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.