Determination of catechol-O-methyltransferase activity in brain tissue by high-performance liquid chromatography with on-line radiochemical detection.
A sensitive assay for catechol-O-methyltransferase (COMT) activity by high-performance liquid chromatography with on-line radiochemical detection was described. The method was based on the measurement of 3H-labeled 3-O- and 4-O-methylated products of the substrate, 3,4-dihydroxybenzoic acid, using S-adenosyl-L-[methyl-3H]methionine as the methyl donor, or the measurement of 14C-labeled 3-O- and 4-O-methylated products of the substrate, [7-14C]dopamine. The reaction products were determined from the incubation mixture after removal of protein by injecting an aliquot into the liquid chromatograph. The detection limit with counting efficiency of 40% was 0.45 pmol 3H-labeled product, and 0.04 pmol 14C-labeled product with 61% counting efficiency. The method is suitable for assaying membrane-bound and soluble COMT activities in the brain tissue and for calculation of meta/para product ratios.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002394:Catechol O-Methyltransferase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D062385:Hydroxybenzoates; D007700:Kinetics; D011854:Radiochemistry; D051381:Rats; D012436:S-Adenosylmethionine
D062385:Hydroxybenzoates; C009091:protocatechuic acid; D012436:S-Adenosylmethionine; D002394:Catechol O-Methyltransferase