Purification and characterization of three forms of microsomal cytochrome P-450 in liver from 3-methylcholanthrene-treated guinea pigs.
One molecular form of cytochrome P-450IIA from liver microsomes of guinea pigs treated with 3-methylcholanthrene was purified to a specific content of 17.4 nmoles/mg of protein. The difference spectrum of reduced hemoprotein-carbon monoxide complex of this cytochrome exhibits an absorption maximum at 448 nm. The absolute absorption spectrum of the oxidized form of this hemoprotein suggests a high-spin state of heme iron. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein shows a single band of polypeptide stained with Coomassie brilliant blue at the position corresponding to Mr 54,000. On the other hand, the other two forms of cytochrome P-450, cytochrome P-450I and P-450IIB, were also separated and purified to specific contents of 8.7 and 5.2 nmoles/mg of protein, respectively. Both cytochrome P-450I and P-450IIB exhibit absorption maxima at 450 nm in the difference spectrum of reduced hemoprotein-carbon monoxide complex, and a low-spin state of ferric iron in the heme. The spectrophotometrical property of cytochrome P-450I and P-450IIB was clearly different from that of cytochrome P-450IIA. Molecular activities of a reconstituted aryl hydrocarbon hydroxylase (EC 1.14.14.1) containing, respectively, cytochrome P-450I, P-450IIA, and P-450IIB were 0.224, 0.250, and 0.395 (moles per minute per mole of cytochrome P-450), and were estimated to be one-tenth that of cytochrome P-448 induced in rat liver by 3-methylcholanthrene, indicating the presence of the low inducibility by 3-methylcholanthrene of aryl hydrocarbon hydroxylase in liver microsomes of guinea pigs.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001564:Benzo(a)pyrene; D001580:Benzopyrenes / Q000378:metabolism; D002845:Chromatography; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006168:Guinea Pigs; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D051381:Rats; D013057:Spectrum Analysis
D001580:Benzopyrenes; D001564:Benzo(a)pyrene; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D009251:NADPH-Ferrihemoprotein Reductase