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. coli and B. megaterium.

The reaction mixture contained crude cell extracts (105,000Xg supernatant fluid) prepared from

quartz powder firinding frozen cells of E. coli or B. megaterium 10-15 mg. protein, ATP 3 /ƒÊmoles, tris buffer, pH 7.4, 50ƒÊmoles, MgC12 10 ƒÊmoles, casamino acids (Difco)+tryptophan 1 mg. and P132 3ƒÊmoles

(0.1 pc), in a total volume of 2.0 ml. After incubating at 25•Ž for 15 minutes with aeration, the reaction was terminated by adding 2 ml of 10% TCA. Separation of ATP and inorganic phosphate was carried

out from the acidified medium according to the method described by Crane and L i p m a n (17).

incorporation into polypeptides by extracts sRNA with each amino acid. It was con prepared from the same organisms, had no cluded from the results that blasticidin S inhibitory effects on the extent of charging neither affects the step of amino acid activa- Blasticidin S, an Inhibitor of Protein Synthesis 671

tion nor that of aminoacylation of SRNA. to the ribosomes where SRNA molecules Inhibition by Blasticidin S of Incorporationof translate the sequence of nucleotides along a S RNA-bound Leucine into Protein in a System of strand of messenger RNA molecule into a E. coli-A number of works on the protein sequence of amino acids and consequently synthesis showed that aminoacyl-sRNA travels polypeptide chains grow from the amino end

TABLE IV Effect of Blasticidin S on Labeling of SRNA with C14-Leucine or with C14-Phenylalanine by Cell Extracts of E. coli and B. megaterium.

The reaction mixture contained: 105,000Xg supernatant fluid of cell extracts of E. coli or Bmegaterium

6 mg. protein, ATP 5/moles, C14-leucine or C14-phenylalanine 0.05 µc, L-amino acids mixture minus

phenylalanine or leucine 0.05 ƒÊmoles each, KCl 50 ƒÊmoles, MgC12 5 ƒÊmoles and Tris buffer, pH 7.6, 10 ƒÊ moles, in a total volume of 0.5 ml. Incubation was carried out at 37°C for 15 minutes. The RNA

fraction was isolated by the method described elsewhere (16 ).

TABLE V Effect of Blasticidin S on Transfer of C14-Leucinefrom C14-Leucyl-SRNA to Polypeptide in a System of E. coli.

The complete reaction mixture consisted of washed ribosome 3.3 mg. protein, supernatant 0.8 mg.

protein, sRNA charged with C14C-leucine 0.3 mg. (3,000 c.p.m.), ATP 0.5 ƒÊmoles, CP 2.5 ƒÊmoles, creatine kinase 50 ƒÊg., GTP 0.15 ƒÊmole, glutathione (GSH) 3 ƒÊmoles, 20 L-amino acids mixture minus leucine 0.1

ƒÊ mole each, KCl 50 ƒÊmoles, MgC12 5 ƒÊmoles and Tris buffer, pH 7.6, 25 ƒÊmoles, in a volume of 0.51 ml. After incubation at 37°C for 15 minutes, the hot acid-insoluble fraction was isolated to measure the radio-

activity. 672 H. YAMAGUCHI, C. YAMAMOTO and N. TANAKA

TABLE VI Effect of Blasticidin S on Release of Labeled Compounds from Prelabeled Ribosomes of E. coli

The complete reaction mixture contained: E. coli ribosomes which were prelabeled with C14-leucine under the condition described in the footnote of Table I and were washed twice with tris buffer, pH 7.6, 10 mg. protein (16,600 c.p.m.), supernatant 6 mg. protein, ATP 0.75 ƒÊmole, CP 3.8 ƒÊmoles, creatine kinase

50 ƒÊg., GTP 0.23 ƒÊmole, 20 L-amino acids mixture 0.1 ƒÊmole each, KCl 75 ƒÊsmoles, MgC12 7.5 ƒÊmoles, Tris HC1 buffr, pH 7.6, 40 ƒÊmoles, in a total volume of 0.75 ml. Incubation was carried out at 37°C for 60 minutes. After the reaction, cold TCA-soluble and hot TCA-insoluble materials were prepared from the

soluble fraction which was separated from ribosomal sediment by centrifugation at 105,000xg for 120 minutes.

(22, 23). similar to that of puromycin observed in Because blasticidin S has a marked in mammalian (4) and bacterial (5) in vitro hibitory activity on over-all leucine incorpora systems. tion into polypeptides without affecting Inhibition by Blasticidin S of Release of activation of amino acids or their transfer to Radioactivityfrom the RibosomesPreviously Labeled S RNA, the site of action of the antibiotic is with C14-Leucineto the Supernatant in an in vitro presumably located in the subsequent steps in Bacterial System-Effects of blasticidin S and protein synthetic pathway. As shown in puromycin on release of labeled leucine from Table V, it was definitely demonstrated that the ribosomes in the forms of acid-soluble or blasticidin S suppressed the incorporation of -insoluble peptides or free amino acid was leucine linked with SRNA into polypeptides comparatively studied in an in vitro system to the same degree as that observed when of E. coli. Since the possibility of breaking free leucine was incorporated in the same in down labeled ribosomes during the incubation vitro system of E. coli (Table I). was excluded in the preliminary experiments, The above results suggested that blasticidin radioactivity of the supernatant fraction was S affects protein synthesis by blocking a not derived from the structural proteins of certain steps following aminoacyl-sRNA for ribosomes, but from the nascent proteins mation. Such an effect of the antibiotic is formed by the previous reaction. As present- Blasticidin S, an Inhibitor of Protein Synthesis 673

TABLE VII Inhibition by Blasticidin S of Poly U-directed Polyphenylalanine Synthesis in a System of E. coli.

Incubated crude extracts (inc. S30) (12) of E. coli 2.0 mg. protein, ATP 0.5 ƒÊmole, CP 2.5 ƒÊmoles, creatine kinase 25 ƒÊg., GTP 0.015 ƒÊmole, E. coli sRNA 1.2 mg., KCI 50 ƒÊmoles, MgCl2 5 ƒÊnnoles, ƒÀ-mer captoethanol 3 ƒÊmoles and Tris-HC1 buffer, pH 7.8, 50 ƒÊmoles were mixed and incubated at 35°C with or

without antibiotics. After 10 minutes preincubation, C14-phenylalanine 0.02 ƒÊmole, 20 L-amino acids minus

phenylalanine 0.025 ƒÊmole each and poly U 5,!g. were added to a final volume of 0.5 ml. and incubation was continued for an additional 40 minutes.

ed in Table VI, approximately half radio- investigated with a crude extract of E. coli. activity of the ribosomes was transferred to As presented in Table VII, poly U-directed the supernatant as acid-insoluble and cold polyphenylalanine synthesis was markedly .acid-soluble materials within 60 minutes in inhibited by blasticidin S which was added cubation at 37°C in the control complete to the reaction mixture and incubated for system, in which release of acid-insoluble 10 minutes prior to the addition of poly U. peptides was partly dependent upon energy In other experiments, the same results were sources, GTP and the supernatant. Puromycin obtained with the system, in which blasticidin was observed to accelerate the release of S and poly U were simultaneously added to nascent peptides from the ribosomes in the the reaction mixture before the incubation. forms of cold acid-soluble and hot acid- Comparing the results in Table VII with insoluble materials. Contrary to the stimulatory those in Table I, the inhibitory effect on poly effect of puromycin, blasticidin S suppressed U-directed polyhenylalanine synthesis appear- this reaction with or without energy sources ed to occur to a similar degree with the effect or the supernatant. The results suggested on leucine incorporation into polypeptides that there seems to exist a different mechanism under the direction of endogenous messengers. of action between these two nucleside anti On the contrary, puromycin possessed a less biotics. activity on poly U-directed peptide synthesis Inhibition of Poly-U-directedPolyphenylalanine than on leucine incorporation. Synthesis by Blasticidin S in a Bacterial System- Time-courseStudy of Inhibition by Blasticidin Effects, of blasticidin S on polypeptide synthesis S of Poly U-directed Polyphenylalanine Synthesis directed by poly U (polyuridylic acid), a in the Bacterial System-The experiment was synthetic messenger for polyphenylalanine, was performed to decide whether blasticidin S had 674 H. YAMAGUCHI, C. YAMAMOTO and N. TANAKA

TABLE VIII Inhibition by Blasticidin S of C14-Leucine Incorporation into Polypeptide in a Cell free System of Mouse Ehrlich Ascites Tumor.

The complete reaction mixture contained (per ml.) : ribosome 0.6 mg. protein, pH 5 fraction 0.6 mg.

protein, ATP 1 ƒÊmole, CP 5 ƒÊmoles, creatine kinase 50 ƒÊg., GTP 0. 3,ƒÊmole, C14-Leucine 0.4 pc, sucrose 250 ƒÊmoles, KC1 50 ƒÊmoles, MgCl2 10 ƒÊmoles, Tris-HC1 buffer, pH 7.6, 50 ƒÊmoles, in a volume of 0.5 ml. Incubation was at 37°C for 40 minutes.

TABLE IX Effect of Blasticidin S on C14-Leucine Incorporation into Polypeptide in a Cellfree System of Normal Rat Liver.

The complete reaction mixture contained (per ml.) : ribosome 0.9 mg. protein, pH 5 fraction 0.3 mg.

ATP I ƒÊmole, CP 5 ƒÊmoles, creatine kinase 25 ƒÊg., GTP 0.3 ƒÊmole, C14-leucine 0.1 pc, sucrose 250 ƒÊmoles,

KC1 25 ƒÊmoles, MgC12 10 ƒÊmoles, Tris-HC1 buffer, pH 7.8, 35 ƒÊmoles, in a volume of volume of 0.5 ml.

Incubation was carried out at 37°C for 60 minutes.

to be added to the reaction mixture prior to 3 minutes. By 3 minutes incubation, approxi the addition of poly U to reveal a sufficient mately one- third as high as maximal incor activity. The polyphenylalanine synthesis was poration was reached. Therefore it seems immediately blocked by blasticidin S, when it plausible that blasticidin S affects the poly was added to a ribosomal system of E. coli peptide synthesis even after the formation of which had been preincubated with poly U for the active complexes containing one or more Blasticidin S, an Inhibitor of Protein Synthesis 675 ribosomes attached to a poly U molecule. The of blasticidin S on leucine incorporation into results are illustrated in Fig. 3. protein was also observed in cell-free systems of mammalian source : mouse Ehrlich ascitic tumor and normal rat liver. In a cell-free system of Ehrlich carcinoma, blasticidin S significantly inhibited leucine incorporation into protein under the direction of natural messengers. The results are presented in Table VIII. Approximately 50% inhibition occurred at the concentration of 2.3x10-6M and the degree of inhibition seemed to be not less than that observed in a cell-free system of B. megaterium. Although blasticidin S also affected leucine incoporation with the ribosomal particles of normal rat liver cells, the extent of inhibition was much lower and 2.3 x 10-4 M of the antibiotic caused 50% inhibition (Table IX). Chioramphenicol and puromycin exhibited similar degrees of activities against both mammalian systems.

DISCUSSION

FIG. 3. Time-course of inhibitory effect of It was reported by H u a n g et al. (24 )

Blasticidin S on poly U-directed polyphenylalanine that in a cell-free system of Piricularia oryzae synthesis in a system of E. coli blasticidin S markedly inhibited C14-amino . The complete reaction mixture was same as acids incorporation into protein with lower described in Table VII. To one series of tubes extent of inhibition of aminoacyl-sRNA for the definite concentration of Blasticidin S (BS) was mation. We obtained the results that in a added simultaneously with poly U and to the system from E. coli the transfer of leucine other 3 minutes after. Radioactivity of hot tri from leucyl-sRNA to ribosomal polypeptides chloroacetic acid precipitate was determined. -•~ Complete was blocked by the antibiotic to the extent

-•œ- plus Blasticidin S 10-3M at 0min. enough to account for inhibition of free leucine -•£- n 10-4M at n incorporation. It is therefore suggested that

-•¡- II 10-5M at ti blasticidin S possesses common sites of action -•ü- plus Blasticidin S 10-3 M at 3 min. in both bacterial and fungal protein synthetic -11- a 10-4M at ti pathways. -• - 10-5M at ti Contrary to the stimulatory effect of puromycin, blasticidin S was found to have Inhibition by Blasticidin S of in vitro Protein an inhibitory effect on the release of peptides Synthesiswith RibosomalParticles of Mammalian from the ribosome. In this respect, the site Origins-The above results revealed that amino of action of blasticidin S seems to be different acid incorporation into polypeptides was from that of puromycin, although both anti markedly inhibited by blasticidin S in bacterial biotics similarly blocked the protein synthesis in vitro systems. Since blasticidin S is known following aminoacyl-sRNA formation. The to exhibit a tumor-inhibitory activity accom steric structure of blasticidin S may be so panied with toxic effects on host animals, it deviated from naturally occurring aminoacyl was expected that metabolic pathways of S RNA as to become its analogue capable of neoplastic and normal mammalian cells were being incorporated into the end of peptide blocked by the antibiotic. Attempts were chains formed on the ribosome, as is the case made to examine whether an inhibitory effect with puromycin (25). 676 H. YAMAGUCHI, C. YAMAMOTO and N. TANAKA

The inhibition by blasticidin S of leucine not be due to the chelating activity. incorporation into protein appeared to be not Since blasticidin S is a basic compound mainly due to such a reduced release of containig a guanido group in the structure peptides from the ribosome, because in the (3), it is plausible to anticipate the binding present in vitro system of E. coli a large portion of the antibiotic molecule with RNA moiety of incorporated C14-amino acid was detected of the ribosome, SRNA linked with amino in the ribosomal protein fraction with a acid and/or messenger RNA to disorder their smaller extent of labeling of the soluble frac functions in protein synthesis. Concerning tion. Although the significance of the this problem, experiments are in progress to inhibitory effect on the release of peptides determine a more precise site of action of the from the ribosome remains to be determined, antibiotic. blasticidin S may principally attack the pro The finding presented in this paper also cess concerning the formation of peptide chains demonstrate that in neoplastic and normal on the ribosome in participation of aminoacyl mammalian cell systems blasticidin S inhibits S RNA, messengers and related enzymes. in vitro protein synthesis as is the case with Whether the ribosome and blasticidin S bacterial systems. The toxicity and tumor- were mixed before addition of poly U or inhibitory activity of the antibiotic (2) may after it, the resultant inhibition of polyphyl be attributed to the inhibitory effect on alanine synthesis occurred in a similar degree. protein synthesis. In two systems of mammalian Moreover the polyphenylalanine synthesis was cells, mouse Ehrlich carcinoma and normal immediately and sufficiently arrested by rat liver, while chloramphenicol and puro addition of the antibiotic within several mycin exhibited a similar extent of inhibition, minutes after initiation of the reaction. These the inhibitory effect of blasticidin S on leucine results indicate that blasticidin S may not incorporation was much higher in the former affect the attachment of ribosomes to a mes system than that in the latter system. senger strand to form an active complex, a It is concluded that blasticidin S is a potent

polyribosome (26), but that the site of action inhibitor of protein synthesis in in vitro of the antibiotic may be located in the steps bacterial, fungal and mammalian systems and following the formation of polyribomes. This that the mechanism of action seems to be assumption is supported by the results that different from that of any other protein leucine incorporation into protein under the inhibitors so far investigated. direction of endogenous messengers which are SUMMARY known to be attached to the ribosomes is inhibited by the antibiotic to a similar extent Effects of blasticidin S, a nucleoside anti- with that of polyphenylalanine synthesis biotic of structural similarity with puromycin, directed by poly U. Such a site of action of on protein synthetic pathways was studied blasticidin S appears to be different from that stepwise to determine the site of action of the of streptomycin reported by F 1 a k s et al. (27 ) antibiotic in a bacterial system. Blasticidin S and by C o x et al. (28). markedly inhibited leucine incorporation into Although blasticidin S is capable of form- polypeptides under the direction of endogenous ing chelates with Mg2+, Mn2+ and other messengers in extracts of E. coli and B. cations, analytical studies of Schlieren diagrams megaterium. No inhibition by the antibiotic of the ribosome of E. coli and B. megaterium of oxidative phosphorylation was detected. suspended in tris buffer containing 0.01 M Without interfering with formation of amino- Mg"" showed no dissociation or degradation acyl-SRNA, it blocked the transfer of leucine of the 30 S, 50 S or 70 S peaks by the incuba from leucyl-SRNA to polypeptides to the extent tion of 60 minutes at 37°C at the concentration enough to account for the inhibition of free as high as 2.3•~10-4 M of the antibiotic. It is leucine incorporation into polypeptides. Unlike therefore suggested that the inhibitory effect puromycin, blasticidin S reduced the release of on amino acid incorporation into protein may peptides from the ribosome to the supernatant. Blasticidin S, an Inhibitor of Protein Synthesis 677

It inhibited poly U-directed polyphenylalanine (9) Hultin, T., Biochim. et Biophys. Acta, 51, 219 synthesis to the degree as high as that of the (1961) inhibition of leucine incorporation into poly (10) Rabinovitz, M., and Fisher, J. M., J. Biol. Chem., 237, 477 (1962) peptides under the direction of natural messengers. It was suggested that the anti- (11) Schweet, R., Bishop, J., and Morris, A., Lab. Invest., 10, 992 (1961) biotic attacked a certain steps following the (12) Nirenberg, M. W., and Matthaei, J. H., Proc. formation of active complexes of ribosomes Natl. Acad. Sci. U. S., 47, 1588 (1961) and messenger strands. Blasticidin S also (13) Kirsch, J. F., Siekevitz, P., and Palade, G. E., inhibited leucine incorporation into poly J. Biol. Chem., 235, 1419 (1960) peptides in cell-free systems of mammalian (14) Littlefield, J. W., and Keller, E. B., J. Biol. cells, more highly in a system of mouse Chem., 224, 13 (1957) Ehrlich carcinoma than in that of normal rat (15) Hoagland, M. B., Stephenson, M. L., Scott, J. liver. F., Hecht, L., I., and Zamecnik, P. C,. J. Biol. Chem., 231, 241 (1958) This investigation was supported in whole by (16) Yamaguchi, H., J. Antibiotics, 14A, 313 (1961) Public Health Service Researdh Grant CA05082-04, (17) Crane, R. K., and Lipmann, F., J, Biol. Chem., 201, 235 (1953) from the U.S. National Cancer Institute. (18) Hoagland, M. B., Zamecnie, P. C., and Stephenson, M. L., Biochim.et Biophys. Acta, 24, REFERENCES 215 (1957) (1) Takeuchi, S., Hirayama, K., Ueda, K.. Sakai, (19) Berg, P., J. Biol. Chem., 222, 1025 (1956) H., and Yonehara, H., J. Antibiotics, 11A, 1 (20) Hecht, L. I., Stephenson, M. L., and Zamecnik, (1958) P. C., Proc. Natl. Acad. Sci. U. S., 45, 505 (1959), (2) Tanaka, N., Sakagami, Y., Nishimura, T., and (21) Berg, P., and Ofengand, E. J., Proc. Natl. Acad. Yamaki, H., J. Antibiotics, 14A, 123 (1961) Sci. U. S., 44, 78 (1958) (3) Yonehara, H., Otake, N., Takeuchi, S., and (22) Bishop, J., Leahy, J. and Schweet, R., Proc. Endo, T., I. A. M. Symposia on Microbiology, 6, Natl. Acad. Sci. U. S., 46, 1030 (1960) 31 (1964) (23) Dintzis, H., Proc. Natl. Acad. Sci. U. S., 47, 247 (4) Yarmolinsky, M. B., and de la Haba, G. L., (1961) Proc. Natl. Acad. Sci. U. S., 45, 1721 (1959) (24) Huang, K. T., Misato, T., and Asuyama, H., (5) Nathans, D., and Lipmann, F., Proc. Natl. Acad. J. Antibiotics, 17A, 65 (1964) Sci. U. S., 47, 497 (1961) (25) Nathans, D., Proc. Natl. Acad. Sci. U. S., 51, (6) Morris, A., Favelukes, S., Arlinghaus, R., and 585 (1964) Schweet, R., Biochem. Biophys. Research. Com (26) Gilbert, W., J. Mol. Biol., 6, 374 (1963) muns.,7, 326 (1962) (27) Flaks, J. G., Cox, E. C., and White, J. R., (7) Allen, D. W., and Zamecnik, P. C., Biochem. et Biochem. Biophys. Research. Communs., 7, 385 Biophys. Acta, 55, 865 (1962) (1962) (8) Morris, A. J., and Schweet, R. S., Bischem. et (28) Cox, E. C., White, J. R., and Flaks, J. G., Biophys. Acta, 47, 415 (1961) Proc. Natl. Acad. Sci. U. S., 51, 703 (1964)