Purification and properties of a NAD: 3 alpha-hydroxy-5 alpha-pregnan-20-one-oxidoreductase from rat liver microsomes.
From rat liver microsomes a NAD: 3 alpha-hydroxy-5 alpha-pregnan-20-one oxidoreductase was isolated and purified up to a specific activity of 73 nmol/min . mg by affinity chromatography and DEAE-cellulose chromatography. Various Km-values have been determined. The enzyme exhibits highest affinity for 5 alpha-pregnane-3,20-dione and NADH. The 3-oxo group of 5 alpha-dihydrocortisone (17,21-dihydroxy-5 alpha-pregnane-3,11,20-trione) was not reduced by the purified enzyme preparation and NADH and no dehydrogenation with NAD was observed of 3 alpha,11 beta,17,21-tetrahydroxy-5 alpha-pregnan-20-one. The optimal pH for the hydrogenation of th 3-oxo group was at pH 5.3 and for the dehydrogenation at pH 8.9. Disc gel electrophoresis in presence of 0.1% sodium dodecylsulfate yielded a homogeneous preparation.
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004589:Electrophoresis, Disc; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats
D015096:3-Hydroxysteroid Dehydrogenases; C027431:NAD 3 alpha-hydroxy-5 alpha-pregnan-20-one-oxidoreductase