Purification and characterization of glutamyl-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8.
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Glutamyl-tRNA synthetase has been isolated from an extreme thermophile, Thermus thermophilus HB 8. The enzyme has been purified to homogeneity by successive chromatography on columns of DEAE-cellulose, DEAE-Sephacel, phosphocellulose and hydroxyapatite. 11.7mg of purified enzyme has been obtained from 2kg of T. thermophilus cells, with a purification factor of 600 with an 11% yield. From gel permeation chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme is found to be a monomer protein with a molecular weight of 50, 000. The optimum temperature for the aminoacylation of T. thermophilus tRNAGlu is 65°C, and the optimum pH range is 8.0-9.0, in the presence of 5mM Mg2+. The Km values for ATP, L-glutamate, and T. thermophilus tRNAGlu are 230 μM, 70 μM, and 0.65 μM, respectively, in the presence of 50mM KCl and 10mM MgCl2 at pH 8.0 at 65°C. Escherichia coli tRNA2Glu is also a good substrate with a Km value of 0.60 μM at 65°C. The mole fractions of Arg and Leu residues are higher and that of Asx residues is lower than those of E. coli glutamyl-tRNA synthetase. Glutamyl-tRNA synthetase from T. thermophilus is remarkably thermostable; even after incubation for 9 h at 65deg;C, 70% of the enzyme activity is retained in the absence of any protecting factors. Such an extremely thermostable enzyme with a low molecular weight will be useful for detailed physicochemical analyses on the molecular mechanism of strict recognition by aminoacyl-tRNA synthetases.
- 社団法人 日本生化学会の論文
社団法人 日本生化学会 | 論文
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