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) (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.

Multiple forms of cytochrome P-450 related to forms induced marginally by phenobarbital. Differences in structure and in the metabolism of alkoxyresorufins

“…It had a different Mr, did not metabolize benzphetamine (result not shown) and had one difference in the N-terminal sequence. The first three N-terminal amino acids of PB2d are the same as 'form f' described by Hanniu et al [26]; however, the sequence is then identical with form h and not form f. Those authors have discussed whether form h was equivalent to that described by Waxman & Walsh [11] as PB1. From these studies, and in substantiation of their hypothesis, it is clear that these represent different proteins which are both expressed simultaneously.…”

“…Although PB1, appears to be another member of this group, it was isolated from a different microsomal preparation by a different purification procedure [12]. The N-terminal sequence of PBia and PBlb are identical with that of the enzyme described by Waxman & Walsh [11] as PB1. These data demonstrate that several gene products ofthis P-450 subfamily are expressed with identical N-terminal sequences.…”

“…One of the gene subfamilies in which we are interested is constitutively expressed and characterized by marginal induction by phenobarbital. Members of this family have been isolated from rat liver and termed 'PB1' by Waxman & Walsh [11] and 'PB-C' by Guengerich et al [6]. We have previously described the isolation and purification of two cytochrome P-450 forms (PB11I and PB2) marginally inducible by phenobarbital and a variety of other reagents such as trans-stilbene oxide and Aroclor 1254 [12].…”

Modulation of cyclophosphamide-based cytochrome P 450 gene therapy using liver P450 inhibitors

“…These latter findings are consistent with the known selectivity of chloramphenicol and metyrapone for inhibition of CYP2B1. 35,36 To further confirm in vivo the effects of these inhibitors on CPA 4 -hydroxylation seen in vitro, four of the P450 inhibitors were administered to uninduced adult rats. Two hours later, livers were excised and CPA 4-hydroxylation was assayed in isolated liver microsomes (Fig 2 ).…”

Potentiation and suppression of mouse liver cytochrome P‐450 isozymes during the acute‐phase response induced by bacterial endotoxin

“…and MClb of family P-4.501. According to the nomenclature of Ryan et al [50] PB3, = form b, MC1, = form d and MClb = form c. PB1 is equivalent to PB1 described by Waxman and Walsh [51]. PBzc appears to be part of the pregnenolone-I 6a-carbonitrile-inducible P-450 family as reported by Scheutz et al [52] and Wrighton et al [53].…”