A method for the measurement of catechol-O-methyltransferase activity using norepinephrine, an endogenous substrate

• Published in: Analyst (London) 2001-01, Vol.126 (5), p.637-640
• Main Authors: TSUNODA, Makoto, TAKEZAWA, Kazuko, IMAI, Kazuhiro
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Catechol O-Methyltransferase - analysis; Chromatography, High Pressure Liquid - methods; Enzymes and enzyme inhibitors; Erythrocytes - enzymology; Fundamental and applied biological sciences. Psychology; Male; Norepinephrine; Rats; Rats, Wistar; Transferases
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/b100119l

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.