Assay of catechol-O-methyltransferase activity in human erythrocytes using norepinephrine as a natural substrate.
BACKGROUND
Catechol-O-methyltransferase (COMT) catalyses the inactivation of catecholamines. It is widely distnbuted in most tissues in soluble (S-COMT) and membrane-bound (MB-COMT) forms. Recently, we used a new assay for COMT activity and demonstrated that COMT plays an important role in blood pressure regulation in spontaneously hypertensive rats. In order to investigate whether this is true for human hypertension, we have evaluated the erythrocyte COMT assay in humans.


METHODS
The assay procedure included the use of norepinephrine (NE) as a natural substrate and the quantification of the reaction product, normetanephrine, followed by high-performance liquid chromatography separation and fluorescence or chemiluminescence detection.


RESULTS
After evaluation of the method, the optimum conditions were obtained for the assay of human erythrocyte COMT. The S- and MB-COMT activities obtained were 50.6 (24.5) and 329.8 (179.4) fmol/min/mg protein, respectively [mean (standard deviation); n = 54]. The Km values for NE were 91.3 (14.1) and 11.7 (1.1) micromol/L for S- and MB-COMT, respectively (n = 6).


CONCLUSIONS
The established assay method used to assess S- and MB-COMT activities in human erythrocytes could be useful to elucidate catecholamine metabolism in the normal physiological state as well as in the pathology of certain diseases.
Masuda
Mayumi
M
Graduate School of Pharmaceutical Sciences, The University of Tokyo 7-3-1, Hongo Bunkyo-ku Tokyo 113-0033, Japan.
Tsunoda
Makoto
M
Yusa
Yukie
Y
Yamada
Shizuo
S
Imai
Kazuhiro
K
D000328:Adult; D000368:Aged; D002394:Catechol O-Methyltransferase / Q000032:analysis* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D009638:Norepinephrine / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity; D013997:Time Factors
D002394:Catechol O-Methyltransferase; D009638:Norepinephrine