Extraction of 11 beta-hydroxysteroid dehydrogenase from rat liver microsomes by detergents.
In these studies our goal was to solubilize the microsomal enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD) as the first step in its purification. Enzyme was extracted from rat liver microsomes with representative detergents (Zwittergents, Tritons, modified sterols). Oxidation-reduction (O-R) ratios of extracts varied with detergent used and ranged from 0.18 (CHAPS) to 3.8 (Zwittergent 3-14) relative to a ratio of 1.7 in intact microsomes. All detergents solubilized 11-HSD using lack of sedimentation during high speed centrifugation as criterion. With Triton DF-18 and Triton X-100, optimum extraction of 11-HSD occurred in the detergent-protein ratio range of 0.1 to 0.2 O-R ratios decreased with increased Triton X-100, but were constant as Triton DF-18 was varied. The pH optimum of enzyme extraction was 9 at a detergent-protein ratio of 0.05 and 7.5-8.0 at a ratio of 0.2. Sodium chloride increased enzyme extraction by detergents; in the absence of detergent, salt extracted protein, but not enzyme. In aqueous solution at 0 degrees C or -15 degrees C, microsomal 11-oxidation activity rose within 24 h, then decreased; reductase activity consistently decreased. Oxidation and reduction activities were inversely related in the microsomal bound enzyme. No relationship between these activities appeared in detergent-solubilized enzymes. Possible mechanisms to account for the unexpected behavior of this enzyme are discussed.
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000818:Animals; D002793:Cholic Acids; D002850:Chromatography, Gel; D003902:Detergents; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D008297:Male; D008722:Methods; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols; D000644:Quaternary Ammonium Compounds; D051381:Rats; D011919:Rats, Inbred Strains; D012965:Sodium Chloride / Q000494:pharmacology; D012995:Solubility; D013501:Surface-Active Agents
D002793:Cholic Acids; D003902:Detergents; D000644:Quaternary Ammonium Compounds; D013501:Surface-Active Agents; C038647:zwittergent 310; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D017830:Octoxynol; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate