Inhibition of Mammalian Glucose-6-phosphate Dehydrogenase by Steroids.

It has been considered for some time that steroid hormones may exert their regulatory effects by influencing certain enzymes.' However, our present knowledge of the mechanisms of steroid action upon enzymes is limited. Estrogens have been shown to stimulate the transfer of hydrogen between diand triphosphopyridine nucleotides,2 ' although there is disagreement as to the role of steroids in this reaction.4' Recently, steroids were found to inhibit reduced diphosphopyridine nucleotide oxidation by enzyme preparations from mammalian and microbial sources.6 I This action appears to be a general property shared by a large number of steroids.7 This study demonstrates a highly specific inhibitory effect on mammalian glucose-6-phosphate dehydrogenase (G-6-PD) of very low concentrations of dehydroisoandrosterone, pregnenolone, and certain related steroids. These steroids are not inhibitors of yeast or spinach G-6-PD or of mammalian 6-phosphogluconic dehydrogenase or isocitric dehydrogenase. Materials and Methods.-Preparation of enzymes: G-6-PD was purified from human erythrocytes by a procedure described in detail elsewhere' involving ammonium sulfate fractionation followed by absorption on calcium phosphate gel, elution in a phosphate buffer, and a second series of ammonium sulfate precipitations. The final product generally had a specific activity 500-fold that of the crude hemolysate in a yield of about 40 per cent. This preparation was essentially free of hemoglobin and 6-phosphogluconic dehydrogenase activity. Crude enzyme preparations were obtained from human and rat tissues and from spinach by homogenizing freshly obtained material in isotonic potassium chloride buffered at pH 7.4 (10%, weight/volume). The homogenate was centrifuged 30 minutes at 18,000 X g to sediment debris. The supernatant fluid was employed for assays of enzyme activity. Partially purified yeast G-6-IPD and pig heart isocitric dehydrogenase were obtained from Sigma Chemical Corporation. IPartially purified 6-phosphogluconic dehydrogenase was prepared from rat liver by the method of Glock and AIcLean.9 Enzyme assays: Purified preparations of G-6-PD were assayed by the method of Kornberg and Horecker.10 In measuring G-6-PD activity in crude tissue preparations, due to the presence of 6-phosphogluconic dehydrogenase, the rate of TPNH formation exceeds the rate of glucose-6-phosphate oxidation. Accordingly, in crude preparations, G-6-PD activity was assayed by the method of Kornberg and Horecker"° determining the initial rate of TPNH formation by measuring the change of absorption at 340 m,4 with an automatic recording Cary spectrophotometer or, alternatively, by the method of Glock and AMcLean.9 In this latter method, G-6-PD activity is taken as the difference in the rate of TP'NH reduction when the reaction mixture contains both 6-phosphogluconate plus glucose-6-phosphate and in the presence of 6-phosphogluconate alone. Comparable results with