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

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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 7 of these forms in rabbits. The 8 P-450 were distinguished by spectral properties of the ferric, ferrous and ferrous carbonyl forms, apparent monomeric MW 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-benzphentamine, 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 8 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 8 P-450 was at least as high as the spectrally determined P-450 content. Individual forms of P-450 can be induced by different compounds, and 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 8 P-450. These studies provide a basis for further studies on the regulation of individual P-450, the physical properties of the different P-450 and the metabolic consequences of changes in the forms of P-450 in rat liver models.