Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: purification, characterization, and interactions with metyrapone and cytochrome b5

Abstract

Cytochrome P-450 isozyme 1 (PB-1) (MW .simeq. 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 .apprx. 2- to 4-fold by PB, as compared to the > 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 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) .simeq. 200 .mu.M], which varied in magnitude with the substrate, with a maximal stimulation of 5- to 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- to 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 membraneous segment evidence the importance of this segment for efficient, b5-mediated electron transfer to P-450 PB-1 in the reconstituted monooxygenase system.