BHMT catalyzes a methyl transfer from betaine to homocysteine forming dimethylglycine and methionine The assay of choice for measuring BHMT activity requires the use of [ 14C-merfry/]-betaine Following incubation of the reaction for 1 to 2 h, labeled betaine is separated from labeled dimethylglycine and methionine by chromatography on Dowex 1 and the conversion measured by scintillation counting. This assay is relatively expensive, primarily due to the cost of the ion exchange resin, scintillation cocktail, and the labeled substrate. Furthermore, 14C-labeled betaine is no longer commercially available and most laboratories produce this substrate by converting [ 14Cmethyl]-cbo\ine to [14C-me(Ay/]-betaine using a bacterial choline oxidase system To circumvent the preparation of labeled betaine and decrease the cost of measuring BHMT activity we have developed a new microbiological assay for this enzyme using an E. coli methionine auxotroph Although not sensitive enough for kinetic analysis, this assay is adequate for measuring activity in crude liver or kidney extracts and in typical purification procedures. When samples are measured using both the radioactive and microbiological assays, the values obtained show a high degree of correlation (r2 > 0 95) The microbiological assay takes approximately the same amount of hands-on time to perform as the radioactive assay and is significantly less expensive Illinois AES.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology