PURIFICATION OF THE NADPH - 5-ALPHA-DIHYDROPROGESTERONE 3-ALPHA-HYDROXYSTEROID OXIDOREDUCTASE FROM FEMALE RAT PITUITARY CYTOSOL

The NADPH: 5-alpha-dihydroprogesterone 3-alpha-hydroxysteroid oxidoreductase (3-alpha-HSOR) [EC 1.1.1.50] which catalyzes the reversible conversion of 5-alpha-pregnane-3,20-dione (5-alpha-dihydroprogesterone; 5-alpha-DHP) to 3-alpha-hydroxy-5-alpha-pregnan-20-one (3-alpha-,5-alpha-tetrahydroprogesterone; 3-alpha,5-alpha-THP) was purified to apparent homogeneity from female rat anterior pituitary cytosol by a three step micro-purification procedure. Specific activity of purified 3-alpha-HSOR was enriched 438-fold from that in pituitary cytosol using successive ion exchange, chromatofocusing and affinity column chromatography purification steps. 3-alpha-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 3-alpha-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 5-alpha-DHP of 82 nM and a V(max) of 1.2-mu-mol of 3-alpha,5-alpha-THP formed per mg protein/30 min. The K(m) for NADPH was 0.71-mu-M. In the oxidative direction, the enzyme in the presence of NADP+ has a K(m) for 3-alpha,5-alpha-THP of 1.4-mu-M and a V(max) of 9.7-mu-mol of 5-alpha-DHP formed per mg protein/30 min. The K(m) for NADP+ was 1.6-mu-M.