Complete Inactivation and Labeling of Methionyl-tRNA Synthetase by Periodate-Treated Initiator tRNA in the Presence of Sodium Cyanohydridoborate

Abstract

Methionyl-tRNA synthetase from Escherichia coli can react with periodate-treated tRNA to form a Schiff''s base through the .epsilon.-amino group of a lysine within the enzymic active center and the 2'',3''-aldehyde groups created at the 3''-terminal ribose of tRNA. At alkaline pH, the Schiff''s base equilibrium can be continuously and specifically displaced by reduction in situ with sodium cyanohydridoborate, which leaves intact the reacting aldehyde groups of oxidized tRNA. The effects of temperature, pH and reducing agent concentration on the rate and extent of reduction of the Schiff''s base are analyzed. Conditions are described (37.degree. C, pH 8.0, in the presence of 1 mM cyanohydridoborate) which allowed rapid and complete conversion of the monomeric trypsin-modified methionyl-tRNA synthetase into its 1:1 covalent complex with .**GRAPHIC**.