PURIFICATION AND PROPERTIES OF HUMAN HEPATIC 3-ALPHA-HYDROXYSTEROID DEHYDROGENASE

3alpha-Hydroxysteroid dehydrogenase (3alpha-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5beta-[H-3]dihydrocortisol (5beta-DHF) as substrate. Electrophoretically homogeneous enzyme was obtained using a procedure that involved ammonium sulfate precipitation and three successive column chromatography steps: DEAE-cellulose, hydroxylapatite and Blue-Sepharose. The enzyme is a monomer since the native molecular weight was found to be 37,000, using a calibrated Sephadex G-75 column, and the denatured subunit molecular weight was determined to be 38,500, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme had a pI of 5.6-5.9. The 3-ketosteroids: cortisol, testosterone, progesterone and androstenedione, were not substrates for 3alpha-HSD indicating that a saturated 4,5 double bond was required for substrate activity. The conformation at the 5 position, however, did not influence substrate activity since 5alpha- and 5beta-DHF and 5alpha-dihydrotestosterone were all reduced at similar rates. The purified enzyme preferred NADPH to NADH as a cofactor and showed a broad peak of activity in the pH range of 6.8-7.4. The apparent K(m) for 5beta-DHF was 18 muM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4-degrees-C. Freezing and thawing of the enzyme resulted in a large loss of activity during early stages of the purification. This is the first report of the purification to homogeneity of 3alpha-HSD from human tissue.