[Purification and properties of rat kidney catechol-O-methyltransferase].
The S-adenosyl-methionine: catechol-O-methyltransferase (EC 2.1.1.6) from rat kidney was purified about 650 fold as compared with the homogenate and the result of disc electrophoresis presented. The purification involved extraction, precipitation at pH 5, ammonium sulfate fractionation, Chromatographies on Biogel 0.5 m, Ultrogel AcA 44 and DE Sephadex A 50. Affinity chromatography was tried but unsuccessful. The enzyme exhibited two pH optima at 7.9 and 9.6 with a minimum at about 8.9. The COMT had a temperature optimum of 50 degrees C, with activation energy of 23.1 Kcal/Mole between 25-35 degrees C, 18.9 Kcal/mole between 35-45 degrees C and the Q10 within the range of 25-35 degrees amounted to 3.5. The molecular weight was estimated to be 21500+/-1000 daltons from its behavior on Ultrogel AcA 44 and the pH1 determined by electrofocalisation was near 5.50. The time of half life of the best purified enzymatic extract was found to be 2 h 10 min. at -20 degrees C. At basic pH the instability of the enzyme was increased. Since O-methylation required the presence of divalent cations, our results show that apparent Michaelis constants for Mg++ and Mn++ were respectively 0.50 X 10(-3) M and 0.33 X 10(-5) M. The study of their Hill's number indicated that there was only one point of fixation on the enzyme. The Km value determined by Florini and Vestling's method were 2.5 X 10(-4) M and 11.9 X 10(-5) M for epinephrine and S-adenosyl-methionine respectively. All results were discussed with respect to other investigations.
D000818:Animals; D001665:Binding Sites; D002394:Catechol O-Methyltransferase / Q000302:isolation & purification*; D002850:Chromatography, Gel; D004355:Drug Stability; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium; D008345:Manganese; D008970:Molecular Weight; D051381:Rats; D013329:Structure-Activity Relationship
D008345:Manganese; D002394:Catechol O-Methyltransferase; D008274:Magnesium
Purification et propriétés de la catéchol-O-méthyltransférase du rein de rat