Partial characterization of the microsomal and solubilized hypothalamic progesterone 5 alpha-reductase.
Microsomal progesterone 5 alpha-reductase activity from female rat hypothalamus has been solubilized and partially characterized in terms of kinetic and physical properties. The solubilization of progesterone 5 alpha-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 Km for progesterone (Microsomal Km = 113 +/- 11 nM; solubilized Km = 144 +/- 20 nM) and their affinity (approximately 7 nM) for the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione, which is a potent inhibitor of progesterone 5 alpha-reduction. Both activities are inhibited by divalent cations (Zn2+ and Cu2+) and the sulfhydryl-blocking agent p-chloromercuribenzoic acid. Studies aimed at optimizing isolation and assay conditions for the hypothalamic progesterone 5 alpha-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 5 alpha-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.
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D005260:Female; D005415:Flavins / Q000494:pharmacology; D007031:Hypothalamus / Q000201:enzymology*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D010084:Oxidation-Reduction / Q000187:drug effects; D010743:Phospholipids / Q000494:pharmacology; D011375:Progesterone Reductase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D012256:Riboflavin / Q000502:physiology; D012995:Solubility
D005415:Flavins; D008670:Metals; D010743:Phospholipids; D011480:Protease Inhibitors; D015096:3-Hydroxysteroid Dehydrogenases; D011375:Progesterone Reductase; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D012256:Riboflavin