Purification and properties of human hepatic 3α-hydroxysteroid dehydrogenase

Abstract 3α-Hydroxysteroid dehydrogenase (3α-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5β-[ 3 H]dihydrocortisol (5β-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 3α-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 5α- and 5β-DHF and 5α-dihydrotesterone 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 5β-DHF was 18 μM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4°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 3α-HSD from human tissue.