Purification and properties of testicular 3β-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase

Through the treatment of rat testicular microsomes with sodium cholate, 3β-hydroxy-5-ene-steroid dehydrogenase and 5-ene-4-ene isomerase (abbreviated as the 3β -hydroxysteroid dehydrogenase and isomerase, respectively) were solubilized, and then purified by DEAE and hydroxylapatite column chromatographies. The findings were as follows: 1. With this purification procedure, the 3β-hydroxysteroid dehydrogenase activity could not be separated from the isomerase. 2. For 3-oxo-4-ene-steroid formation from 3β-hydroxy-5-ene-steroids, NAD + was required as a cofactor. While the 3β-hydroxysteroid dehydrogenase required NAD +, the isomerase also required NAD + or its reduced form, in contrast to the microbial enzyme. 3. On treatment of the purified enzyme with 5'- p-fluorosulfonyl-benzoyladenosine (FSBA), both enzyme activities were markedly reduced. 4. The enzyme, affinity labeled with [adenine-8- 14C]FSBA, showed a mol. wt of 46.8 K. 5. During 4-androstenedione production from DHA, 5-androstenedione was detected as an intermediate.