Purification of a 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase from pig liver microsomes active in major and alternative pathways of bile acid biosynthesis

A 3β-hydroxy-Δ⁵-C₂₇-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 7α-hydroxycholesterol, 7α,25-dihydroxycholesterol, 7α,27- dihydroxycholesterol, and 3β,7α-dihydroxy-5-cholestenoic acid into the corresponding 3-oxo-Δ⁴ compounds. The enzyme was inactive with C₁₉ and C₂₁ steroids as substrates. The enzyme was also inactive with C₂₇ steroids having the 7-hydroxy group in β- instead of α-position. The K(m) was found to be 0.30 and 0.32 μM with 7α-hydroxycholesterol and 7α,27- dihydroxycholesterol as substrates, respectively. NAD⁺ was the preferred cofactor. A monoclonal antibody raised against the 3β-hydroxy-Δ⁵-C₂₇- steroid dehydrogenase was prepared. After coupling to Sepharose, the antibody was able to bind the dehydrogenase and to decrease the conversion of 7α- hydroxycholesterol into 7α-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 3β-hydroxy-Δ⁵-steroid dehydrogenases active in steroid hormone biosynthesis. Thus, the purified enzyme active toward C₂₇ steroids in bile acid biosynthesis appears to represent a novel type of 3β-hydroxy-Δ⁵-steroid dehydrogenase.