title: Purification and characterization of NADP+-dependent 3 alpha-hydroxysteroid dehydrogenase from mouse liver cytosol.
abstract: A monomeric 3 alpha-hydroxysteroid dehydrogenase with a molecular weight of 34,000 was purified to apparent homogeneity from mouse liver cytosol. The enzyme catalyzed the reversible oxidation of the 3 alpha-hydroxy group of C19-, C21-, and C24-steroids, reduced a variety of carbonyl compounds, and was inhibited by SH-reagents, synthetic estrogens, anti-inflammatory drugs, prostaglandins, and delta 4-3-ketosteroids. Although these properties are similar to those of the enzyme from rat liver cytosol, the mouse enzyme exhibited low dehydrogenase activity toward benzene dihydrodiol and some alicyclic alcohols, it showed a strict cofactor specificity for NADP(H), and high substrate inhibition was observed in the reverse reaction. In addition, dexamethasone, deoxycorticosterone, and medroxyprogesterone acetate inhibited the mouse enzyme competitively at low concentrations and noncompetitively at high concentrations, whereas hexestrol, indomethacin, and prostaglandin A1 were competitive inhibitors. Steady-state kinetic measurements in both directions indicated that the reaction proceeds through an ordered bi bi mechanism with the cofactors binding to the free enzyme. The 3-ketosteroid substrates inhibited the enzyme uncompetitively at elevated concentrations, suggesting that the substrates bind to the enzyme.NADPH complex and to the enzyme NADP+ complex.
authors.affiliation: Department of Biochemistry, Gifu Pharmaceutical University.
authors.forename: A
authors.initials: A
authors.lastname: Hara
authors.forename: Y
authors.initials: Y
authors.lastname: Inoue
authors.forename: M
authors.initials: M
authors.lastname: Nakagawa
authors.forename: F
authors.initials: F
authors.lastname: Naganeo
authors.forename: H
authors.initials: H
authors.lastname: Sawada
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D000818:Animals; D003600:Cytosol / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D051379:Mice; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D009249:NADP; D015096:3-Hydroxysteroid Dehydrogenases