Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: purification, characterization, and interactions with metyrapone and cytochrome b5.
Cytochrome P-450 isozyme 1 (PB-1) (Mr congruent to 53 000) was purified to apparent homogeneity from phenobarbital (PB)-induced rat liver microsomes, and its spectral, structural, immunochemical, and catalytic properties were determined. PB-1, present in significant amounts in uninduced rat liver microsomes, is induced approximately 2-4-fold by phenobarbital, as compared to the greater than 30-fold induction typical of the major PB isozymes characterized previously. PB-1 was distinguished from the major PB-induced isozymes PB-4 and PB-5 [Waxman, D. J., & Walsh, C. (1982) J. Biol. Chem. 257, 10446-10457] by the absence of a Fe2+-metyrapone P446 complex, by its unique NH2-terminal sequence and distinct peptide maps, by the lack of immuno-cross-reactivity to PB-4, and by its characteristic substrate-specificity profile. Metyrapone effected a saturable enhancement of several PB-1-catalyzed reactions in the reconstituted system [Km(metyrapone) congruent to 200 microM], which varied in magnitude with the substrate, with a maximal stimulation of 5-8-fold in the case of acetanilide 4-hydroxylation. That metyrapone enhanced the corresponding microsomal activities only in cases where the metyrapone-sensitive PB-4 did not catalyze the same reaction at significant rates suggested that PB-1 is probably responsible for the substrate-dependent stimulatory effects of metyrapone on microsomal monooxygenations. In contrast to PB-4 and PB-5, PB-1 was characterized by a marked, but not absolute, dependence on cytochrome b5 (b5) for catalytic activity, with 4-7-fold stimulations typically effected by inclusion of stoichiometric b5 in the reconstituted system. That these b5-stimulations were lipid dependent and were abolished with specific proteolytic fragments lacking b5's COOH-terminal membranous segment evidenced the importance of this segment for efficient, b5-mediated electron transfer to P-450 PB-1 in the reconstituted monooxygenase system.
D000595:Amino Acid Sequence; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008797:Metyrapone / Q000494:pharmacology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010634:Phenobarbital / Q000494:pharmacology*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D013379:Substrate Specificity
D003573:Cytochrome b Group; D007527:Isoenzymes; D010446:Peptide Fragments; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital; D008797:Metyrapone