Leucyl-Transfer Ribonucleic Acid Synthetase from a Wild-Type and Temperature-Sensitive Mutant of Salmonella typhimurium1
AUTOR(ES)
Mikulka, T. W.
RESUMO
Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium. The partially purified enzyme had the following Km values: leucine, 1.1 × 10−5m; adenosine triphosphate, 6.5 × 10−4m; tRNAILeu, 4.1 × 10−8m; tRNAIILeu, 4.3 × 10−8m; tRNAIIILeu, 5.3 × 10−8m; and tRNAIVLeu, 2.9 × 10−8m. The tRNALeu fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a pH optimum of 8.5 and an activation energy of 10,400 cal per mole, and was inactivated exponentially at 49.5 C with a first-order rate constant of 0.064 min−1. Strain CV356 (leuS3 leuABCD702 ara-9 gal-205) was isolated as a mutant resistant to dl-4-azaleucine and able to grow at 27 C but not at 37 C. Extracts of strain CV356 had no leucyl-tRNA synthetase activity (charging assay) when assayed at 27 or 37 C. Temperature sensitivity and enzyme deficiency were caused by mutation in the structural gene locus specifying leucyl-tRNA synthetase. A prototrophic derivative of strain CV356 (CV357) excreted branched-chain amino acids and had high pathway-specific enzyme levels when grown at temperatures where its doubling time was near normal. At growth-restricting temperatures, both amino acid excretion and enzyme levels were further elevated. The properties of strain CV357 indicate that there is only a single leucyl-tRNA synthetase in S. typhimurium.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=285181Documentos Relacionados
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