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.
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 F32 episome), 0.25 mg X methionyl-tRNA synthetase was obtained at 90% purity per ml of agarose-hexyl-adenosine-5'-phosphate gel.
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D008715:Methionine / Q000031:analogs & derivatives; D008718:Methionine-tRNA Ligase / Q000302:isolation & purification*; D008970:Molecular Weight; D012685:Sepharose; D014357:Trypsin
D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D012685:Sepharose; D008715:Methionine; D014357:Trypsin; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase