Valyl-tRNA synthetase from Bacillus stearothermophilus. Purification and Binding with the Substrates L-Valine and ATP(Biological Chemistry)

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Valyl-tRNA synthetase (L-valine:tRNA^<val> ligase (AMP forming); EC 6.1.1.9) (VRS) was purified from Bacillus stearothermophilus NCA 1503 by streptomycin treatment, ammonium sulfate fractionation, and batch adsorption on DEAE-Sephadex A-50, then column chromatographies on hydroxylapatite, DEAE-Toyopearl 650S, BUTYL-Toyopearl 650S, and Sephacryl S-300 superfine. The enzyme preparation, purified approximately 1100-fold, was homogeneous on polyacrylamide gel disc electrophoresis and consisted of a single chain polypeptide. The molecular weight was about 100,000 and 110,000 by SDS-polyacrylamide gel electrophoresis and Sephacryl S-300 gel filtration, respectively. Enzyme activity in tRNA aminoacylation reaction increased as the temperature increased up to 52℃ at pH 7.6 and pH 8.5. pH-optima of the aminoacylation activity were 8.5 at 30℃ and 8.0 at 52℃ under our conditions. Ligand-induced decrease of the enzyme protein fluorescence was observed and used as a probe for studying the binding of substrates (L-valine and ATP) to VRS. Substrate bindings were also studied by equilibrium dialysis; two moles of L-valine or ATP were bound to a mole of enzyme when each substrate was examined separately. These two binding equilibria of L-valine or ATP seemed equivalent when each substrate is bound alone.
社団法人日本農芸化学会の論文
1986-10-23