Purification and characterization of liver microsomal cytochromes P-450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or .beta.-naphthoflavone

Abstract

Eight different forms of cytochrome P-450 (P-450) were purified to electrophoretic homogeneity by a common procedure from liver microsomes of rats treated with phenobarbital or beta-naphthoflavone. Antibodies were prepared to seven of these forms in rabbits. The eight P-450s were distinguished by spectral properties of the ferric, ferrous, and ferrous carbonyl forms, apparent monomeric molecular weights, peptide mapping, immunological reactivity as discerned by double-diffusion immunoprecipitin analysis and crossed immunoelectrophoresis, and catalytic activities toward the substrates acetanilide, aminopyrine, aniline, benzo[a]-pyrene, d-benzphetamine, N,N-dimethylnitrosamine, 7-ethoxycoumarin, 7-ethoxyresorufin, ethylmorphine, p-nitroanisole, testosterone, and (R)- and (S)-warfarin. Crossed sodium dodecyl sulfate-polyacrylamide gel immunoelectrophoresis was used to estimate the levels of each of the eight forms of P-450 present in the liver microsomes of untreated rats and rats treated with phenobarbital, 5,6-benzoflavone, pregnenolone-16 alpha-carbonitrile, isosafrole, or the polychlorinated biphenyl mixture Aroclor 1254. In each situation, the sum of the levels of these eight P-450s was at least as high as the spectrally determined P-450 content. The results clearly demonstrate that individual forms of P-450 can be induced by different compounds and that a single compound can lower the level of one form of P-450 while inducing one or more other forms of P-450. Catalytic activities toward each of the substrates observed with microsomal preparations are compared to rates predicted on the basis of the content of each of the eight P-450s. These studies provide a basis for further studies on the regulation of individual P-450s, the physical properties of the different P-450s, and the metabolic consequences of changes in the forms of P-450 in rat liver models.

Anticarcinogenic Effect and Modification of Cytochrome P450 2E1 by Dietary Garlic Powder in Diethylnitrosamine-Initiated Rat Hepatocarcinogenesis

“…Many of the CYP enzymes are inducible by a variety of chemicals. This alteration of the composition of these enzyme systems may have profound effects on chemical toxicity and carcinogenesis (Guengerich et al, 1982).…”

“…Many of the CYP enzymes are inducible by a variety of chemicals. This alteration of the composition of these enzyme systems may have profound effects on chemical toxicity and carcinogenesis (Guengerich et al, 1982).Dially sulfide (DAS), one of the garlic OSCs, inhibits chemical toxicity and tumorigenesis in several animal models, presumably by acting as an inhibitor of the CYP-mediated metabolic activation of the toxic and carcinogenic chemicals. DAS seems to cause a selective alteration to certain forms of CYP enzymes in rat liver.…”

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

“…This family has been split into several subfamilies [3,4]. Those involved in foreigncompound metabolism have been classified by their inducibility in the liver by specific compounds including 3-methylcholanthrene [1,3,4], high and low inducibility with phenobarbital [1,3,5] and by inducibility with pregnenolene-16a.-carbonitrile [6,7]. Members within the same subfamily can show extensive sequence homology (up to 97% [8,9]), yet large differences in substrate specificity have been observed [10].…”

“…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].…”

Complexation of Membrane-Bound Enzyme Systems

“…Recombinant human CYP3A4NF14 and human NADPH-P450 reduc tase (reductase) were purchased from Oxford Bio chemical Research, Inc., Oxford, MI (USA). Rat CYP2B1 and CYP1A1 and rat and rabbit NADPH-P450 reductase were purified to electro phoretic homogeneity using procedures described by Guengerich et al (1982).…”