A rapid assay method for catechol-O-methyltransferase activity by flow injection analysis.
A rapid assay employing flow injection analysis (FIA) to determine the activity of purified catechol-O-methyltransferase (COMT) from porcine liver is described. The method was based on the determination of normetanephrine, the 3-O-methyl metabolite of the substrate norepinephrine. Excess norepinephrine was removed from the incubation mixture by alumina extraction twice to allow normetanephrine to be subjected to flow injection analysis, coulometrical oxidation, fluorogenic reaction with ethylenediamine and fluorescence detection. K(m) and V(max) values for COMT obtained with the system were 503 microM and 4.51 nmol/min/mg protein, respectively. The method is suitable for screening of COMT inhibitors or activators, as a large number of samples, up to 200, can be processed in one working day.
Aoyama
Nozomi
N
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Tsunoda
Makoto
M
Nakagomi
Kazuya
K
Imai
Kazuhiro
K
D000818:Animals; D002394:Catechol O-Methyltransferase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D017022:Flow Injection Analysis / Q000379:methods*; D008099:Liver / Q000201:enzymology; D009638:Norepinephrine / Q000032:analysis; D013552:Swine
D002394:Catechol O-Methyltransferase; D009638:Norepinephrine