Radiochemical high-performance liquid chromatographic assay for the determination of catechol O-methyltransferase activity towards various substrates.
A new chromatographic catechol O-methyltransferase (COMT) assay based on S-adenosyl-L-[methyl-14C]methionine and on-line radioactivity detection was developed. With minor modifications in the mobile phase composition the methylation velocities for 30 structurally diverse compounds including simple catechols, neurotransmitters, catecholestrogens and catecholic drugs could be measured using human and rat recombinant soluble COMT. The enzymes showed very similar substrate selectivities. The radiochemical method was validated using 3,4-dihydroxybenzoic acid as a model substrate and it was shown that accurate and reproducible methylation velocity values could be achieved for both of the catecholic hydroxyls. The method proved to be suited for determining the enzyme kinetic parameters and can probably be further used for gathering enzyme kinetic data on differentially substituted catechols in order to construct proper structure-activity relationships for COMT.
Lautala
P
P
Department of Pharmacy, University of Helsinki, Finland. Pia.Lautala@helsinki.fi
Ulmanen
I
I
Taskinen
J
J
D000818:Animals; D016588:Anticarcinogenic Agents / Q000378:metabolism; D002250:Carbon Radioisotopes; D002394:Catechol O-Methyltransferase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D062385:Hydroxybenzoates / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D016588:Anticarcinogenic Agents; D002250:Carbon Radioisotopes; D062385:Hydroxybenzoates; C009091:protocatechuic acid; D002394:Catechol O-Methyltransferase