Biochemical characterization of highly purified cytochrome P-450 and other components of the mixed function oxidase system of liver microsomal membranes.
Cytochrome P-450 has been purified from liver microsomes of phenobarbital-induced rabbits in the presence of ionic and nonionic detergents to concentrations over 17 nmoles per mg of protein. The purified cytochrome P-450 LM gives a single major band on SDS-polyacrylamide gel electrophoresis representing about 90 per cent of the total protein. The polypeptide chain has a molecular weight of about 49,000 daltons. NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-induced rats in the presence of ionic and nonionic detergents to a stage where it catalyzes the reduction of 33,000 nmoles of cytochrome c per min per mg of protein. The ratio of activities toward cytochrome P-450 and cytochrome c is constant throughout purification. The purified reductase contains equimolar amounts of FMN and FAD and gives a single major band on SDA-polyacrylamide gel electrophoresis accounting for about 70 per cent of the total protein; the molecular weight is about 80,000 daltons. The purified cytochrome P-450 is free of cytochrome b5 but contains another electron acceptor, provisionally called Factor C, which is equivalent in amount to the heme present. Two electrons are taken up per molecule of cytochrome P-450 from dithionite or from NADPH in the presence of catalytic amounts of the reductase, and both electrons are readily transferred from the reduced cytochrome P-450 to molecular oxygen or artificial electron acceptors. The reconstituted enzyme system containing purified cytochrome P-450, purified NADPH-cytochrome P-450 reductase, and phosphatidylcholine retains the ability to catalyze the hydroxylation of drugs, fatty acids, hydrocarbons, and aniline in the presence of NADPH and molecular oxygen.
D000693:Anaerobiosis; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D011756:Diphosphates / Q000494:pharmacology; D004227:Dithionite / Q000494:pharmacology; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D011817:Rabbits; D012995:Solubility; D013057:Spectrum Analysis
D003574:Cytochrome c Group; D011756:Diphosphates; D010713:Phosphatidylcholines; D004227:Dithionite; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital