Competitive Inhibition of Adrenal Δ5-3β-Hydroxysteroid Dehydrogenase and Δ5-3-Ketosteroid Isomerase Activities by Adenosine 3',5'-Monophosphate

Abstract

Abstract Microsomal and mitochondrial preparations from rat adrenal glands converted pregnenolone (3β-hydroxypregn-5-ene-20-one) to progesterone, a two step reaction which requires the participation of a DPN+-dependent Δ5-3β-hydroxysteroid dehydrogenase and a Δ5-3-ketosteroid isomerase. The mitochondrial enzymes could be prepared as a membrane fraction after sonic treatment of the mitochondria. This preparation revealed a lower apparent Km for DPN+ than the corresponding microsomal system. The submitochondrial fraction rapidly oxidized DPNH formed during the reaction, whereas the microsomal fraction was much less active in this regard. When a DPN+-regenerating system (lactic dehydrogenase + pyruvate) was added to the microsomal system, the apparent Km for DPN+ was indistinguishable from that of the submitochondrial preparation. The conversion of pregnenolone to progesterone was inhibited by cyclic adenosine 3',5'-monophosphate (3',5'-AMP) in both subcellular systems, but was more sensitive to inhibition in submitochondrial particles than in the microsomal preparation. However, when the DPN+-regenerating system was added to the microsomal fraction, utilization of pregnenolone to progesterone was inhibited by 3',5'-AMP to a comparable extent in both subcellular preparations. Inhibition of pregnenolone utilization by 3',5'-AMP was strictly competitive with respect to DPN+ in each instance. Among various adenine compounds tested, 3',5'-AMP inhibited the conversion of pregnenolone to progesterone to the greatest extent. Adenosine also produced a significant inhibition. Adenosine 5'-monophosphate and adenosine 5'-diphosphate appeared to be slightly inhibitory. Adenosine 3'-monophosphate, adenosine 5'-triphosphate, and adenosine 2',3'-monophosphate were inactive. Activation of lactic dehydrogenase by DPNH was also inhibited competitively by 3',5'-AMP. This action of the cyclic nucleotide was less specific than its effect on pregnenolone utilization, since 5'-AMP, 5'-ADP, and 5'-ATP inhibited nearly as well. Moreover, adenosine had no effect in this instance. Steroid dehydrogenase and isomerase activities in adrenal subcellular fractions were also studied individually, using substrates which were specific either for Δ4-3β-hydroxysteroid dehydrogenase (pregn-4-ene-3β,20β-diol and androst-4-ene-3β,17β-diol) or for Δ5-3-ketosteroid isomerase (pregn-5-ene-3,20-dione and androst-5-ene-3,17-dione). Both enzyme activities were stimulated by DPN+ and competitively inhibited by 3',5'-AMP. At all levels of DPN+ and 3',5'-AMP, the activity of these dehydrogenase systems, as well as that of the conversion of pregnenolone to progesterone, was lower than that of the isomerase systems. These results suggested that Δ5-3β-hydroxysteroid dehydrogenase activity may be rate-limiting in the over-all reaction and that the inhibitory effects of the cyclic nucleotide on conversion of pregnenolone to progesterone in adrenal subcellular preparations were primarily related to actions on the dehydrogenase.