THIOL METHYLTRANSFERASE ACTIVITY IN COLONOCYTES AND ERYTHROCYTE-MEMBRANES

Aims-To verify the improved thiol methyltransferase (TMT) assay and me asure activity in isolated colonocytes and erythrocyte membranes of th e same subjects.Methods-High performance liquid chromatography with ra dioactivity detection was used to measure C-14-methylmercaptoethanol f ormation, the reaction product of cell extracts incubated with mercapt oethanol and C-14-S-adenosylmethionine. Results-Verification of radiol abelled C-14-methylmercaptoethanol was by exogenous addition of methyl mercaptoethanol and simultaneous ultraviolet detection at 214 nm. Usin g a substrate concentration of 10 mM mercaptoethanol, the Km for S-ade nosylmethionine 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/h our/mg protein for colonocytes and 73+/-7 pmol/hour/mg protein for ery throcyte membranes. Conclusions-The direct assay of TMT activity is se nsitive, specific and eliminates concern over non-enzymatic methylatio n of thiol compounds. High activities in colonic epithelial cells dese rve evaluation in disease states.