Conversion of Cholesterol to Bile Acids in Rat: Purification and Properties of a Δ4-3-Ketosteroid 5β-Reductase and a 3α-Hydroxysteroid Dehydrogenase

A Δ4-3-ketosteroid 5β-reductase active on 7α-hydroxycholest-4-en-3-one and 7α,12α-di-hydroxycholest-4-en-3-one and a 3α-hydroxysteroid dehydrogenase active on 7α-hydroxy-5β-cholestan-3-one and 7α,12α-dihydroxy-5β-cholestan-3-one have been isolated from the soluble fraction of a rat liver homogenate and have been partially purified by chromatography on TEAE-cellulose and Sephadex G-75. The Δ4-3-ketosteroid 5β-reductase required NADPH as cofactor and was inhibited reversibly by p-chloromercuribenzoate. The enzyme preparation catalyzed the reduction of the double bond in a number of Δ4-3-ketosteroids of the C19, C21, C24, and C27 series. The reduction of 7α,12α-dihydroxycholest-4-en-3-one in the presence of the Δ4-3-ketosteroid 5β-reductase preparation was not inhibited by addition of 7α-hydroxycholest-4-en-3-one and the reduction of 7α-hydroxycholest-4-en-3-one was not inhibited by 7α,12α-dihydroxycholest-4-en-3-one indicating that these substrates were reduced by different reductases present in the Δ4-3-ketosteroid 5β-reductase preparation. The 3α-hydroxysteroid dehydrogenase required reduced pyridine nucleotide, NADPH being preferred, and was inhibited reversibly by p-chloromercuribenzoate. The enzyme preparation catalyzed the reduction of the 3-keto group in a number of 3-ketosteroids of the C19, C21, C24, and C27 series. The rate of reduction was faster with 3-keto-5β-steroids than with the corresponding 3-keto-5α-steroids.