Effect of Lincomycin and Clindamycin on Peptide

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1975, p Copyright American Society for Microbiology Vol. 7, No. 1 Printed in U.S.A. Effect of Lincomycin and Clindamycin on Peptide Chain Initiation FRITZ REUSSER Research Laboratories, The Upjohn Company, Kalamazoo, Michigan Received for publication 29 July 1974 Lincomycin does not affect initiation factor-dependent formation of 70S initiation complexes formed with fmet-trnaf, the initiation triplet A-U-G, and 70S ribosomes, whereas its 7-chloro-derivative clindamycin substantially stimulates this process. Conversely, lincomycin stimulates nonenzymatic formation of the 70S complex, but clindamycin does not. Both antibiotics stimulate the assembly of non-enzymatically formed 70S initiation complexes, with R,7 phage ribonucleic acid and exert little effect on those formed in the presence of initiation factors. The formation of 30S initiation complexes is stimulated or remains unaffected by lincomycin or clindamycin except when initiation occurs in the presence of very low Mg2+ concentrations. In this case, both antibiotics inhibit the assembly of the 30S complexes regardless of the messenger present. Lincomycin has been identified for some time as an inhibitor of bacterial protein synthesis (4). Subsequently, numerous authors concluded that lincomycin inhibits the peptidyltransferase function of the 50S ribosomal subunit (reviews in references 6 and 11). More recent reports suggest that lincomycin interacts with the process of chain initiation when studied in protoplast or cell lysate systems (2, 7). The present paper shows that lincomycin as well as its chlorinated analogue clindamycin affect chain initiation when studied in certain cell-free initiation systems. Streptomycin, a known inhibitor of initiation effective on the 30S subunit (10), was included in these studies for comparative purposes. MATERIALS AND METHODS Salt-washed ribosomes and 30S and 50S ribosomal subunits were prepared as described previously (8, 9) except that the 30S subunits were subjected to five sucrose gradient centrifugation steps instead of the customary three. Escherichia coli trnafmt was obtained from the Oak Ridge National Laboratories. Synthetase-transformylase enzyme required for the synthesis of fmettrnaf was isolated by the procedure of Dubnoff and Maitra (3). [thifmet-trnaf was prepared as described by Caskey et al. (1). Initiation factors were isolated from the first ribosomal salt wash fluid (3) The puromycin reaction was studied in a system measuring the synthesis of fmet-puromycin from fmet-trnaf prebound to 705 ribosomes. (See Table 7 for the composition of the reaction mixtures.) The trinucleotide A-U-G was obtained from Collaborative Research, Inc., Waltham, Mass. R17 phage ribonucleic acid (RNA) was kindly supplied by J. Davies. IJniversity of Wisconsin, Madison, Wis. Binding of fmettrnaf to 70S ribosomes and 30S ribosomal subunits was assessed by the nitro-cellulose filtration technique. RESULTS Binding of fmet-trnaf. Control experiments showed that no fmet-trnaf was bound to salt-washed ribosomes in the presence of mrna but in absence of initiation factors if the Mg2+ concentration in the reaction mixtures amounted to 2 to 4 mm. Hence, our system was strictly dependent on the participation of initiation factors to form an initiation complex in the presence of low Mg2+ concentrations. If the Mg2+ concentration in the test system was raised to 15 to 20 mm, the participation of initiation factors was no longer required to form initiation complexes. Binding carried out at low Mg2+ concentrations (2 to 4 mm) with the participation of initiation factors is referred to as enzymati initiation and initiation carried out in the presence of niigh Mg2+ concentrations (10 to 20 mm) not requiring initiation factors is referred to as nonenzymatic or Mg2+-induced initiation. We recently demonstrated that the antibiotic rubradirin specifically prevents the formation of enzymatic initiation complexes but does not affect the formation of nonenzymatic ones (9). Hence, these two types of initiation complexes must differ in some significant aspect. 32

2 VOL. 7, 1975 Initiation complexes formed with the codon A-U-G. Clindamycin markedly stimulated the enzymatic binding of fmet-trnaf to 70S ribosomes (Table 1). The extent of stimulation was approximately 2.7-fold in the presence of 2 mm Mg2+ and 1.4-fold in the presence of 4 mm Mg2+. The extent of stimulation was thus inversely related to the amount of Mg2+ present in the system. At high Mg2+ concentrations (15 or 20 mm) where nonenzymatic initiation conditions exist, clindamycin did not affect the formation of the 70S initiation complex. Clindamycin inhibited the incorporation of fmet-trnaf into 30S initiation complexes (48% TABLE 1. LINCOMYCIN-CLINDAMYCIN EFFECT ON PEPTIDE CHAINS inhibition) if the reaction mixtures contained 2 mm Mg2+ (Table 1). In the presence of 4, 15, or 20 mm Mg2+, some stimulation occurred, the extent of which decreased in relation to the increasing Mg2+ concentration. It is of interest to note that only the formation of the enzymatically induced 70S initiation complexes was stimulated by clindamycin, as discussed above. Conversely, 30S initiation complex formation was stimulated under enzymatic and nonenzymatic initiation conditions. Lincomycin stimulated the enzymatic formation of the 70S complex (Table 2). In several other experiments, stimulation of this system Effect of clindamycin on formation of 70S and 30S initiation complexes with A-U-Ga Samples Mg2+ concn fmet-trnaf Controls fmet-trnaf Controls (MM) bound Cotosbound Cnrl (counts/min) (%) (counts/md) (%) Control Clindamycin 2 1, Control 4 2, Clindamycin 4 3, Control 15 6, , Clindamycin 15 6, , Control 20 10, , Clindamycin 20 9, , The reaction mixtures contained in a total of 0.15 ml: 50 mm Tris(hydroxymethyl)aminomethane-hydrochloride, ph 7.8; 100 mm NH4Cl; Mg. acetate as indicated; 4 mm mercaptoethanol; 2 mm guanosine 5'-triphosphate; 0.04 units of optical density at 260 nm of (OD2,0) A-U-G; 4.5 OD2.0 units of 70S ribosomes, or 1.5 units of 30S ribosomal subunits; 0.11 OD2,,0 units of [3H]fmet-tRNAF containing -30,000 counts/min; and 25,g of crude initiation factors. Binding was measured after incubation of the reaction mixtures for 15 min at 37 C. Antibiotic concentration equaled 0.05 Mmol/ml. TABLE 2. Effect of lincomycin on formation of 70S and 30S initiation complexes with A-U-Ga Samples Mg2+ concn (MM) fmet-trnaf bound Controls fmet-trnaf Cotosbound Cnrl Controls (counts/min) (counts/mmn) Control 2 1, Lincomycin 2 1, Control 4 2, , Lincomycin 4 3, , Control 15 6, , Lincomycin 15 11, , Control 20 8, , Lincomycin 20 15, , a For reaction mixtures see Table 1. tjv

3 34 REUSSER was actually found to be absent. Under Mg2+induced initiation conditions, stimulation by lincomycin amounted to over 60%. Contrary to the results obtained with clindamycin, the extent of stimulation induced by lincomycin in this 70S initiation system was directly related to the Mg2+ concentration present in the reaction mixtures. Lincomycin inhibits the enzymatic formation of the 30S initiation complex to an extent of 37% at the low Mg2+ concentration of 2 mm (Table 2). In the presence of 4, 15, or 20 mm Mg2+, lincomycin marginally stimulated the binding of fmet-trnaf to 30S ribosomal subunits. The extent of stimulation decreased as the Mg2+ concentration was increased. Both antibiotics thus affected these 30S initiation systems alike, although clindamycin proved to be a more TABLE 3. ANTIMICROB. AGENTS CHEMOTHER. potent effector in each case. Streptomycin inhibited the enzymatic and nonenzymatic binding of fmet-trnaf to 70S ribosomes (Table 3). Formation of the 30S initiation complex was stimulated in the presence of 2 mm Mg2+. In the presence of 4, 15, or 20 mm Mg2+, this process was inhibited by streptomycin. Initiation complexes formed with R17 phage RNA. Clindamycin did not appreciably affect the enzymatic formation of the 70S initiation complexes (Table 4). Conversely, nonenzymatic formation of the 70S complexes (15 or 20 mm Mg2+) was stimulated more than threefold by clindamycin. Formation of the 30S initiation complex was inhibited by clindamycin in the presence of 2 mm Mg2+ only and remained essentially unaf- Effect of streptomycin on formation of 70S and 30S initiation complexes with A-U-Ga Samples Mg'+ concn fmet-trnaf Controls fmet-trnaf Controls (MM) bound Cotosbound (counts/min) ( ) (counts/min) Control 2 1, Streptomycin 2 1, Control 4 3, , Streptomycin 4 2, , Control 15 6, , Streptomycin 15 3, , Control 20 6, , Streptomycin 20 5, , a For reaction mixtures see Table 1. TABLE 4. Effect of clindamycin on formation of 70S and 30S initiation complexes with phage RNAa Samples Mg'+ concn fmet-trnaf Controls fmet-trnaf Controls (MM) bound Cotosbound Cnrl (counts/min) ( (counts/min) ( Control 2 4, Clindamycin 2 4, Control 4 5, , Clindamycin 4 4, , Control 15 1, , Clindamycin 15 5, , Control 20 2, , Clindamycin 20 9, , a The reaction mixtures were the same as described in Table 1 except that they contained 0.5 OD3*o units of R17 phage RNA in place of 0.04 OD2,,0 units of A-U-G.

4 VOL. 7, 1975 TABLE 5. LINCOMYCIN-CLINDAMYCIN EFFECT ON PEPTIDE CHAINS fected at higher Mg2+ concentrations (Table 4). The effects observed with lincomycin are smaller but parallel those obtained with clindamycin in the phage RNA-directed systems (Table 5). Enzymatic 70S complex formation was stimulated in the presence of 2 mm Mg2+ (25% stimulation) and inhibited to an extent of 16% at 4 mm Mg2+. Nonenzymatic induction of the 70S complex was stimulated more than twofold by lincomycin. Formation of the 30S complex was inhibited to the extent of 18% at 2 mm Mg2+ and remained essentially unaffected by lincomycin at higher Mg2+ concentrations. In the 70S initiation systems with A-U-G serving as the initiating condon (see above), lincomycin and clindamycin were found to differ in their effects in that lincomycin stimulated the nonenzymatic process almost exclusively, whereas clindamycin stimulated the enzymatic process. With R,7 phage RNA as a messenger, both antibiotics inhibited the Mg2+induced process only. These stimulatory effects differ depending on the messenger used and therefore are messenger dependent. Streptomycin proved to be a strong inhibitor of the formation of the 70S complex regardless of whether initiation occurred enzymatically or nonenzymatically (Table 6). Inhibition amounted to 70% in the initiation factor-induced systems and from 34 to 58% in the Mg2+-induced systems. Formation of the 30S initiation complex was not inhibited by streptomycin in the presence of 2 mm Mg2+. In the presence of 4, 15, and 20 mm Mg2+, inhibition did occur and amounted from 40 to 43% (Table 6). Thus streptomycin which was used as a control in our systems was found to inhibit both the 70S and 30S initiation systems. In addition, Effect of lincomycin on formation of 70S and 30S initiation complexes with phage RNAa Samples Mg2+ (MM) concn fmet-trnaf Controls fmet-trnaf Controls bound Cotosbound Cnrl (counts/m)in) (counts/minm) Control 2 3, Lincomycin 2 4, Control 4 4, Lincomycin 4 4, Control 15 1, , Lincomycin 15 4, , Control 20 3, , Lincomycin 20 7, , a For reaction mixtures see Table 4. TABLE 6. Effect of streptomycin on formation of 70S and 30S initiation complexes with phage RNAa Samples Mg2+ concn fmet-trnaf Controls fmet-trnaf Contrs (MM) bound Cotosbound Cnrl (counts/min) ( (counts/min) ) Control 2 2, Streptomycin Control 4 3, , Streptomycin 4 1, Control 15 1, , Streptomycin , Control 20 3, , Streptomycin 20 1, , a For reaction mixtures see Table 4. 35

5 36 REUSSER complexes formed with viral RNA proved more sensitive to streptomycin inhibition than the ones formed with A-U-G. These results essentially corroborate the ones of previous investigators (6, 10). Effect of clindamycin and lincomycin on the puromycin reaction. Previous investigators have demonstrated that lincomycin inhibits peptide bond formation, as studied with the puromycin reaction (11). The concentrations of this antibiotic required to effect inhibition of this reaction are higher (approximately 0.5,umol/ml) than the ones required to stimulate initiation (0.05 Asmolml). We thus studied the effect of low antibiotic concentrations (0.05 gmol/ml) on the puromycin reaction in a system which measures the formation of fmetpuromycin from fmet-trnaf prebound to 70S ribosomes. The results obtained show that neither lincomycin nor clindamycin had any effect on the puromycin reaction when present in concentrations (in the case of lincomycin) sufficient to stimulate fmet-trnaf binding to 70S ribosomes (Table 7). DISCUSSION The differential effects observed between clindamycin (stimulation of enzymatic initiation) and lincomycin (stimulation of nonenzymatic initiation) are confined to 70S initiation complexes induced by the triplet A-U-G which represents a somewhat non-physiological test system. No such differential effects are apparent with the R17 phage-induced 70S system. In TABLE 7. Effect of lincomycin and clindamycin on puromycin reactiona fmet-trnaf Cha Sample bound nges (counts/min) Control, 20 min 1,160 Control, 30 min 1, Puromycin only Lincomycin only 1, Lincomycin and puromycin Clindamycin only 1, Clindamycin and puromycin a Reaction mixtures were as described in the footnote to Table 1 (15 mm Mg2+). The 70S initiation complex was formed in the absence of drugs by incubating the reaction mixtures for 20 min. Antibiotics were then added, and the mixtures were incubated for an additional 10 min. Amounts used: puromycin, 1 pmol/ml; lincomycin or clindamycin, 0.05 gmol/ml. ANTIMICROB. AGENTS CHEmOTHER. this latter case, both antibiotics exert identical effects and stimulate the nonenzymatic initiation systems only. The differential effects in the 70S initiation systems induced by A-U-G are reproducible, but it is somewhat aiuficult to judge whether these effects are trivial or whether they also manifest themselves in whole cells. However, the possibility has to be considered that the mode of action of lincomycin and clindamycin might not necessarily be identical in all aetaui at the rirosomal level. The formation of the 30S initiation systems studied remains either unaffected or is slightly stimulated by clindamycin and lincomycin except when initiation occurs in the presence of very low Mg2+ levels (2 mm Mg2+). Under these latter initiation conditions, moderate inhibition results with either antibiotic. The magnitude of this inhibitory effect is small but significant as it is observed with both antibiotics and is independent of the messenger used (A-U-G or phage RNA). Lincomycin so far has been considered to be an effector of the 50S ribosomal subunit (6). Our data suggest that lincomycin as well as clindamycin also interact with an initiation function associated with the formation of the 30S complex. This effect is probably secondary in nature as it is small. This conclusion is substantiated by the observation that lincomycin and clindamycin, under certain conditions, substantially stimulate the formation of the 70S complex, which functionally follows the formation of the 30S complex. The described stimulatory effects of the two antibiotics on peptide chain initiation are measurable at concentrations well below the ones required to inhibit peptide bond formation as studied with the puromycin reaction. In the case of lincomycin, it has been well established that this antibiotic binds preferentially to the 50S ribosomal subunit. In conjunction with our results, this suggests that lincomycin and clindamycin interact with the 50S ribosomal subunit, which results in a modification of the P-site to produce an enhanced binding capacity for fmet-trnaf under certain initiation conditions in cell-free systems. It is difficult to extrapolate the various stimulatory effects of clindamycin and lincomycin observed in cellfree systems to phenomena which might occur in whole bacterial cells. However, other wellknown inhibitors of bacterial protein synthesis such as the tetracyclines and pactamycin also stimulate certain cell-free systems. The tetracyclines stimulate the enzymatic formation of 705 initiation complexes directed by R17 phage-rna but not the ones directed with A-U-G (5); pactamycin stimulates the polyuridylic acid-

6 VOL. 7, 1975 LINCOMYCIN-CLINDAMYCIN EFFECT ON PEPTIDE CHAINS 37 directed synthesis of polyphenylalanine in the presence of high Mg2+ concentrations (8). In general, our results obtained while working with cell-free initiation systems with washed ribosomes and defined messengers suggest that lincomycin and clindamycin affect the process of peptide chain initiation. This notion is supported by the results of Cundliffe (2) and Pestka (7), who by the use of protoplast or cell lysate systems also concluded that these two antibiotics affect the initiation process. ACKNOWLEDGMENTS I am indebted to J. Davies for his gift of R,, phage RNA. The technical assistance of J. L. Kay is gratefully acknowledged. LITERATURE CITED 1. Caskey, C. T., E. Scolnick, R. Tomkins, G. Milman, and J. Goldstein Release factors: in vitro assay and purification. Methods Enzymol. 20: Cundliffe, E Antibiotics and polyribosomes. II. Some effects of lincomycin, spiramycin, and streptogramin A in vivo. Biochemistry 8: Dubnoff, J. S., and U. Maitra Isolation and properties of protein factors involved in peptide chain initiation in E. coli. Methods Enzymol. 20: Josten, J. J., and P. M. Allen The mode of action of lincomvcin. Biochem. Biophvs. Res. Commun. 14: Modolell, J Action of streptomycin and tetracycline on polypeptide synthesis initiation. Int. Congr. Biochem. 8th, Switzerland. 6. Pestka, S Inhibit6rs of ribosome functions. Annu. Rev. Microbiol. 25: Pestka, S Studies of transfer ribonucleic acidribosome complexes. J. Biol. Chem. 247: Reusser, F Rubradirin, an inhibitor of ribosomal polypeptide biosynthesis. Biochemistry 12: Reusser, F Rubradirin, a selective inhibitor of initiation factor dependent peptide chain initiation. Biochemistry 12: Schreiner, B., and K. H. Nierhaus Protein involved in the binding of dihydrostreptomycin to ribosomes in E. coli. J. Mol. Biol. 81: Weisblum, B., and J. Davies Antibiotic inhibitors of the bacterial ribosome. Bacteriol. Rev. 32: Downloaded from on October 12, 2018 by guest