Purification and Characterization of the Corticosteroid 1lβ-Dehydrogenase Component of the Rat Liver 1lβ-Hydroxysteroid Dehydrogenase Complex*

Abstract

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 .mu.M for corticosterone and 17.3 .+-. 2.24 .mu.M for cortisol. First order rate constants were 10 times greater for corticosterone than cortisol, and maximum velocities were similar.