Abstract
To verify the improved thiol methyltransferase (TMT) assay and measure activity in isolated colonocytes and erythrocyte membranes of the same subjects. High performance liquid chromatography with radioactivity detection was used to measure 14C-methylmercaptoethanol formation, the reaction product of cell extracts incubated with mercaptoethanol and 14C-S-adenosylmethionine. Verification of radiolabelled 14C-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 microM. 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. 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.
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