Fusidic Acid and Related Antibiotics Inhibit Protein Synthesis In

The Journal of Biochemistry, Vol. 67, No. 3, 1970

Inhibition by Fusidic Acid of Transferase II in Reticulocyte Protein Synthesis

By NOBUO TANAKA, TOSHIO NISHIMURA and TADATOSHI KINOSHITA

(From Institute of Applied Microbiology, University of Tokyo, Tokyo)

(Received for publication, September 8, 1969)

Protein synthesis with endogenous mRNA and polyphenylalanine synthesis with poly U were inhibited by fusidic acid in a highly fractionated reticulocyte system. It interfered with ribosome-dependent GTPase activity of TF-II. The grade of inhibition by fusidic acid of the polypeptide synthesis and the GTPase reaction was parallel and it was comparable to what was observed in the bacterial system. The antibiotic did not significantly affect the puromycin reaction with polyphenylalanyl-tRNA or acetyl phenylalanyl-tRNA, presumably attached to the donor site of ribosomes. However, puromycin reaction, enhanced by GTP and TF-II, was inhibited by fusidic acid. It suggested that the antibiotic selectively inhibited the translocation of peptidyl-tRNA from the acceptor site to the donor site on the reticulocyte ribosomes. The results were in accordance with those obtained with bacterial G factor, and indicated that TF-II may be a mammalian equivalent of G factor.

Fusidic acid and related antibiotics inhibit munication (9). A similar result was reported protein synthesis in the bacterial system in vivo independently by MALKIN and LIPMANN(10). and in vitro (1-3 ). In an E. coli system, they inhibit ribosome-dependent GTPase activity MATERIALS AND METHODS of G factor, and the grade of inhibition is Sodium fucidate was kindly supplied by Dr. W.O. parallel to that of polypeptide synthesis (4). Godtfredsen, Leo Pharmaceutical Products, Ballerup, The antibiotic seems to be a specific inhibitor Denmark. L-Phenylalanine-14C (297 mCi/mmole) and of G factor and to inhibit translocation of L-phenylalanine-3H (2 Ci/mmole) were purchased from Daiichi Chemical Co., Tokyo. 14C-„@mino acid mix peptidyl-tRNA on the ribosomes (4-8). ture of Chlorella protein hydrolysate was given by Prof. For the purpose of elucidating the bio- B. Maruo, Institute of Applied Microbiology, Univer chemical basis of the selective toxicity, the sity of Tokyo. Poly U was obtained from Calbiochem., sensitivity of mammalian protein-synthesizing Los Angeles, California. systems to fusidic acid has been investigated. The reticulocytes were collected from rabbits,

It has been observed that fusidic acid inhibits which had been injected with phenylhydrazine, fol ribosome-dependent GTPase activity of TF-II lowing the method of ALLEN and SCHWEET (12). KCI- and polypeptide synthesis, using a purified washed ribosomes, deoxycholate-treated ribosomes reticulocyte system. Puromycin-dependent (3„VDOC ribosomes), and transferases (TF-I and TF- release of peptide from the ribosomes is not II) were prepared by the method of ARLINGHAUS et al. significantly affected by fusidic acid in the ( 13, 14). NH4CI-washed ribosomes were obtained by the method of FELICETTI and LIPMANN (15). Reti absence of GTP and TF-II ; but the puromycin culocyte sRNA was labelled with 14C-phenylalanine reaction, enhanced by GTP and TF-II, is and 14C-amino acid mixture of Chlorella protein, fol inhibited by the antibiotic. It indicates that lowing the method of HOAGLAND et al. (16). Acetyl- fusidic acid inhibits translocation of peptidyl- 3H-phenylalanyl-sRNA was prepared by the method tRNA on the ribosomes. The results are pre of HAENNI and CHAPEVILLE (11), using E. coli B sented in this publication. A part of the pres sRNA. GTPase assay was performed by the method ent work has been reported as a short com- of FELICETTi and LIPMANN (15). GTP-ƒÁ-32P was pre- 459 460 N. TANAKA, T. NISFIIMURA and T. KINOSHITA pared as described previously (6). studied in a highly fractionated reticulocyte system with endogenous mRNA. The anti- RESULTS biotic was observed to interfere with the amino The effects of fusidic acid on amino acid acid polymerization. The extent of the inhibi transfer from aminoacyl-sRNA to protein was tion was 83% at the fusidic acid concentra-

TABLE I Inhibition by antibiotics of protein synthesis in a reticulocyte system

The assay for protein synthesis was performed in the reaction mixture, containing (per ml) : 0.1 M

KCI-washed ribosomes 500,ƒÊg, TF-I 100ƒÊg, TF-II 130ƒÊg, GTP 0.05 /moles, MgCl2, 6.7 ƒÊmoles, KCl 6.7ƒÊ moles, Tris-HCI, pH 7.5, 33 ,moles, GSH 10,moles, aminoacyl-tRNA of 1°C-amino acid mixture of

C/ilorellaa protein (83,000 cpm/mg RNA) 4,340 cpm, in a total volume of 0.5 ml. It was incubated for 10 min at 37°C. The radioactivity of the hot TCA-insoluble fraction was determined in a windowless gas flow counter.

TABLE II Inhibition by antibiotics of polyphenylalanine synthesis in a reticulocyte system

The reaction mixture contained (per ml): L-phenylalanine 0.067ƒÊmoles, GTP 67ƒÊmoles, poly U 100ƒÊg, TF-I 200ƒÊg, TF-II 132ƒÊg, 3„V DOC ribosomes 800„Vg, 14C-phenylalanyl-tRNA 125„Vg (19,800epm), GSH

10 smoles, MgC12, 6.7„Vmoles, KCI 67„Vmoles, Tris-HCI 33„Vmoles, pH 7.5, in a volume of 0.3 m1. It was incubated at 37°C for 15 min. The radioactivity of the hot TCA-insoluble fraction was determined in a windowless gas flow counter. Fusidic Acid on TF-II 461 tion of 370ƒÊM, 60°,'o at 37ƒÊM, and 38% at alanine from the ribosomes was not significantly 3.7ƒÊM. The results are presented in Table I. affected by fusidic acid in the absence of GTP As summarized in Table II, polyphenyl and TF-II. But the puromycin reaction, stim alanine synthesis with poly U was also in ulated by GTP and TF-II, was definitely in hibited by fusidic acid in the reticulocyte sys hibited by fusidic acid at the concentration tem. Approximately 50% inhibition was dem of 370 poi. The results showed that the puro onstrated at the concentration of 3.7 pi. mycin reaction with polyphenylalanyl-tRNA, The ribosome-dependent GTPase activity presumably attached to the donor site of of TF-II was significantly inhibited by fusidic ribosomes, was not affected by fusidic acid; acid. Approximately 50% inhibition was ob- but the translocation of polyphenylalanyl- served at the concentration of 3.7ƒÊM. The tRNA from the acceptor site to the donor site on the ribosomes was inhibited by fusidic grade of inhibition was found to be parallel to that of polypeptide synthesis. Phenomycin acid. A similar result was obtained with the (17,18) and blasticidin S (19, 20) which were inhibitors of protein synthesis in the mam synthesis of acetyl 3H-phenylalanyl-puromycin. malian system did not affect the GTPase reac The puromycin reaction with acetylphenyl tion. The results are shown in Table III. alanyl-tRNA, presumably attached to the donor The effects of fusidic acid on puromycin- site, was not significantly affected by fusidic dependent release of peptide from the poly acid in the absence of GTP and TF-II. The phenylalanyl-poly U-ribosome complex were puromycin reaction, enhanced by GTP and investigated in the absence and presence of TF-II, was depressed by the antibiotic. It GTP and TF-II. As illustrated in Fig. 1, indicated again that the translocation of acetyl puromycin-dependent release of 1°C-phenyl- phenylalanyl-tRNA from the acceptor site to the donor site was inhibited by fusidic acid. The results are summarized in Table ‡W. TABLE III

Effects of antibiotics on ribosome-dependent DISCUSSION GTPase activity of TF-II Polypeptide synthesis is inhibited by fusidic The ribosome-dependent GTPase activity of acid in a highly fractionated reticulocyte sys- TF-II was assayed by measuring liberation of tem. The antibiotic interferes with ribosome- radioactive inorganic phosphate from GTP-ƒÁ-32P dependent GTPase activity of TF-II. The The reaction mixture contained (per ml) : 0.5M grade of inhibition by fusidic acid of both NH4CI-washed ribosomes 585ƒÊg, TF-II 146 jig, reactions is parallel and it is comparable to GTP-ƒÁ-32P (2•~l06 cpm/ƒÊmole) 0.1ƒÊmole, Dƒ²ƒ² what is observed with the bacterial system 16ƒÊmoles, KCI 80ƒÊmoles, MgCl, 10ƒÊmoles, and Tris-HCI 50ƒÊmoles, pH 7.4. It was incubated (4-6). The puromycin reaction with pep

for 15 min at 37°C. The radioactivity was deter- tidyl-tRNA, presumably attached to the donor mined in a GM counter. site of the ribosomes, is not significantly affected by fusidic acid; but the puromycin reac tion, enhanced by GTP and TF-II, is inhib ited by the antibiotic. It indicates that fusidic acid may not affect peptidyl transferase reac tion but may inhibit the translocation of pep tidyl-tRNA from the acceptor site to the donor site on the ribosomes. The present study suggests that TF-II and GTP are essential for the translocation of pep tidyl-tRNA on the ribosomes. And the basic mechanism of amino acid polymerization or the function of polymerization factors is similar in the bacterial and mammalian systems; i.e. 1) 100=8 ,240 cpm 0.2 m1. 462 N. TANAKA, T. NISHIMURA and T. KINOSHITA

FIG. 1. Effects of fusidic acid on puromycin-dependent release of peptide from the ribosomes in the absence and presence of GTP and TF-II.

The reaction mixture contained (per ml): 14C-phenylalanyl-tRNA-charged ribosomes 1.5 mg, poly U 100ƒÊg, TF-II 130ƒÊg, GTP 67 mƒÊmoles, puromycin 212mƒÊmoles, KCI 67ƒÊmoles, MgCI2 6.7ƒÊmoles, and Tris-HCI 33,ƒÊmoles, pH

7.5, in a total volume of 0.3 ml. It was incubated at 37°C for 10 min and sucrose density gradient, 7.5 to 30%"0, centrifugation analysis was performed at 40,000rpm for 90min at 5°C. Each 15 drop-fraction was assayed for O.A.260 and for TCA-insoluble radioactivity. The radioactivity was determined in a windowless gas flow counter.

(a) Control.

(b) With puromycin 212ƒÊM. (c) With puromycin 212ƒÊM and fusidic acid 370ƒÊM. (d) With TF-II 130ƒÊg/ml and GTP 67ƒÊM.

(e) d+puromycin 212ƒÊM.

(f) d+puromycin 212ƒÊM+fusidic acid 370ƒÊM. Fusidic Acid on TF-II 463

TABLE I V Effects of antibiotics on the synthesis of acetyl-3H-phenylalanyl-puromycin in the presence and absence of GTP and TF-II in a highly fractionated reticulocyte system

The reaction mixture contained (per ml) : 3„V DOC ribosomes 1.2 mg, TF-II 80ƒÊg, poly U 100ƒÊg,

DTT 2ƒÊmoles, acetyl-3H-phenylalanyl-tRNA 455ƒÊg RNA (57,000 cpm), puromycin 212 mƒÊmoles, GTP 100 mƒÊmoles, KC1 67ƒÊmoles, MgCl2 13ƒÊmoles, and Tris-HCI 33ƒÊmoles, pH 7.5, in a total volume of 0.2 ml. The reaction mixture without GTP, TF-II, and antibiotics was incubated for 20min at 30°C, and then

GTP, TF-II and the antibiotic were added and incubated for 8 min at 30°C. Puromycin was then intro

duced to the mixture and incubated for 10 min at 30°C. Acetyl-3H-phenylalanyl-puromycin was extracted by ethylacetate, following the method of LEVER and BURSZTYN (21), and counted in a windowless gas flow counter.

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