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Moted Lysine Incorporation Was Susceptible to Hy 54 BIOCHEMISTRY: KAZIRO ET AL. PROC. N. A. S. Applied Chemistry Symposium on Protein Structure, ed. A. Neuberger (New York: John Wiley and Sons, 1958). 7 Craig, L. C., T. P. King, and W. Konigsberg, Ann. N.Y. Acad. Sci., 88, 571 (1960). 8 Craig, L. C., T. P. King, and A. Crestfield, Biopolymers, in press. 9 Craig, L. C., and W. Konigsberg, J. Phys. Chem., 65, 166 (1961). 10 Hill, R. J., W. Konigsberg, G. Guidotti, and L. C. Craig, J. Biol. Chem., 237, 1549 (1962). 11 Hasserodt, U., and J. Vinograd, these PROCEEDINGS, 45, 12 (1959). 12 Rossi-Fanelli, A., E. Antonini, and A. Caputo, J. Biol. Chem., 236, 391 (1961). Is Benesch, R. E., R. Benesch, and M. E. Williamson, these PROCEEDINGS, 48, 2071 (1962). 14 Antonini, E., J. Wyman, E. Bucci, C. Fronticelli, and A. Rossi-Fanelli, J. Mol. Biol., 4, 368 (1962). 16 Vinograd, J., and W. D. Hutchinson, Nature, 187, 216 (1960). 16Stracher, A., and L. C. Craig, J. Am. Chem. Soc., 81, 696 (1959). 17 Bromer, W. W., L. G. Sinn, A. Staub, and 0. K. Behrens, J. Am. Chem. Soc., 78, 3858 (1956). IsSquire, P. G., and C. H. Li, J. Am. Chem. Soc., 83, 3521 (1961). IDENTIFICATION OF PEPTIDES SYNTHESIZED BY THE CELL-FREE E. COLI SYSTEM WITH POLYNUCLEOTIDE MESSENGERS* BY YOSHITO KAZIRO, ALBERT GROSSMAN, AND SEVERO OCHOA DEPARTMENT OF BIOCHEMISTRY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE Communicated May 23, 1963 Characterization of the products formed by cell-free systems of protein synthesis as a result of the incorporation of amino acids into acid-insoluble products, pro- moted by synthetic polyribonucleotides, has lagged behind the use of these com- pounds in studies on the genetic code.1' 2 Nirenberg and Matthaei' partially characterized the product of phenylalanine incorporation by the Escherichia coli system, in the presence of poly U, as poly L-phenylalanine. This characterization was based on comparison with an authentic sample of the chemically synthesized compound as regards solubility and susceptibility to acid hydrolysis. However, the insolubility of the product in aqueous solvents precludes identification through cleavage with proteolytic enzymes. This is also true of polypeptides made with copolynucleotide messengers rich in uridylic acid. The insolubility of these-- prod- ucts also interferes with the determination of their chain length, or the identifica- tion of the C- and N-terminal amino acids, by conventional methods of end-group assay.4 The finding5 that poly A directs the synthesis of polylysine which, although in- soluble in tungstic acid, is soluble in trichloroacetic acid and in aqueous solvents, opened the way for enzymatic and chemical studies of the products synthesized by cell-free systems with poly A or with adenylic acid-rich copolynucleotide mes- sengers. Like chemically synthesized polylysine, the product of the poly A-pro- moted lysine incorporation was susceptible to hydrolysis by trypsin, but resistant to the action of chymotrypsin.5 Further work, to be reported in this paper, has shown that the product of lysine incorporation with poly A as messenger- yields di- and trilysine on tryptic digestion, This conclusively identifies this. product as poly-L-lysine. Downloaded by guest on September 25, 2021 VOL. 50, 1963 BIOCHEMISTRY: KAZIRO ET AL. 55 All of the eight triplets occurring in AU copolymers have been assigned as fol- lows:" AAA, lysine; 2AlU, asparagine, isoleucine, lysine; 1A2U, isoleucine, leucine, tyrosine; UUU, phenylalanine. Thus, AU copolymers promote the incorpora- tion of the six amino acids, asparagine, isoleucine, leucine, lysine, phenylalanine, and tyrosine. As reported in this paper, high-voltage electrophoresis of tryptic digests of the products formed, in the presence of poly AU (5:1), with either (a) lysine-C'4, (b) isoleucine-C14, (c) lysine-C14 + isoleucine-C14, or (d) lysine-C14 + asparagine-C14, and the remaining four or five amino acids without radioactive label, yields a series of radioactive peptide peaks. The major peaks are six in number when lysine-C14 is present in the incubation mixture, but only four when the C14 label is in amino acids other than lysine. Peaks 5 and 6 move in the posi- tion of di- and trilysine. Upon acid hydrolysis, peaks 1 through 4 all yield radio- active isoleucine + lysine and asparagine + lysine in experiments (c) and (d), respectively. These results show that synthetic copolynucleotide messengers give rise to copolypeptides in the E. coli system. Methods.-Composition of reaction mixtures: All reaction mixtures contained per ml (in umoles unless otherwise stated) Tris-HCl buffer, pH 7.9, 37; Lubrol, 1.8 mg; KC1, 55; MgCl2, 13; mercaptoethylamine, 11; ATP, 0.9; GTP, 0.22; creatine phosphate, 12; creatine kinase, 44 1g; E. coli transfer RNA, 3 mg; E. coli ribosomes, 2 mg protein; and E. coli supernatant, 4 mg protein. In addition, in experiments with poly A the samples contained polymer, 40 ,ug, and lysine C14, (7 uc/Mmole), 0.2. In experiments with poly AU (5: 1) they contained polymer, 80 ug, and aspara- gine, isoleucine, leucine, lysine, phenylalanine, and tyrosine, each 0.05. In this case one or more amino acids were labeled with C14 and were used at specific radioactivities between 50 and 100 Mc/Amole. Incubation was for 30 min at 370. Isolatton of lysine-rich polypeptides by differential precipitation: One advantage of dealing with polylysine or lysine-rich polypeptides is that, due to their solubility in trichloroacetic acid,6 they can be largely separated from proteins in the reaction mixture including C14-labeled, trichloroacetic acid-insoluble material which is always formed by the system in small amounts, whether in the presence or absence of synthetic polynucleotides, probably as a result of the presence of residual messenger RNA in the E. coli fractions. This separation was accomplished as follows. An equal volume of 10% trichloroacetic acid was added to the reaction mixture after incubation. The pre- cipitate was collected by centrifugation, washed three times with 2 ml of 5% trichloroacetic acid, each time with heating for 15 min at 900, and dissolved in 0.5 N KOH This fraction (Fraction A) contained some radioactive protein. After digestion of this material with trypsin followed by high- voltage paper electrophoresis, there was lit e or no migration of radioactivity from the origin. To the supernatant, which was combined with hings of the trichloroacetic acid precipitate, was added 1.0 mg of carrier polylysine followed by sodi tungstate to a final concentration of 0.25%. The precipitate was recovered by centrifugation, shed three times with a solution of 0.25% sodium tungstate in 5% trichloroacetic acid, adjus to pH 2.0, each time with heating for 15 min at 900, and finally taken up in 1.6 N ammonium hydroxide. Insoluble material having little radioactivity was removed by centrifugation and discarded. The clear ammonium hydroxide solution (Fraction B) contains the bulk of the polylysine or lysine-rich polypeptides. In an experiment with poly AU (5:1) (isoleucine-C14, lysine-C 4, cold asparagine, leucine, phenylalanine, and tyrosine), 25,000 cpm were recovered in Fraction A and 477,000 cpm in Fraction B. Trypsin digestion and separation of peptides: Fraction B was evaporated to dryness in a flash evaporator and the residue taken up in about 2 ml of water. To the suspension was added 0.5 ml of 10% ammonium bicarbonate, 1.0 mg of carrier polylysine, and drystalline trypsin (0.05 mg in experiments with poly A, 0.2 mg in experiments with poly AU). The volume was made up with water to 5.0 ml and the mixture incubated for 16 hr at 37°. The tryptic digest was evap- orated to dryness and dissolved in a few ml of water. The evaporation and solution were repeated several times to ensure complete removal of the ammonium bicarbonate. The final residue was dissolved in as little water as possible, and this solution subjected to either paper chromatography or paper eleotrophoreuis. Downloaded by guest on September 25, 2021 56 BIOCHEMISTRY: KAZIRO ET AL. PROC. N. A. S. In experiments with poly A, the products of tryptic digestion were separated by descending chromatography for 48-72 hr in n-butanol: acetic acid: water: pyridine (30: 6: 24: 20 v/v) accord- ing to Waley and Watson.7 Under these conditions, lysine, dilysine, and trilysine moved about 15, 9, and 6 cm, respectively, from the origin in 48 hr. In experiments with poly AU, separation of the products of tryptic digestion was accomplished by high-voltage paper electrophoresis.8 In most cases electrophoresis was conducted for 2.5 hr at 4,000 volts in pyridine: acetate: water (10:1:69 v/v, pH 6.0) and the samples were banded near the anode. Under these conditions, lysine, dilysine, and trilysine moved 57, 66, and 71 cm, respectively, toward the cathode. All neutral amino acids migrated 8 cm in the same direction. Authentic samples of amino acids and a tryptic digest of authentic poly-L-lysine, containing di- and trilysine and some tetralysine, were used as markers. The spots were located with ninhydrin spray, and radioactivity was detected with use of a strip counter (Nuclear-Chicago Actigraph) supplemented occasionally by autoradiography. Other methods: In experiments with poly AU, following separation of the tryptic peptides by paper electrophoresis, paper strips corresponding to radioactive peaks were cut out, shredded into small pieces, and eluted with 10 ml of water overnight. Approximately 60% of the radio- activity was recovered in this way. After decanting the fluid, the paper residue was washed twice with 3-5 ml of water, and the washings were combined with the eluate. The solution was dried by flash evaporation, the residue dissolved in 5.7 N hydrochloric acid of constant boiling point, and the peptides were hydrolyzed in a sealed tube for 17 hr at 106°.
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