Purification and properties of an N-formylmethionyl-tRNA hydrolase.
The isolation and properties of a novel N-formylmethionyl-tRNA hydrolase (hydrolase II) from Escherichia coli are described. This enzyme is difficult to detect in crude extracts; purification, however, unmasks the activity. Sedimentation and gel filtration parameters of this enzyme differ from those of the previously described peptidyl-tRNA hydrolase (hydrolase I), and preparations can be obtained where the two activities are free of each other. A mutant of hydrolase I has wild-type levels of hydrolase II. These data indicate that hydrolase II is a different enzyme, or an altogether different form of hydrolase I. The bulk of the enzymic activity occurs in the ribosome-free cytoplasm; the remainder is found on intact or dissociated 70-S ribosomes. Purified preparations of hydrolase II analyzed by two-dimensional gel electrophoresis contain 2 protein bands. These 2 proteins do not coincide in electrophoretic mobility with any known ribosomal proteins. Analysis after mixing experiments verifies this conclusion. The purified enzyme (hydrolase II) is inhibited by ribosomes bearing bound N-formylmethionyl-tRNA. The inhibition is potentiated by sparsomycin and other antibiotics that block specifically peptide-bond synthesis. The relationship of this enzyme to other hydrolytic activities, including a newly described ribosome-dependent hydrolase, are discussed.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D009239:N-Formylmethionine; D010442:Peptide Chain Initiation, Translational; D012329:RNA, Bacterial; D012343:RNA, Transfer; D012270:Ribosomes / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D009239:N-Formylmethionine; D012343:RNA, Transfer; D002265:Carboxylic Ester Hydrolases; C023105:N-formylmethionyl-tRNA hydrolase