Purification and characterization of the corticosteroid 11 beta-dehydrogenase component of the rat liver 11 beta-hydroxysteroid dehydrogenase complex.
We have proposed that 11 beta-hydroxysteroid dehydrogenase is composed of structurally independent units with 11 beta-dehydrogenase and 11-reductase activities. We now report the purification of rat liver 11 beta-dehydrogenase to apparent homogeneity. Starting with microsomes, 800-fold purification was achieved with agarose-NADP affinity chromatography. No 11-reductase accompanied the purification. Homogeneity of 11 beta-dehydrogenase was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid end-group analysis and immunoprecipitation. The terminal amino acid was methionine. Monomer mol wt was 34,000. The enzyme was found to be a glycoprotein. A sequence of 40 amino acid units was identified from the amino end. The amino-terminal region was found to be highly nonpolar. Unlike unpurified microsomal 11 beta-dehydrogenase, which showed curvilinear Eadie plots, homogeneous enzyme gave rectilinear plots. Michaelis constants were 1.83 +/- 0.06 microM for corticosterone and 17.3 +/- 2.24 microM for cortisol. First order rate constants were 10 times greater for corticosterone than cortisol, and maximum velocities were similar.
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000305:Adrenal Cortex Hormones / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D005779:Immunodiffusion; D007525:Isoelectric Focusing; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains
D000305:Adrenal Cortex Hormones; D000596:Amino Acids; D002241:Carbohydrates; D009249:NADP; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases