THIOL METHYLTRANSFERASE ACTIVITY IN COLONOCYTES AND ERYTHROCYTE-MEMBRANES

Aims-To verify the improved thiol methyltransferase (TMT) assay and measure activity in isolated colonocytes and erythrocyte membranes of the same subjects. Methods-High performance liquid chromatography with radioactivity detection was used to measure C-14-methylmercaptoethanol formation, the reaction product of cell extracts incubated with mercaptoethanol and C-14-S-adenosylmethionine. Results-Verification of radiolabelled C-14-methylmercaptoethanol was by exogenous addition of methylmercaptoethanol and simultaneous ultraviolet detection at 214 nm. Using a substrate concentration of 10 mM mercaptoethanol, the Km for S-adenosylmethionine was 25 mu M. The sensitivity of the radioactive method was 2 pmol, with coefficients of variation of 7% within assay and 6.4% between assay. TMT activities (mean+/-SE; n=17) were 471+/-64 pmol/hour/mg protein for colonocytes and 73+/-7 pmol/hour/mg protein for erythrocyte membranes. Conclusions-The direct assay of TMT activity is sensitive, specific and eliminates concern over non-enzymatic methylation of thiol compounds. High activities in colonic epithelial cells deserve evaluation in disease states.