R. Knuppen

Reinigung und charakterisierung einer S-adenosyl-methionin:catechol-O-methyltransferase der menschlichen placenta

Abstract An S-adenosylmethionine:catechol-O-methyltransferase (EC 2.1.1.6) is localised in the 150 000 × g supernatant of human placenta. The enzyme was purified 66-fold by (NH4)2SO4 precipitation and by repeated filtration of the (NH4)2SO4 precipitate through a Sephadex G-100 column. After incubation of epinephrine and S-[Me-14C]-adenosylmethionine, the main metabolite was identified as metanephrine (3-O-methyl-adrenalin). In the pH range of 6–8, the activity of the enzyme increased steadily. The Michaelis-Menten constants were found to be 43.5·10−5 M for epinephrine and 7.8·10−5 M for S-adenosylmethionine. The purified catechol O-methyltransferase was active for more than 3 months when stored at −20°. The enzyme shows a temperature optimum at 50° and an activation energy of 17.3 kcal/mole within the range of 24–42°. By gel filtration, using Sephadex G-100, the molecular weight of the placental catechol O-methyltransferase was found to be 52 000. The purified enzyme preparation was only active in the presence of cysteine (20–80 mM). In addition to epinephrine, other catechols were also methylated. When 2-hydroxyoestradiol-17β was used as substrate, 2-methoxyoestradiol-17β as well as 2-hydroxyoestradiol-17β 3-methyl ether were formed as metabolites. The methylation of epinephrine to metanephrine was inhibited competitively by 2-hydroxyoestradiol-17β.