Elongation factor T from Bacillus stearothermophilus and Escherichia coli. Purification and some properties of EF-Tu and EF-Ts from Bacillus stearothermophilus

Abstract

Homogeneous preparations of elongation factors EF-Tu and EF-Ts from Bacillus stearothermophilus have been obtained with specific activities of 20000 +/- 2000 and 500000 +/- 50000 units/mg, respectively. By dodecylsulphate-polyacrylamide gel electrophoresis the molecular weight of EF-Tu was found to be 49000 +/- 2000 and of EF-Ts 35500 +/- 1000. Nucleotide-free EF-Tu was prepared by using ITP as a GDP-binding-site-directed analogue. EF-Tu was shown to contain two sulphydryl groups, one reacting fast and one slowly with N-ethylmaleimide and 5,5'-dithio-bis(2-nitrobenzoic acid) under non-denaturing conditions. The same reagents were shown to react with the three sulphydryl groups of EF-Ts in the native state. The heat stabilities of EF-Tu and EF-Ts are reversed with respect to the Escherichia coli factors, EF-Tu being the more stable protein; even nucleotide-free EF-Tu is relatively stable with a half-life at room temperature of about 35 h.