Étude de la méthionyl‐tRNA Synthétase d'Escherichia coli

1 Methionyl-tRNA synthetase from Escherichia coli extracts conserved at 0° has an apparent molecular weight of 175,000 estimated by gel filtration through Sephadex G-200, and a sedimentation coefficient of 6.5 S determined by sucrose lensity gradient centrifugation. 2 Incubation of the extract at 37° results in a time-dependent modification of methionyl-tRNA synthetase into an enzymatically active form, which has an apparent molecular weight of 54,000 and a sedimentation coefficient of 3.7 S. Under the same conditions of incubation, the Sephadex G-200 elution profile of several other aminoacyl-tRNA synthetases remains unaltered. 3 This modification of methionyl-tRNA synthetase is observed upon incubation of extracts derived from E. coli K12 or B cells grown under a variety of conditions (in minimal or enriched medium, at exponential or stationary phase). 4 The modified enzyme, which was partially purified by chromatography on hydroxylapatite, catalyses the formation of aminoacyl adenylate and also of aminoacyl-tRNA, using tRNAF as well as tRNAM. Furthermore, the Km values for various substrates of the two reactions do not differ significantly from those of the native enzyme. However, conversion into the modified enzyme is accompanied by a decrease in specific activity, and by an increase in the ratio of ATP-PPi exchange capacity to aminoacyl-tRNA forming capacity. 5 Incubation of the modified enzyme at 37° in the presence of the substrates for each of the two reactions, followed by sucrose density gradient centrifugation in the presence of these substrates, does not lead to reversion to the native form. Thus, the ability to catalyze both reactions appears to be an intrinsic property of the modified enzyme. 6 In contrast to the behaviour of methionyl-tRNA synthetase in crude extracts, the partially or totally purified enzyme remains unaltered upon incubation at 37°. This observation has led to the demonstration that the extract contains a macromolecular component, readily separable from methionyl-tRNA synthetase, which is responsible for the conversion of the native to the modified enzyme. 7 Earlier studies on the purified enzyme [1] have shown that methionyl-tRNA synthetase from E. coli, with a molecular weight of 173,000, undergoes dissociation in the presence of 8 M urea or 6 M guanidine into four subunits which have a molecular weight of 43,000, and which appear to be homogenous by sedimentation analysis and polyacrylamide gel electrophoresis. In the light of those observations, the modification of methionyl-tRNA synthetase reported in this paper is tentatively interpreted as the conversion, mediated by a macromolecular component in the extract, of the native enzyme into enzymatically active subunits. Work is in progress to elucidate the structural relationship between these enzymatically active subunits, and the subunits produced upon treatment of the purified enzyme with dissociating reagents.