Partial characterization of the microsomal and solubilized hypothalamic progesterone 5α-reductase

Microsomal progesterone 5α-reductase activity from female rat hypothalamus has been solubilized and partially characterized in terms of kinetic and physical properties. The solubilization of progesterone 5α-reductase has been accomplished through the use of a digitonin/KCL-extraction. Both the microsomal and solubilized enzyme activities exhibit similar kinetic and physical characteristics. These include their apparent K m for progesterone (Microsomal K m = 113 ± 11 nM; solubilized K m = 144 ± 20 nM) and their affinity (~7nM) for the 5α-dihydroprogesterone analog, 4-aza-4-methyl-5α-pregnane-3,20-dione, which is a potent inhibitor of progesterone 5α-reduction. Both activities are inhibited by divalent cations (Zn 2+ and Cu 2+) and the sulfhydryl-blocking agent p-chloromercuribenzoic acid. Studies aimed at optimizing isolation and assay conditions for the hypothalamic progesterone 5α-reductase indicate that the microsomal activity is enhanced in the presence of monovalent cations (particularly K + and Li +) and the metal chelator EDTA, but is unaffected by the sulfhydryl reducing agent dithiothreitol. The activity of the solubilized enzyme is also enhanced by EDTA but slightly stimulated by dithiothreitol. Analysis of hypothalamic progesterone 5α-reduction for possible flavin involvement (as a hydride carrier between NADPH and the steroid) indicates that the enzyme activity is decreased by high levels of flavins, flavin analogs and riboflavin deficiency.