Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical microsomes.
The steroid 21-hydroxylase system was purified from bovine adrenocortical microsomes. The physicochemical properties of NADPH-cytochrome P-450s21 were studied. The properties of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) of bovine adrenocortical microsomes have been reported previously (Hiwatashi, A. and Ichikawa, Y. (1979) Biochim. Biophys. Acta 580, 44-63). 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 per mg protein was an electrophoretically pure glycoprotein. The molecular weight of the cytochrome P-450s21 was estimated to be 47 500 by SDS-polyacrylamide gel electrophoresis. Although cytochrome P-450scc or cytochrome P-45011 beta of bovine adrenocortical mitochondria did not react with antibody to cytochrome P-450BPA 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.
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification*; D007120:Immunochemistry; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000302:isolation & purification*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification*; D013255:Steroid 21-Hydroxylase / Q000302:isolation & purification*; D013250:Steroid Hydroxylases / Q000302:isolation & purification*; D013379:Substrate Specificity
D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D013255:Steroid 21-Hydroxylase