 Homoserine Leucine Tyrosine The carboxypeptidase A was pretreated with M DFP overnight at 0 in 0.7 M NaCi, 0.3 M ammonium acetate, ph 9.0. Fragments A and B were preincubated for 5 hr at ph 9 to favor the opening of the lactone ring. sequences would be found in chymotrypsin B, elastase, and other related "serine" enzymes. Recent studies29 30 clearly establish that one of the two histidines of chymotrypsin is an essential part of the active center of the molecule. Since the two histidines are brought in close proximity by a disulfide bridge, both imidazole groups must be either a part of the active site or, at the least, one would be adjacent to an element of the active site. The discovery that two histidines in trypsinogen are in a strikingly identical decapeptide sequence also bridged by a disulfide linkage strongly suggests a common functional role of this unique structure, and leads us to propose that not one, but two histidines are involved in the mechanism of catalysis-a proposition which appears compatible with certain recent observations in the field of enzymology. Crestfield, Stein, and Moore3" have provided evidence that in ribonuclease, a hydrolytic enzyme, two histidines at positions 12 and 119 in the linear structure of the molecule, are essential for the enzymatic function, and that these two imidazoles must be within 5 Angstrom units of each other in the three-dimensional structure of the protein. Bruice and Topping32 have recently proposed that in the imidazole-catalyzed transamination reaction between pyridoxal and a-aminophenylacetic acid, not one but two imidazole molecules participate in a catalytic mechanism in which imidazole acts as a general base and imidazolium as a general acid. While the mechanism is specifically applied to the reaction just mentioned, there does not seem to be any violation of the principle of such a "concerted" attack by its extension to hydrolytic reactions of the type catalyzed by "serine" proteases. Several mechanisms have been proposed in recent years to explain the mode of action of trypsin and chymotrypsin (Cunningham,33 Westheimer,34 Spencer and Sturtevant,35 Bruice,36 and Bender28). All of them involve an 0-acyl intermediate, and the participation of a histidine residue in several ways in both the acylation and deacylation steps somewhere in the sequence of reactions between substrate and product. Bender has suggested that histidine participates in a symmetrical mechanism in the acylation and deacylation reactions, and the ph dependence of these two steps has been described by all proponents as reflecting the effect of ph on the ionization of the imidazolyl group of histidine. The hypothesis proposed in the present report,

7 VOL. 51, 1964 BIOCHEMISTRY: WALSH ET AL. 307 namely, that two different histidines are involved, requires little extension of any one of several schemes previously proposed and receives experimental support through the direct demonstration of their presence in the structural element which must be included if the hypothesis of the involvement of histidine in the catalytic process is correct in the first place. The authors would like to express particular appreciation to Dr. T. Hofmann for his valuable collaboration in concurrent studies of the cleavage of tryp~inogen with cyanogen bromide. The capable assistance of Mr. P. Schneider, Mrs. H. Froste, and Mr. L. H. Ericsson in many phases of this work is gratefully acknowledged. * This work was supported in part by the Public Health Service (GM-04617), by the American Cancer Society (P-79), and by the Office of Naval Research (NonR477-04). 1 Dixon, G. H., D. L. Kauffman, and H. Neurath, J. Biol. Chem., 233, 1373 (1958). 2 Walsh, K. A., D. L. Kauffman, and H. Neurath, Biochem., 1, 893 (1962). 3Walsh, K. A., D. L. Kauffman, and H. Neurath, Biochim. Biophys. Acta, 65, 540 (1962). 4Hofmann, T., K. A. Walsh, D. L. Kauffman, and H. Neurath, Fed. Proc., 22, 528 (1963). 5 Mikes, O., V. Holeysovsky, V. Tomasek, and F. Sorm, Collection Czech. Chem. Commun., 26, 1048 (1961). 6 Mikes, O., V. Holeysovsky, V. Tomasek, B. Keil, and F. Sorm, Collection Czech. Chem. Commun., 27, 1964 (1962). 7 Tomasek, V., 0. Mikes, V. Holeysovsky, B. Keil, and F. Sorm, Biochim. Biophys. Acta, 69, 186 (1963). 8 Hofmann, T., Biochem., in press. 9 The following abbreviations are used: DIP, Asn, Gln, HSer, and AEC for diisopropyl phosphoryl, asparaginyl, glutaminyl, homoseryl, and S-,6-aminoethylcysteinyl, respectively. The abbreviation Lu indicates that leucine would not have been distinguished from isoleucine in the analytical method employed. The abbreviation Glx indicates that a residue is either glutaminyl or glutamyl; Asx indicates either an asparaginyl or an aspartyl residue. 10 Goldberger, R. F., and C. B. Anfinsen, Biochem., 1, 401 (1962). 1 Lindley, H., Nature, 178, 647 (1956). 12 Gross, E., and B. Witkop, J. Am. Chem. Soc., 83, 1510 (1961). 13 A peptide C-Ha with the structure Ileu. Lys(Gln, Thr, Ala, Ileu)Ser. Asn was isolated from a chymotryptic digest of S-sulfotrypsinogen and has not previously been reported. 14 Pech~re, J.-F., G. Dixon, R. Maybury, and H. Neurath, J. Biol. Chem., 233, 1364 (1958). '5Peptide C-12a was obtained from a chymotryptic digest of S-sulfotrypsinogen and was erroneously reported as (Asp2, Lu)3 where methionine had apparently become oxidized to the sulfone during analysis of the hydrolysate on paper. Its true sequence is Asn.Asn.Asp.Ileu. Met.Leu. 16 Davie, E. W., and H. Neurath, J. Am. Chem. Soc., 74, 6305 (1952). 17 Neurath, H., Fed. Proc., in press. 18 Neurath, H., and G. H. Dixon, Fed. Proc., 16, 791 (1957). 19 Neurath, H., and B. S. Hartley, J. Cell. Comp. Physiol., 54, 179 (1959). 20 Bender, M. L., and E. T. Kaiser, J. Am. Chem. Soc., 84, 2557 (1962). 21 Hartley, B. S., in Enzyme Models and Enzyme Structure, Brookhaven Symposia in Biology, No. 15 (1962), p Oosterbaan, R. A., P. Kunst, J. van Rotterdam, and J. A. Cohen, Biochim. Biophys. Acta, 27, 549, 556 (1958). 23Brown, J. R., and B. S. Hartley, Biochem. J., 89, 59p (1963). 24 Keil, B., Z. Prusik, L. Moravek, and F. Sorm, Collection Czech. Chem. Commun., 27, 2946 (1962). 25Keil, B., Z. Prusik, and F. Sorm, Biochim. Biophys. Acta, 78, 559 (1963). 26 Kostka, V., B. Meloun, and F. Sorm, CoUiction Czech. Chem. Commun., 28, 2779 (1963). 27 Sanger, F., J. Polymer Sci., 49, 3 (1961). 28Bender, M. L., J. Am. Chem. Soc., 84, 2582 (1962).

8 308 BIOCHEMISTRY: HASELKORN AND FRIED Pnoc. N. A. S. 29 Schoellmann, G., and E. Shaw, Biochem., 2, 252 (1963). 30 Koshland, D. E., Jr., D. H. Strumeyer, and W. J. Ray, Jr., in Enzyme Models and Enzyme Structure, Brookhaven Symposia in Biology, No. 15 (1962), p Crestfield, A. M., W. H. Stein, and S. Moore, J. Biol. Chem., 238, 2421 (1963). 32 Bruice, T. C., and R. M. Topping, J. Am. Chem. Soc., 85, 1488 (1963). 33Cunningham, L. W., Science, 125, 1145 (1957). 34 Westheimer, F. H., these PROCEEDINGS, 43, 969 (1957). 35 Spencer, T., and J. M. Sturtevant, J. Am. Chem. Soc., 81, 1874 (1959). 36 Bruice, T. C., these PROCEEDINGS, 47, 1924 (1961). CELL-FREE PROTEIN SYNTHESIS: THE NATURE OF THE ACTIVE COMPLEX* BY R. HASELKORN AND V. A. FRIED COMMITTEE ON BIOPHYSICS, UNIVERSITY OF CHICAGO Communicated by Raymond E. Zirkle, December 30, 1963 In a previous communication' we presented evidence indicating that the addition of RNA from turnip yellow mosaic virus (TYi\IV) to purified E. coli ribosomes results in the formation of a complex containing one 70s ribosome and one molecule of RNA. We shall call such complexes monosomes. Indirect evidence, consisting of a correlation between the number of monosomes formed as a function of the RNA-ribosome ratio, and the extent of amino acid incorporation measured in parallel experiments, suggested that monosomes were the active complexes for protein synthesis in the cell-free system. However, the direct experiments were restricted to the demonstration that before protein synthesis had occurred, viral RNA was to be found only in monosomes. In this paper it will be shown that after protein synthesis has occurred, the newly formed protein appears predominantly in monosomes, and while viral RNA-directed protein synthesis is taking place, the only activity for amino acid incorporation is found in monosomes.2 On the other hand, poly U promotes the formation of polysomes;3-5 these are compared with monosomes with respect to the rate and extent of amino acid incorporation. Materials and Methods.-Ribosomes and supernatant were obtained by alumina grinding of freshly grown log phase E. coli B, as described previously,6 except that the buffer used to prepare the crude extract was changed to M Mg++, 0.06 M KCl, 0.01 M Tris ph 7.6, M mercaptoethanol.7 The crude extract was centrifuged for 20 min at 16,000 rpm, and the supernatant recentrifuged for 30 min at 16,000 rpm. To deplete endogenous messenger RNA, the supernatant thus obtained was supplemented with ATP, GTP, PEP, PK, and cold amino acids, and incubated for 80 min at 361C.8 After incubation the ribosomes were purified by several cycles of centrifugation at 38,000 rpm. The supernatant from the first of these ribosome centrifugations was concentrated by dialysis against polyethylene glycol, and then dialyzed against several changes of 0.01 M Tris, M mercaptoethanol, 0.01 M Mg++, 0.02 M KC1. P32-labeled TYMV-RNA was obtained as described previously.' The poly U sample was part of a gift from L. Heppel to E. P. Geiduschek; it had S20 = 5.3 in 0.1 M NaCl. TMV-RNA was prepared by phenol extraction9 of TMV (strain Ul) purified by differential centrifugation in the presence of 0.01 Al EDTA, ph 8.'o The complete system for amino acid incorporation contained, in 0.5 ml, 25 j.moles Tris ph 7.5, 25 Mmoles KCl, 5 Mmoles Mg++, 5 Mmoles mercaptoethanol, 2 Mmoles PEP, 0.02 mg PK, 1 umole ATP, 0.2 jamoles GTP, 0.25 Moles of each amino acid except the label, ribosomes, and RNA as