17β-Estradiol Dehydrogenase of Ovine Ovaries

Abstract An estradiol dehydrogenase (17β-hydroxysteroid:NAD(P)+ oxidoreductase) which catalyzed the reversible oxidation of 17β-estradiol to estrone was purified more than 800-fold from ovine ovary homogenates with a 9.9% yield. The specific activity in the best preparation was 136 nmoles of NADH produced per min per mg of protein at 30°. The preparations approached homogeneity by sedimentation analysis and by discontinuous polyacrylamide gel electrophoresis at pH 9.5 and 4.3. The molecular weight was 104,000 from sucrose density gradient centrifugation, and the Stokes radius was 50.5 A from gel filtration on Sephadex G-150. The preferred steroid substrate was 17β-estradiol; the preferred cofactor was NAD+. The enzyme also accepted 3-O-methyl-17β-estradiol as a substrate but did not dehydrogenate ethanol or 1,2-propanediol. Under the conditions of assay used (20% organic solvent), the Michaelis constant for NAD+ was 5.2 x 10-4 m, and for 17β-estradiol it was 3.0 x 10-5 m. Kinetic studies of the forward reaction showed that the mechanism was sequential. The purified enzyme was unstable in simple buffers and was stabilized by the presence of certain inorganic ions, 20% glycerol, and 0.05 m 2-mercaptoethanol.