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A. MORRIS, R. ARLINGHAUS,
s. FAVELUKES,
AND R. SCHWEET
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Biochemistry
von Ehrenstein, G., and Lipmann, F. (1961), Proc. Nut. Acad. Sci. U . S. 47, 941. Warner, J. R., Knopf, P. M., andRich, A. (1963), Proc. Nut. Acad. Sci. U . S. 49, 122. Warner, J. R., Rich, A., and Hall, C. E. (1962), Science 138, 1399. Webster, G. C. (1957), J. Bid. Chern. 229, 535. Weinstein, I. B., and Schechter, A. N. (1962), Proc. Nut. Acud. Sci. U . S. 48, 1686. Young, R . J., Kihara, H. K., and Halvorson, H. 0. (1961), Proc. Nut. Acad. Sci. U. S. 47, 1415.
Inhibition of Hemoglobin Synthesis by Puromycin* ALLANMORRIS, t RALPHARLINGHAUS,t SUSANNA FAVELUKES, AND RICHARD SCHWEET $ From the Department of Biochemistry, University of Kentucky College of Medicine, Lexington Received April 11, 1963 The inhibition of hemoglobin synthesis by puromycin was proportional to inhibitor concentration. However, inhibition a t low levels of puromycin increased with time. Preincubation studies showed that a small amount of protein synthesis in the presence of puromycin resulted in almost complete inhibition a t 2 x 10-5 M concentration of inhibitor. The inhibitor caused the release of labeled protein from the ribosomes, and this release was related to the degree of inhibition. The released material contained incomplete globin chains, as shown by solubility, chromatographic behavior, and N-terminal analysis. About 25% of the released material contained chains which were newly started in the cell-free system. Release of incomplete chains took place a t 4 O in the absence of added enzyme, although this was a slow reaction. The mechanism of puromycin action suggested was that the inhibitor substitutes for the next incoming amino acid a t the growing point of the peptide chain (the carboxyl end). This displaces the chain. Further synthesis can occur, but yields only small peptides which are displaced, and are acid soluble. The inhibition of protein synthesis by puromycin, first reported by Yarmolinsky and de la Haba (1959), has been confirmed in various systems (Nathans and Lipmann, 1961; Morris and Schweet, 1961; Hultin, 1961). More complex effects of puromycin have been reported by Nemeth and de la Haba (1962), Mueller et al. (1961), and Rabinovitz and Fisher (1962). A direct effect of puromycin on ribosomes resulting in the release of soluble protein was found by Morris and Schweet (1961), Hultin (1961), Morris et al. (1962), Allenand Zamecnik (1962),and Lamborg (1962). Detailed studies of the mechanism of puromycin action on reticulocyte ribosomes are reported here.
* This study was supported by a grant (H-5293) from the U. S. Public Health Service and a grant (G-21026) from the
Preparation of Labeled Ribosomes.--Incubations of intact cells were carried out by a modification of the procedure of Borsook et al. (1957). For each 2 ml of washed reticulocytes was added 0.33 ml of rabbit plasma, 1.84 ml. of twice-concentrated NKMl salt solution, 0.067 ml. of 1 M Tris buffer, p H 7.5, 1.0 ml of amino acid mixture less valine, 0.067 ml. of 0.01 M ferrous ammonium sulfate, 0.15 ml of 1 x M Cl4-~-valine, and 1.5 ml of water. The mixture was incubated 15 minutes a t 37" and the cells were then centrifuged, resuspended in cold NKM solution, and recentrifuged. Labeled ribosomes were then isolated in the usual manner, except that unlabeled high-speed supernatant (approximately 4 mg protein/mg ribosomal protein) was added to the ribosome suspension after the first sedimentation of the particles in order to dilute any adsorbed or occluded labeled hemoglobin. For the labeling of ribosomes in the cell-free system, the ribosome pellet after the first centrifugation (Allen and Schweet, 1962) was suspended in 0.25 M sucrose to a final concentration of approximately 14 mg of ribosomes per ml (6-7 mg of ribosomal protein) and incubated 10 minutes a t 37" in the usual complete system with C14-leucine or other C14-amino acid and other components of the cell-free system (Allen and Schweet, 1962). The reaction was terminated by the addition of 6-10 volumes of cold medium B (0.25 M sucrose, 0.0175 M KHCO,, 0.002 M MgC12)containing a 50-fold excess of C1*-leucine. The labeled ribosomes were then reisolated by centrifugation. Studies of Released Components.-The release of labeled material from labeled ribosomes either with or
National Science Foundation. t Postdoctoral fellow of the National Institutes of Health, U. S. Public Health Service. $ Career Research Awardee of the U. S. Public Health Service.
1 Abbreviations used in this work: 'TCA, trichloroacetic acid; chloramine T, sodium pura-toluenesulfonchloramine; DIFP, diisopropylfluorophosphate; NKM, 0.13 M NaCl, 0.005 M KC1,0.0075 M MgC12.
EXPERIMENTAL Materials.-DL-l-C I4-leucine was purchased from the California Corporation for Biochemical Research and had a specific activity of 10.3 pc/pmole. Uniformly labeled L-valine, L-arginine, and L-lysine were obtained from the Nuclear-Chicago Corporation and had specific activities of 6.5, 2.5, and 8.3 pc/pmole, respectively. Puromycin hydrochloride was kindly donated by Dr. E. Stokstad of Lederle Laboratories. Rabbit reticulocytes, enzyme fractions, and other components of the cell-free system have been described (Allen and Schweet, 1962). The data have been calculated for C14-amino acid a t a specific activity of 7 pc/pmole, which gave 2.3 X l@ cpm per pmole in the thin-window Geiger counter used.
Vol. 2, No. 5, Sept.-Oct., 1963 without puromycin was studied as follows. After incubation, the contents of a single incubation tube (usually 1.4 ml) were cooled and transferred quantitatively to 4-ml centrifuge tubes (Spinco centrifuge rotor No. 40) with the aid of 2 ml of 0.25 M sucrose. The ribosomes were removed by centrifugation at 40,000 rpm for 60 minutes. The supernatant solution containing the released material was removed and the ribosome pellet was rinsed with small amounts of sucrose solution. Casein solution (15 mg/ml) was added to the supernatant plus washings to a total protein content of 15 mg, and the mixtures were precipitated with trichloroacetic acid (TCA) a t a final concentration of 5%. The ribosome pellet was made into a paste with a glass rod and then suspended by the addition of 1 ml of 0.1 M Tris-HC1 buffer, p H 7.5. The suspension was then decanted into a tube containing casein in an amount calculated to give approximately 15 mg of total protein. Quantitative transfer of the ribosomes was ensured by washes with 0.5 ml of 1 N NaOH and two small volumes of 5% TCA. The ribosome and supernatant solutions were then washed and plated as described for the usual incubation mixture. Certain types of incubations resulted in the conversion of TCAprecipitable radioactivity originally present in labeled ribosomes into TCA-soluble radioactivity. The acidsoluble radioactivity was calculated by the difference in TCA-precipitable radioactivity before and after incubation. In all cases, the TCA-precipitable material was taken through the complete washing procedure before determination of radioactivity. The radioactivity of the acid-soluble materials could be determined directly only after dialysis of labeled ribosomes to remove adsorbed C14-leucine. Ribosomes were suspended in a medium containing 0.25 M sucrose, 0.0175 M KHCO,, 0.001 M MgC12, and 2 X l o - 4 ~ C12-leucineand dialyzed for 12 hours against the same medium without the leucine. N-Terminal valine analysis followed the procedure of Bishop et al. (1960). Protein (10 mg) was precipitated with 5% TCA, washed twice with 1% TCA, dissolved in 0.1 N NaOH, and immediately adjusted to p H 9.5 as described. Quantitative decarboxylation with chloramine-?' was used to determine the percentage of C14-leucine present as free amino acid in the acid-soluble fraction, which also contained labeled peptides (see below). The following procedure was used. Acid insoluble material was precipitated with 5% TCA and centri fuged. The supernatant was decanted, and the in soluble material was washed twice with small amounts of 5% TCA. The combined supernatants and washes were filtered through Whatman No. 4 paper and TCA was removed by extraction with ether. Residual ether was then removed by bubbling nitrogen through the solution, and aliquots were used for the decarboxylation analysis. The reaction was carried out in a vessel which allowed 1 ml of 6 % (w/v) solution of chlor amine-T to be placed in one part of the container, the analytical sample plus 3 mg of C12-glycineplus 0.4 ml of citrate buffer (0.1 M, p H 2.5) in another portion of the vessel, and 0.3 ml of 1 M hyamine base in a small cup in the center of the reaction chamber. After a partial vacuum was applied to the system, the vessels were closed to the outside atmosphere and the chloramine and C14-sampleswere mixed and incubated for 30 minutes a t 30". The vessels were then opened and the hyamine C14-carbonates were dissolved in 20-ml counting vials containing 15 ml of counting fluid (Bishop et al., 1961). The radioactivity was then determined in a Packard Liquid Scintillation Spectrometer. Fingerprint studies were conducted according to the
PUROMYCIN INHIBITION
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