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Mechanism of action of streptomycin : studies with polynucleotide phosphorylase and ribosomes Willick, Gordon Edward


Some of the previous work on the mechanism of action of streptomycin has indicated the general area of nucleic acid metabolism as being a possible site of action. Specifically, a report has appeared that streptomycin inhibits the "exchange reaction" catalyzed by polynucleotide phosphorylase. Therefore, the possible action of streptomycin on polynucleotide phosphorylase from a streptomycin sensitive strain of Escherichia coli has been reinvestigated. In preparation for the study on polynucleotide phosphorylase, the nucleoside diphosphates of adenosine, guanosine, cytidine, and uridine were synthesized in sufficient yield and of a satisfactory purity by the recently developed method of Moffatt and Khorana. It was not possible to confirm the inhibition of the polynucleotide phosphorylase catalyzed "exchange reaction" reported by Kornberg, using enzyme from our streptomycin sensitive E. coli SA. Nor was it possible to demonstrate any inhibition of the polymerization reaction catalyzed by this enzyme, or any effect on the sedimentation properties of the polymer so formed. However, the diamines putrescine, cadaverine, spermine, and spermidine, while having no effect on the time course of the polymerization reaction, did lower the sedimentation coefficient of the polymer formed and cause a more heterogeneous polymer to be formed. Streptomycin has been suggested as an inhibitor of protein synthesis. Therefore, a study of the possible effects of streptomycin on ribosomes from E. coli was made. These subcellular particles have been shown to be a site of protein synthesis. Dihydrostreptomycin (DHSM) interacted strongly with the ribosomes. It was found that most of the ribonucleo-protein precipitated when ribosomes were dialyzed overnight at 4° against buffer containing about 500 μg./ml. of DHSM. A study of ultracentrifuge patterns of dialysates of ribosomes against lower levels of DHSM indicated that disruption, with a loss of discrete sedimentation coefficients, occurred when the level of DHSM was about 350 μg./ml. A study of elution patterns from DEAE-cellulose columns indicated only a partial change in the pattern after breakdown. Examination of sedimentation coefficients of ribosome dialysates at lower DHSM levels indicated no specific effect of DHSM. The autodegradation of ribosomes by polynucleotide phosphorylase was studied. DHSM, at low levels, had an effect on the time course of the auto-degradation. A study of the distribution of induced β-D-galactosidase associated with the ribosomes indicated that it was associated with that protein of the ribosomes not precipitated by DHSM. It can be concluded that streptomycin interacts strongly with ribosomes. This gives support to recent claims that streptomycin inhibits ‘in vitro' protein synthesis, and that the site of this inhibition is the ribosome.

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