Thiol methyltransferase activity in colonocytes and erythrocyte membranes

J Clin Pathol. 1995 Jul;48(7):641-4. doi: 10.1136/jcp.48.7.641.

Abstract

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.

MeSH terms

  • Biomarkers / analysis
  • Carbon Radioisotopes
  • Chromatography, High Pressure Liquid / methods
  • Colon / enzymology*
  • Erythrocyte Membrane / enzymology*
  • Female
  • Humans
  • Male
  • Methylation
  • Methyltransferases / analysis*
  • Methyltransferases / blood
  • Sensitivity and Specificity

Substances

  • Biomarkers
  • Carbon Radioisotopes
  • Methyltransferases
  • thiol S-methyltransferase