Preparation and properties of side-chain cleavage cytochrome P-450 from bovine adrenal cortex by affinity chromatography with pregnenolone as ligand.
A method is described for preparing cytochrome P-450 (side-chain cleavage) from bovine adrenocortical mitochondria, by affinity chromatography on pregnenolong-Sepharose beads. The cytochrome P-450 appears in two fractions, a large form of heterogeneous molecular weight (large P-450) and a form of molecular weight 850 000 composed of 16 apparently identical subunits (molecular weight 52 000-53 000); this form is referred to as protein 16. Electrophoresis on polyacrylamide gel yields one main band and two minor bands; the appearance of the gels is identical whether the starting material is large P-450 or protein 16 or protein 16 prepared by an entirely different method. Yields of protein 16 can be increased by rechromatography on pregnenolone-Sepharose of large P-450 made 0.1 mM in NADPH. Large P-450 shows greater than 10 heme groups per 16 subunits and is less active enzymatically than protein 16. Chromatography on Sepharose and analytical ultracentrifugation show that large P-450 is heterogeneous with respect to molecular weight. Protein 16 shows a heme content of 8 nmol/mg protein and for both large P-450 and protein 16 heme content by CO-difference spectroscopy is in agreement with values by pyridine hemochromogen. This method of preparing P-450 is convenient and both large P-450 and protein 16 are highly purified.
D000302:Adrenal Cortex / Q000201:enzymology*; D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D002248:Carbon Monoxide; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006418:Heme / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification; D010713:Phosphatidylcholines / Q000494:pharmacology; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D010743:Phospholipids / Q000502:physiology; D011284:Pregnenolone; D013053:Spectrophotometry
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D006418:Heme; D011284:Pregnenolone; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D002786:Cholesterol Side-Chain Cleavage Enzyme