title: A method for the measurement of catechol-O-methyltransferase activity using norepinephrine, an endogenous substrate.
abstract: We propose a highly sensitive method for the measurement of catechol-O-methyltransferase (COMT) activity with norepinephrine (NE), an endogenous native substrate. The product, normetanephrine, was determined by high-performance liquid chromatography (HPLC)-peroxyoxalate chemiluminescence reaction detection or, if required, less sensitive fluorescence detection. For the measurement of membrane-bound (MB)-COMT activity in the rat erythrocyte, the HPLC-peroxyoxalate chemiluminescence reaction detection was employed. Soluble (S)- and MB-COMT activities in the rat erythrocyte were 22.9 +/- 2.5 and 4.62 +/- 1.23 pmol min-1 (mg protein)-1, respectively (n = 5). The Km values obtained for S- and MB-COMT were 366 +/- 31 mumol l-1 and 12.0 +/- 1.1 mumol l-1, respectively (n = 5), suggesting that the use of NE as a substrate would give more precise information on the role of both isoenzymes. However, with dihydroxybenzoic acid as an artificial substrate, the Km values for S- and MB-COMT were similar, with values of 69.2 +/- 11.4 mumol l-1 and 72.2 +/- 9.2 mumol l-1, respectively. The proposed method is thought to be useful for the measurement of both S-COMT and MB-COMT activities, and would give us critical information on the role of metabolism of catecholamines in rat tissues.
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Tsunoda
authors.forename: K
authors.initials: K
authors.lastname: Takezawa
authors.forename: K
authors.initials: K
authors.lastname: Imai
mesh_terms: D000818:Animals; D002394:Catechol O-Methyltransferase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology*; D008297:Male; D009638:Norepinephrine; D051381:Rats; D017208:Rats, Wistar
chemical_list: D002394:Catechol O-Methyltransferase; D009638:Norepinephrine