Purification and partial characterization of mitochondrial ribosomes from a HeLa cell line resistant to chloramphenicol

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Miller, Kenton
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Biochemistry, Biophysics and Molecular Biology

Techniques are described for the purification of mitochondrial ribosomes from two lines of HeLa cells, S(,3) and 296-1, one of which (296-1) is resistant to chloramphenicol in vivo. The in vitro protein synthetic activity of purified mitochondrial ribosomes from both strains are compared for their sensitivity to a variety of drugs including chloramphenicol;For the preparation of mitochondrial ribosomes from either cell line, crude mitochondria are washed several times in buffer containing 0.002M EDTA, and mitoplasts are prepared by treatment with digitonin at a digitonin to mitochondrial protein ratio of 0.15. The mitoplasts are lysed by the addition of Brij-58 and Nonidet P-40 at a final concentration of 0.5% each (w/v) and mitochondrial ribosomes are collected by pelleting through a 1M sucrose pad (105,000 x G(,ave) for 16 hours);HeLa cell mitochondrial ribosomes prepared in this manner are free of contaminating cytoplasmic ribosomes as judged by sucrose gradient centrifugation and by extraction and analysis of the ribosomal RNA in polyacrylamide gels;The preparations are active in both poly(U) and poly(C,U)-directed polypeptide synthesis when supplemented with tRNA and supernatant factors from E. coli. However, mitochondrial supernatant factors (S-100) from rat, rabbit and HeLa cells were not able to replace those from E. coli. Despite repeated attempts to subfractionate these S-100 preparations, a potent inhibitor of in vitro polypeptide synthesis remained. At least one of these preparations was shown to contain significant RNase activity;Mitochondrial ribosomes from these cell lines differ in their sensitivity to chloramphenicol. When assayed for poly(C,U)-directed polypeptide synthesis, HeLa S(,3) mitochondrial ribosomes are inhibited 50% by 200 (mu)g/mL chloramphenicol. Mitochondrial ribosomes from HeLa 296-1 are unaffected at concentrations up to 400 (mu)g/mL. From these results, it is clear that for HeLa 296-1, the cytoplasmically inherited mutation to chloramphenicol resistance has affected a component of the mitochondrial ribosome. In vitro, polypeptide synthesis on HeLa 296-1 ribosomes is also resistant to inhibition by lincomycin at concentrations which were found to significantly inhibit HeLa S(,3) mitochondrial ribosomes. This is presently the only report of cross-resistance between these two drugs.

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Thu Jan 01 00:00:00 UTC 1981