Metabolism of androst-4-ene-3,17-dione by subcellular fractions of rat adrenal tissue with particular reference to microsomal C19-steroid 5alpha-reductase

The location and some characteristics of rat adrenal C(19)-steroid 5alpha-reductase were investigated by using [7alpha-(3)H]androst-4-ene-3,17-dione and [7alpha-(3)H]testosterone as substrates. The enzymes system was shown to be NADPH-dependent and associated with the microsomal fraction. In addition, some evidence was also obtained for the existence of a separate NADH-dependent system in the soluble fraction. Further investigation of androst-4-ene-3,17-dione metabolism by subcellular fractions indicated the presence of NADH-dependent 3alpha- and 3beta-hydroxy steroid dehydrogenase systems in the microsomal pellet. This pellet also appeared to contain an NADH-dependent 17beta-hydroxy steroid dehydrogenase system, and a similar though separate system was detected in the cytosol. Malate (20mm) effectively inhibited the microsomal C(19)-steroid 5alpha-reductase, which showed similar values for K(m) and V(max.) when either androst-4-ene-3,17-dione or testosterone was used as substrate. Cytochrome c was added to all incubation mixtures used for the determination of these values to inhibit the formation of metabolites other than 5alpha-androstane-3,17-dione and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) respectively. It was also found that corticosterone did not inhibit the 5alpha-reduction of androst-4-ene-3,17-dione under these conditions, indicating that separate enzymes exist for the 5alpha-reduction of C(19)- and C(21)-steroids in the rat adrenal.