Purification and characterization of cholesterol 7 alpha-hydroxylase cytochrome P-450 of untreated rabbit liver microsomes.
Cytochrome P-450 which catalyzes the 7 alpha-hydroxylation of cholesterol was purified from liver microsomes of untreated rabbits. The minimum molecular weight of the cytochrome P-450 was estimated to be 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation contained 7 nmol of cytochrome per mg of protein. The oxidized form of the P-450 showed absorption maxima at 568, 535, and 417 nm, which are characteristic of a low spin hemoprotein, while the reduced form showed maxima at 545 and 413 nm. The carbon monoxide complex of the reduced form showed maxima at 550 and 447 nm. The cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes was reconstituted with the purified P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5. The P-450 catalyzed the 7 alpha-hydroxylation of cholesterol 500 times more efficiently than the starting microsomes. The reconstituted hydroxylase system showed a substantial salt dependency. In the presence of cytochrome b5 the activity was maximum at 0.4 M KCl (4.55 nmol product formed/mg of protein per min), whereas in the absence of cytochrome b5 the activity was marginal (0.65 nmol product formed/mg of protein per min) and inhibited by KCl. Thus, cytochrome b5 stimulated the hydroxylase activity by one order of magnitude. These results indicate that cytochrome b5 is an essential component of the cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes.
D000818:Animals; D002790:Cholesterol 7-alpha-Hydroxylase / Q000302:isolation & purification* / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism; D015786:Cytochromes b5; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000302:isolation & purification*
D003573:Cytochrome b Group; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase