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Inhibition of Protein Synthesis by Blasticidin S I. Studies with Cell-Free

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Inhibition of Protein Synthesis by Blasticidin S I. Studies with Cell-Free The Journal of Biochemistry Vol. 57, No. 5, 1965 Inhibition of Protein Synthesis by Blasticidin S I. Studies with Cell-free Systems from Bacterial and Mammalian Cells By HIDEYO YAMAGUCHI, CHIEKO YAMAMOTO and NOBuo TANAKA (From the Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo) (Received for publication, December 14, 1964) It has been demonstrated that blasticidin S, a useful fungicidal antibiotics, also exhibits MATERIALS AND METHODS antibacterial activity (1), toxicity to mam Chemicals-Purified blasticidin S in a crystal form malians and tumor-inhibitory activity against was kindly supplied by Prof. Hiroshi Yonehara, transplantable animal tumors (2). Although Institute of Applied Microbiology, University of the structure of blasticidin S is not finally Tokyo. L-Leucine-C14 (U) and L-phenylalanine-C14(U) determined, the antibiotic is proved to consist were purchased from the Radiochemical Centre, of cytosine and a pentose linked with a sort Amersham, England, and poly U from CALBIOCHEM, Los Angeles, Colifornia. of amino acid (3). In this respect, blasticidin Preparation of Extracts of Bacterial Cells-E. coli S belongs to the group of nucleoside anti strain B was grown at 37°C in a nutrient broth sup- biotics, of which puromycin is most extensively plimented with 0.5% glucose with reciprocal shaking. investigated concerning the mechanism of The cells were harvested in logarithmic growth phase, action. It is established that puromycin washed twice with standard buffer containing 10-IM inhibits protein synthesis by acting on the potassium chloride, 10-2M magnesium chloride, process following aminoacyl-sRNA formation •~10-2M Tris buffer, pH 7.8, and 6•~10-1M ƒÀ- (4, 5), and by stimulating the release of mercaptoethanolamine, and were stocked in a frozen incomplete peptide chains from ribosomal state until used. The cells of B. megaterium strain particles (6-11). IAM 1030 grown in a nutrient broth at 30°C were The present study was undertaken to harvested, washed and stocked in a similar manner. determine whether or not blasticidin S ex Crude extracts, ribosomes and supernatant frac tions were prepared by grinding frozen cells with hibits a similar mode of action with puromycin quartz powder and by centrifugation in a similar way in protein synthetic pathways. The evidence as described by Nirenberg and Matthaei (72). presented here indicates that blasticidin S Preparation of E. coli sRNA and C14-Leucyl-sRNA markdly inhibits amino acid incorporation -E. coli sRNA was obtained from 105,000xg into polypeptides under the directions of supernatant of quartz-grinding frozen cells by phenol natural or synthetic messengers in cell-free treatment according to the method of N i r e n b e r g systems prepared from bacterial and mam and M a t t h a e i (12). C14-Leucyl-sRNA was prepar malian cells, and that it blocks a certain step ed by the method of N a t h a n s and L i p m a n n (5). Preparation of Cell Free Systems of Mammalian following aminoacyl-sRNA formation with Ceels-In the present study normal rat liver and inhibiting release of peptides from the ribo mouse Ehrlich ascitic tumor were used. Ribosomal some. Blasticidin S appears to inhibit poly particles of rat liver cells were obtained from quickly U-directed polyphenylalanine synthesis not by excised livers of male Wistar rats (120-180 g. body affecting the attachment of ribosomes to weights) by homogenization, deoxycholatetreatment messenger strands, but by acting on the and centrifugal fractionation according to the method active polysomal complexes. It is also sug described by Kirsch et al. (13). gested that the mechanism of action of The Ehrlich carcinoma cells were collected by blasticidin S is different from that of puro centrifugation of the ascitic fluid of dd strain. mice mycin in details. inoculated with 5 X 106 tumor cells 7 days before. 667 668 H. YAMAGUCHI, C. YAMAMOTO and N. TANAKA The ribonucleoprotein particles were obtained from the cell pellet by a similar method as described by Littlefield and Keller (14). The pH 5 enzymes [mixture of L-aminoacide ; SRNA ligases (AMP), EC 6.1.1 group] of both kinds of mammalian cells were prepared by the method of Hoagland et al. (15). Chemical Procedures-Preparations of RNA and protein fractions from the reaction mixtures, quantita tive estimation and determination of radioactivity were carried out according to the method described in the previous paper (16). RESULTS Antibacterial Activity of Blasticidin S-When 5.8•~ 10-5 M of blasticidin S was added to the logarithmic phase culture of E. coli, the cell FIG. 2. Effect of Blasticidin'S/on the'growth of B. megaterium. Same methods as described in Fig. 1. growth was slightly inhibited within 3 hours. Increase of the cell number was completely suppressed at 2.3 X10-4 M and the viable cell number was markedly decreased at 9.2 x 10-4M. A similar inhibition was observed, when cells of B. megaterium was grown in the presence of blasticidin S, except that 2.3 x 10-4M of the antibiotic exhibited a bactericidal activity (Figs. I and 2). From these results, 2.3 X 10-4M was regarded as the minimal growth-inhibitory concentration against these two species of microogramisms. Inhibition by Blascidin S of Leucine Incorpo ration into Polypeptidesin Cell-free Systemsof E. coli and B. megaterium-As presented in Tables I and II, C14-leucine incorporation into poly peptides by extracts of E. coli and of B. FIG. 1. Effect of Blasticidin S on the growth megateriumunder the direction of endogenous of E. coli. messengers was markedly affected by blasticidin One volume of overnight culture of E. coli in S. In the system of E. coli, approximately a nutrient broth was mixed with 1,000 volumes of the same medium and shaken at 37°C vigorously 50% inhibition of leucine incorporation was for 3 hours. The definite concentrations of observed at the concentration of 2.3 x 10-7M blasticidin S (BS) was subsequently added and the of the antibiotic, which corresponds to the viable cell number of each tube was counted after concentration 1,000 times as low as that another 3 hours shaking by the method described required for growth inhibition. Althougn elsewhere (16 ). less inhibitory activity was demonstrated in Blasticidin S, an Inhibitor of Protein Synthesis 669 TABLE I Inhibition by Blasticidin of C14-Leucine Incorporation into Polypeptide in a Cellfree System of E. coli. The complete reaction medium contained (per ml.) : 2-4 mg. protein of ribosome, 1-2 mg. protein of 105,000Xg supernatant, 1 u mole of ATP, 5 ƒÊmoles of creatine phosphate (CP), 50 ƒÊg. of creatine kinase [EC 2.7. 3.2], 0.2 pc of C14-L-leucine, 0.3 limole each of 20 L-amino acids minus leucine, 100 ƒÊmoles of KC1, 10 ƒÊmoles of MgC12 and 50 ƒÊmoles of Tris buffer, pH 7.6, in a total volume of 0.3-0.5 ml. After incubating for 30 minutes at 37°C, 0.5 ml. of cold 10% trichloracetic acid was added to terminate the reaction. The resultant precipitates were washed twice with 5% trichloroacetic acid (TCA), extracted with 5% TCA at 90°C for 20 minutes, again washed with ethanol and ethanol-ether (3: 1), suspended in N-NH4OH and counted in a windowless gas flow counter, with correction for self-absorption. the system of B. megaterium, blasticidin S at tion into polypeptides (Table III). Therefore 2.3xl0-5M, which was comparable to 1/100 it is not plausible that the inhibitory effect of of the minimal growth-inhibitory concentra blasticidin S is attributed to the activity on tion, exhibited 50% inhibition. Moreover energy generation. inhibitory effects of the antibiotic were Ineffectivenessof Blasticidin S on Charging of demonstrated even at the concentrations less sRNA with Leucineand Phenylalanine by Extracts than 5x10-8M in both bacterial systems. of E. coli and B. megaterium-According to the These results indicated that in a certain outlines of the process of protein synthesis so bacterial cell-free systems blasticidin S ex far confirmed, amino acid is first activated hibited much higher activity against protein (18, 19) and then transferred by a specific synthesis than that against cell growth. enzyme to SRNA which is specific for each Inllenceof Blasticidin S on ATP Generation amino acid (20, 21 ). -When 15,000x g supernatant fluid obtained The extent of labeling of RNA of 105,000 from quartz powder-grinding frozen cells of Xg supernatant, containing activating enzymes E. coli or B. megateriumwas used as an enzyme and SRNA, with C14-Leucine and C14-phe source, the formation of P32-ATP dependent nylalanine in the presence or absence of the on oxidative phosphorylation was not affected antibiotic was comparatively studied. As in the presence of high levels of blasticidin S, presented in Table IV, high concentrations of which markedly inhibited leucine incorpora- blasticidin S, sufficiently to inhibit leucine 670 H. YAMAGUCHI, C. YAMAMOTO and N. TANAKA TABLE II Inhibition by Blasticidin S of C14-Leucine Incorporation into Polypeptide in a Cellfree System of B. megaterium. The complete reaction mixture contained: ribosome 1.6-1.0mg. protein, 105,000Xg supernatant 1.6-1.0 protein, ATP 0.3 ƒÊemole, CP 1.5 ƒÊmoles, creatine kinase 30 ƒÊg., GTP 0.1 ƒÊmole, KC1 30 ƒÊmoles, MgC12 3ƒÊmoles, Tris buffer, pH 7.8, 15ƒÊmoles, C14-leucine 0.1 pc and 20 L-amino acid mixture minus leucine 0.01 ƒÊmole each, in a total volume of 0.3 ml. Incubation was carried out at 35•Ž for 60 minutes. TABLE III Effect of Blasticidin S on Formation of P32-ATP from P,,32 by Crude Enzymes of E.
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