Purification and Properties of the Δ5-3β-Hydroxysteroid Dehydrogenase-Isomerase System of Sheep Adrenal Cortical Microsomes

A5-3/3-Hydroxysteroid dehydrogenase and A5-isomerase have been extracted from exhaustively washed sheep adrenal cortical microsomes by treatment with the nonionic detergent Triton X-100 and purified by ion exchange chromatography to near homogeneity.No dissociation of dehydrogenase activity on the basis of substrate specificity (3&hydroxyandrost-S-en-17-one and 3P-hydroxypregn-5-en-20-one) was observed.The purified enzymatically active material requires detergent for aqueous solubility.A mixture of [3H]acetyl-Triton X-100 and the purified enzymatic activities is eluted as a single band on gel filtration on Bio-Gel A-0.5m; the elution volume is exactly that required for elution of [aH]acetyl-Triton X-100 alone and corresponds to that observed for globular proteins of molecular weights in the range of 300,000.The behavior of the purified material on gel filtration probably reflects interaction of the detergent with the protein, resulting in micelle formation.Sodium dodecyl sulfate treatment of the purified material followed by electrophoresis results in a single protein band with a mobility corresponding to a molecular weight of about 40,000.The protein band was eluted from the gels, and amino acid sequence determination was performed from the NHz-terminal end.Alanine was found as the sole NH2terminal amino acid present, followed by glycine as the sole penultimate amino acid.The observation that steroid 3/3-01 dehydrogenase and As-isomerase copurify to a state approaching protein homogeneity as assessed by several methods suggests that the two enzymatic activities are closely associated.The results