Pituitary progesterone 5 alpha-reductase: solubilization and partial characterization.
The microsomal progesterone 5 alpha-reductase activity from female rat anterior pituitary has been solubilized and partially characterized with regard to some of its kinetic and physical properties. The solubilization of progesterone 5 alpha-reductase has been achieved through the use of either an n-octyl glucoside (OG)-KCl- or a digitonin-KCl-extraction. The total yield and specific activity of solubilized enzyme activity is greater using the OG-KCl method. Kinetic analyses of microsomal and OG-KCl-solubilized progesterone 5 alpha-reductase have indicated that both of these preparations exhibit a similar apparent Km for progesterone (microsomal Km = 117 +/- 12 nM; solubilized Km = 123 +/- 11 nM), suggesting that the solubilization procedure does not appreciably alter the kinetic behavior of this enzyme activity. The OG-KCl-extracted progesterone 5 alpha-reductase activity also appears quite stable, since essentially no enzyme activity is lost following dialysis at 4 degrees C for 22 h. In addition, the activity of the solubilized-dialyzed enzyme preparation can be slightly stimulated via the addition of phospholipids. Studies on the properties of the microsomal enzyme activity have indicated that this preparation is unaffected by metal chelators (EDTA or EGTA) but can be completely inhibited by the powerful sulfhydryl blocking agent p-chloromercuribenzoic acid. An evaluation of the possible role of flavins (as a hydride carrier between NADPH and the steroid) has shown that progesterone 5 alpha-reduction is inhibited by high levels of flavins and flavin analogs.
D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D003902:Detergents; D004072:Digitonin; D005260:Female; D005415:Flavins / Q000494:pharmacology; D005960:Glucosides; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D011189:Potassium Chloride; D051381:Rats; D012995:Solubility; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002614:Chelating Agents; D003902:Detergents; D005415:Flavins; D005960:Glucosides; D010743:Phospholipids; D013439:Sulfhydryl Reagents; C018619:octyl-beta-D-glucoside; D011189:Potassium Chloride; D010088:Oxidoreductases; C022447:3-oxo-5-alpha-steroid 4-dehydrogenase (NADP(+)); D004072:Digitonin