Isolation of a nad:3β(α)-hydroxy-5β-androstane dehydrogenase from rat liver microsomes

A rat liver microsomal NAD:3β(α)-hydroxy-5β-androstane dehydrogenase was solubilized by the detergent Lubrol WX and purified by DEAE-cellulose chromatography and affinity chromatography. The homogeneous preparation (disc gel electrophoresis) catalyzed the reduction of 17β-hydroxy-5β-androstan-3-one to the corresponding 3α- and 3β-hydroxysteroids with NADH as hydrogen donor. The 3-oxo group of 17β-hydroxy-5α-androstane-3-one and 5β-pregnane-3,20-dione was not reduced. The purified enzyme preparation had a specific activity of 2.84 μmol 3β-hydroxysteroid formed/min mg and 1.61μmol 3α-hydroxysteroid formed/min mg. Phosphatidylcholine micelles (20mg/mg protein) were essential for activity. Molecular weight determination by Sephadex G-200 gave a value of 56,200.