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.

Reduced Membrane-Bound Catechol-O-Methyltransferase in the Liver of Spontaneously Hypertensive Rats

“…There were no differences in the affinity for NE in both isozymes. In both SHR and WKY rats, the Km values for NE in S-COMT were about 80 times higher than those in MB-COMT, which is in agreement with the previous reports (13,14,17). The Vmax/Km values, i.e., the specificity constant values were calculated based on the above data.…”

“…The general procedure for the COMT activity measurement in tissues and erythrocytes was similar to that previously described (13,14). Tissues were removed from anesthetized rats, weighed, and homogenized in homogenizing buffer (50 mmol/l sodium phosphate buffer (pH 7.5) with 0.5 mmol/l dithiothreitol).…”

Rat liver and kidney catechol‐O‐methyltransferase activity measured by high‐performance liquid chromatography with fluorescence detection

“…It is plausible that these organs may play an important role in the removal of circulating catecholamines and/or other catechol compounds. Previously, to obtain information on the metabolism of catecholamines in vivo, we have developed a method for COMT activity measurement in rat erythrocytes and human erythrocytes with HPLCfluorescence or chemiluminescence detection (Tsunoda et al, 2001a;Masuda et al, 2002). Here, using this method, we report the optimized conditions for COMT activity measurement in rat liver and kidney.…”

“…In a previous paper, we reported a sensitive determination method of COMT activity in rat erythrocytes and human erythrocytes (Tsunoda et al, 2001a;Masuda et al, 2002). The method included on-line extraction of amines, separation on an ODS column, electrochemical oxidation to their respective o-quinones, fluorogenic derivatization with ethylenediamine and fluorescence or peroxyoxalate chemiluminescence reaction detection.…”

Assay of Catechol-O-Methyltransferase Activity in Human Erythrocytes Using Norepinephrine as a Natural Substrate

“…13 In contrast, other studies using artificial substrates such as DBA and 2-(3,4-dihydroxyphenyl)naphtha-[1,2-d]thiazole showed that there was no difference in affinities for the substrates between S-COMT and MB-COMT.7.9.1(' Therefore, it is reasonable to expect that the results obtained by our present assay using NE, a natural substrate, reflected more physiological performance of S-and MB-COMT in the human body.…”