Leucyl-Transfer Ribonucleic Acid Synthetase from a Wild-Type and Temperature-Sensitive Mutant of Salmonella typhimurium

Abstract

Leucyl-transfer ribonucleic acid (tRNA) synthetase was purified 100-fold from extracts of Salmonella typhimurium . The partially purified enzyme had the following K m values: leucine, 1.1 × 10 −5 m ; adenosine triphosphate, 6.5 × 10 −4 m ; tRNA I Leu , 4.1 × 10 −8 m ; tRNA II Leu , 4.3 × 10 −8 m ; tRNA III Leu , 5.3 × 10 −8 m ; and tRNA IV Leu , 2.9 × 10 −8 m . The tRNA Leu fractions were isolated from Salmonella bulk tRNA by chromatography on reversed-phase columns and benzoylated diethylaminoethyl cellulose. The enzyme had a p H 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 .

flrB, a Regulatory Locus Controlling Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium

“…Excretion of leucine was determined qualitatively by an auxanographic technique (10). Quantitative determination of amino acids in culture filtrates was carried out with an amino acid analyzer (21).…”

“…Enzyme assays. The growth, harvesting, and disruption of cells (21) and the assays for a-isopropylmalate synthetase (8), f-isopropylmalate dehydrogenase (7), a-acetohydroxy acid synthase (27), and threonine deaminase (14) have been described previously. Cells were stored frozen prior to the assay for the first two enzymes.…”

“…tRNA was chromatographed on reversed-phase columns either at pH 7 (28) or after charging with leucine at pH 4.5 (15). When columns were eluted at pH 7, the concentration of tRNALeU in effluent fractions was determined by complete esterification with ["C]leucine as described previously (21). Prior to chromatography at pH 4.5, tRNA was charged as follows.…”

Cold-sensitive Mutants of Neurospora crassa

“…Cs 78 responded weakly and uniformly (18-20 mg) to various concentrations of guanine (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) Atmoles/25 ml) at 10 C. However, a pulse of 6 to 9 h at 25 C at mid-growth phase resulted in amplification of growth mg) in response to increasing concentrations of the purine. It would appear that the mutant is unable to utilize guanine at 10 C (T. K. Rao, unpublished observations).…”

"Self-catabolite repression" of pectate lyase in Erwinia carotovora

“…Ca2" has been shown to be the activator of pectate lyase (10). Thus, when the optimum concentration (10-4 M) of Ca2", together with pectate (supplier of the inducer) was added to an EC-1 culture in minimal medium (8), the induction started earlier than the control (i.e., no addition of Ca2") ( Fig. 1).…”