Competitive Inhibition of Adrenal A5-3P-Hydroxysteroid Dehydrogenase and A5-3-Ketosteroid Isomerase Activities by Adenosine 3’, 5’-Monophosphate*

Microsomal and mitochondrial preparations from rat adrenal glands converted pregnenolone (3@-hydroxypregn& ene-20-one) to progesterone, a two step reaction which requires the participation of a DPN+-dependent A&-3phydroxysteroid dehydrogenase and a A6-3-ketosteroid isomerase. The mitochondrial enzymes could be prepared as a membrane fraction after sonic treatment of the mitochondria. This preparation revealed a lower apparent K, 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 K,,, 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