Catechol-O-methyltransferase activity in human erythrocytes: methodological aspects

Abstract

Different methodological aspects on the assay of human erythrocyte catechol-O-methyltransferase (COMT) activity were studied. No temporal variations were found either over a 24 hour period or over one month. Erythrocytes from whole blood collected with any of the anticoagulants heparin, EDTA or citrate could be used as the enzyme source provided the cells were washed in saline. The COMT activity in lysed erythrocytes was rapidly lost when the lysate was stored at +4 degrees C and -20 degrees C. Intact erythrocytes could be stored up to one week in +4 degrees C without considerable loss of activity. The COMT activity was stable for at least two years when storing the cells at -85 degrees C. Freeze-thawing and hypotonic disruption of the erythrocytes resulted in the same activity and neither freeze-thawing nor sonication altered the apparent Km for the substrate. Noradrenaline and 3,4-dihydroxybenozic acid (DBA) could both be used as substrates although DBA gave higher activity values and had a higher affinity to the enzyme. The COMT activity increased with increasing concentration of the methyl-donor S-adenosyl-1-methionine up to approximately 0.1 mM. Preincubation at 47 degrees C decreased the COMT activity whereas the apparent Km values remained unchanged. The present COMT assay was convenient and reproducible and could be used with small amounts of blood with different kinds of anticoagulants. Interactions with plasma factors were avoided by washing the erythrocytes with isotonic sodium chloride.