Determination of catechol-O-methyltransferase activity in brain tissue by high-performance liquid chromatography with on-line radiochemical detection |
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| Authors: | E Nissinen |
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| Affiliation: | 1. Department of Chemistry & Centre of Advanced Studies in Chemistry, Panjab University, Chandigarh 160014, India;2. Department of Biochemistry, Panjab University, Chandigarh 160014, India;3. Florida Polytechnic University, Lakeland 33805, Polk County, FL, USA;1. Department of Chemistry, Faculty of Art and Science, Harran University, Sanliurfa TR-63190, Turkey;2. Research Centre for Science and Technology, Harran University, Sanliurfa 63190, Turkey;3. Department of Biology, Faculty of Art and Science, Siirt University, Siirt 56100, Turkey |
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| Abstract: | 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. |
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