Assay for Methionine adenosyltransferase Activity of Vitamin B_<12> Deficient Rat's Tissue by High Performance Liquid Chromatography

Abstract

An assay method for methionine adenosyltransferase (MAT) activity using high performance liquid chromatography (HPLC) was newly developed. The MAT reaction was carried out in the presence of K^+, Mg^<2+>, DTT and stopped by adding ethanol to a final concentration of 50% (v/v). S-adenosylmethionine (SAM) produced was separated and quantified by HPLC. The conditions of HPLC for separation of SAM were as follows: column, Partisil-10 SCX; mobile phase, 0.22M CH_3COONa pH 4.0; detection wave-length, 254nm. This assay method was used for determination of the MAT activity of rat's tissue, fed on vitamin B_<12>-deprived diet for 90 days. Hepatic MAT activity of B_<12>-deficient rat decreased to 1/4 of that of the B_<12>-supplemented rat. When DMSO was added to the reaction mixture, the enzyme activity of B_<12>-deficient rat decreased to 70% of that of B_<12>-supplemented rat.