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

The Analyst
M TsunodaK Imai

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 meta...Continue Reading

Citations

Apr 5, 2002·Biomedical Chromatography : BMC·Nozomi AoyamaKazuhiro Imai
Aug 23, 2006·Analytical and Bioanalytical Chemistry·M Tsunoda
Nov 27, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Pia Pihlavisto, Ilkka Reenilä
Nov 25, 2003·Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan·Kazuhiro Imai
Nov 5, 2008·Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan·M Tsunoda
Jan 13, 2004·Hypertension Research : Official Journal of the Japanese Society of Hypertension·M TsunodaKazuhiro Imai
Mar 31, 2005·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Yoko HiranoKazuhiro Imai

Related Concepts

Metazoa
Catechol O-Methyltransferase
High Pressure Liquid Chromatography Procedure
Erythrocytes
Norepinephrine, (+, -)-Isomer
Rats, Wistar
Rats, Laboratory

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