RT Journal Article
SR Electronic
T1 Thiol methyltransferase activity in colonocytes and erythrocyte membranes.
JF Journal of Clinical Pathology
JO J Clin Pathol
FD BMJ Publishing Group Ltd and Association of Clinical Pathologists
SP 641
OP 644
DO 10.1136/jcp.48.7.641
VO 48
IS 7
A1 W J Babidge
A1 S H Millard
A1 W E Roediger
YR 1995
UL http://jcp.bmj.com/content/48/7/641.abstract
AB 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 14C-methylmercaptoethanol formation, the reaction product of cell extracts incubated with mercaptoethanol and 14C-S-adenosylmethionine. RESULTS--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. 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.