Purification of a 3 beta-hydroxy-Delta(5)-C-27-steroid dehydrogenase from pig liver microsomes active in major and alternative pathways of bile acid biosynthesis

A 3 beta-hydroxy-Delta(5)-C-27-steroid dehydrogenase active in bile acid biosynthesis was purified from pig liver microsomes by solubilization with sodium cholate and by chromatography on DEAE-Sepharose, aminohexyl-Sepharose, and blue Sepharose. The last step in the purification procedure was preparative isoelectric focusing in a Rotofor cell. The final enzyme preparation showed only one protein band upon SDS-polyacrylamide gel electrophoresis. The isoelectric point was estimated to about 7.0 and the apparent M(r) was 36,000. The purified enzyme catalyzed the conversion of 7 alpha-hydroxycholesterol, 7 alpha,25-dihydroxycholesterol, 7 alpha,27-dihydroxycholesterol, and 3 beta,7 alpha-dihydroxy-5-cholestenoic acid into the corresponding 3-oxo-Delta(4) compounds. The enzyme was inactive with C-19 and C-21 steroids as substrates. The enzyme was also inactive with C-27 steroids having the 7-hydroxy group in beta- instead of alpha-position. The K-m was found to be 0.30 and 0.32 mu M with 7 alpha-hydroxycholesterol and 7 alpha 27-dihydroxycholesterol as substrates, respectively. NAD(+) was the preferred cofactor. A monoclonal antibody raised against the 3 beta-hydroxy-Delta(5)-C-27,,-steroid dehydrogenase was prepared. After coupling to Sepharose, the antibody was able to bind the dehydrogenase and to decrease the conversion of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-4-cholest-3-one by more than 90%. The N-terminal amino acid sequence was determined and found to be similar but not identical with those of known 3 beta-hydroxy-Delta(5)-steroid dehydrogenases active in steroid hormone biosynthesis. Thus, the purified enzyme active toward C-27 steroids in bile acid biosynthesis appears to represent a novel type of 3 beta-hydroxy-Delta(5)-steroid dehydrogenase.