Purification of the NADPH:5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductase from female rat pituitary cytosol

Abstract

The NADPH:5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductase (3α- HSOR) [EC 1.1.1.50] which catalyzes the reversible conversion of 5α-pregnane-3,20-dione (5α-dihydroprogesterone; 5α-DHP) to 3α-hydroxy-5α-pregnan-20-one (3α-,5α-tetrahydro-progesterone; 3α,5α-THP) was purified to apparent homogeneity from female rat anterior pituitary cytosol by a three step micro-purification. Specific activity of purified 3α-HSOR was enriched 438-fold from that in pituitary cytosol using successive ion exchange, chromatofocusing and affinity column chromatography purification steps. 3α-HSOR appears to be a monomer with an approximate molecular weight of 36 kDa and an isoelectric point of about 5.75. The purified enzyme appears as a single protein staining band (36 kDa) when examined by polyacrylamide gel electrophoresis and with both silver or Coomassie blue staining. Under non-dissociating electrophoretic conditions, all of the 3α-HSOR activity co-migrated with the 36 kDa protein staining band. The purified enzyme in the presence of the preferred cofactor, NADPH, has an apparent K m for 5α-DHP of 82 nM and a V max of 1.2 μmol of 3α, 5α-THP formed per mg protein/30 min. The K m for NADPH was 0.71 μM. In the oxidative direction, the enzyme in the presence of NADP + has a K m for 3α,5α-THP of 1.4 μM and a V max of 9.7 μmol of 5α-DHP formed per mg protein/30 min. The K m for NADP + was 1.6 μM.