Isolation of 3β,20α-hydroxysteroid oxidoreductase from sheep fetal blood

Article abstract of DOI:10.1016/0039-128X(87)90089-4

3β,20α-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20α-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnan-20α-ol-3-one or 5α-dihydrotestosterone to 5α-androstan-3β,17β-diol with kinetic characteristics of Km = 30.8 μM and Vmax = 0.7 nmol min^-1 (nmol of enzyme)^-1 or Km = 74 μM and Vmax = 1.3 nmol min^-1 (nmol of enzyme)^-1, respectively. 5α-Dihydrotestosterone competitively inhibits 20α-reductase activity with a Ki value of 102 μM.