PURIFICATION AND RECONSTITUTION OF THE STEROID 21-HYDROXYLASE SYSTEM (CYTOCHROME-P-450-LINKED MIXED-FUNCTION OXIDASE SYSTEM) OF BOVINE ADRENOCORTICAL MICROSOMES

Abstract

The steroid 21-hydroxylase system was purified from bovine adrenocortical microsomes. The physicochemical properties of the cytochrome P-450s21 [cytochrome P-450 steroid 21-hydroxylation] were studied. The properties of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) of bovine adrenocortical microsomes were reported previously. The steroid 21-hydroxylase system was reconstituted by cytochrome P-450s21 and NADPH-cytochrome P-450 reductase in the presence of detergent. The substrate specificity of the cytochrome P-450s21 was examined with the reconstituted system. The enzyme system was active in the 21-hydroxylations of 17.alpha.-hydroxyprogesterone and progesterone and the N-demethylation of (+)-benzphetamine. The cytochrome P-450s21 purified to as high as 16-17 nmol/mg protein was an electrophoretically pure glycoprotein. The MW of the cytochrome P-450s21 was estimated to be 47,500 by SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis. Although cytochrome P-450scc [cytochrome P-450 catalyzing the side chain (C20-C22) cleavage of cholesterol] or cytochrome P-45011.beta. [cytochrome P-450 catalyzing 11.beta.-hydroxylation] of bovine adrenocortical mitochondria did not react with antibody to cytochrome P-450BPA [cytochrome P-450 catalyzing N-demethylation of (+)-benzphetamine] of bovine liver microsomes, the cytochrome P-450s21 formed an immunoprecipitin line against the antibody to cytochrome P-450BPA in the Ouchterlony double diffusion test.