Affinity Chromatography on Agarose-hexyl-adenosine-5'-phosphate of Methionyl-tRNA Synthetase from Escherichia coli. Application of the Couplings between the Methionine and ATP Sites

Open Access
Publisher Website
Google Scholar
Abstract
Recent studies by us [Biochemistry (1977) 16, 2570–2579] have shown that l‐methioninol, a methionine analog lacking the carboxylate negative charge, enhances the affinity of AMP for methionyl‐tRNA synthetase while l‐methionine antagonizes the nucleotide binding. Such couplings between ligands of the enzyme have now been applied to affinity chromatography of methionyl‐tRNA synthetase on an agarose‐hexyl‐adenosine‐5′‐phosphate gel (the spacer is attached to AMP at the adenine C‐8 position). Retention of the enzyme on this gel column was shown to be dependent on the presence of appropriate concentrations of magnesium and of l‐methioninol in the equilibration buffer. The enzyme was then specifically recovered from the column by omitting the amino alcohol or by adding an excess of l‐methionine which antagonizes the cooperative effect of l‐methioninol. This approach has provided the basis for a new purification procedure of methionyl‐tRNA synthetase which leads to a 200‐fold purification in a single chromatographic step. In this manner, after 30–50% ammonium sulfate fractionation of extracts of Escherichia coli EM 20031 (carrying the F 32 episome), 0.25 mg × methionyl‐tRNA synthetase was obtained at 90% purity per ml of agarose‐hexyl‐adenosine‐5′‐phosphate gel.

This publication has 19 references indexed in Scilit: