Purification and characterization of seven distinct forms of liver microsomal cytochrome P-450 from untreated and inducer-treated male Wistar rats.

Abstract

A total of nine forms of cytochrome P-450 were purified to homogeneity from liver microsomes of male Wistar rats. They were P-451 I and P-451 II from untreated rats, P-450 II and P-450 III from phenobarbital-treated rats, MC-P-448 L and MC-P-448 H from 3-methylcholanthrene-treated rats, and P-452, P-448 L, and P-448 H from 3,4,5,3',4'-pentachlorobiphenyl-treated rats. Among them, MC-P-448 L and MC-P-448 H were indistinguishable from P-448 L and P-448 H, respectively, with regard to electrophoretic, spectral, catalytic and immunochemical properties, and thus seven forms were distinct hemoproteins. The minimal molecular weight of each form was as follows: P-451 I (49,000), P-451 II (52,000), P-450 II (52,000), P-450 III (53,500), P-452 (48,000), P-448 L (56,000), P-448 H (54,000). Judging from the oxidized absolute spectra, P-448 H was a high-spin form and the others were of low-spin type. In a reconstituted system, N-demethylations of benzphetamine and aminopyrine were catalyzed by most of the forms at comparable rates. On the other hand, the activities for the oxidations of benzo[a]pyrene, 7-ethoxycoumarin, biphenyl, and estradiol-17 beta varied greatly among the forms of cytochrome P-450. The most efficient catalysts were as follows: P-448 L and P-451 II for benzo[a]pyrene 3-hydroxylation; P-448 L for 7-ethoxycoumarin O-deethylation; P-448 L, P-451 II, and P-448 H for biphenyl 4-hydroxylation; P-448 L and P-448 H for biphenyl 2-hydroxylation; and P-451 II and P-448 H for estradiol 2-hydroxylation. P-451 I, P-450 II, and P-450 III were somewhat poorer catalysts in metabolizing all the substrates except for benzphetamine and aminopyrine, but their substrate specificities were still distinguishable from one another. Of all the purified cytochrome P-450's, P-452 showed the least ability to metabolize all the substrates. Judging from the properties, it appears that six forms in male Wistar rats correspond to the distinct forms of cytochrome P-450 in Long-Evans and/or Sprague-Dawley rats reported by other workers, but P-451 I is a new constitutive isozyme in Wistar rats.