Position-specific oxygenation of benzo(a)pyrene by different forms of purified cytochrome P-450 from rabbit liver.

High-pressure liquid chromatography was used to detect oxygenated products of benzo[a]pyrene formed in a reconstituted microsomal mixed-function oxidase system containing cytochrome P-450 (P-450LM), phospholipid, and NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4). Three cytochrome fractions purified from a single source, hepatic microsomes from phenobarbital-treated rabbits, were studied; the various forms of the cytochrome are designated by their relative electrophoretic mobilities. The total benzo[a]pyrene oxygenation rate was greatest for P-450LM1,7, intermediate for P-450LM2, and least for P-450LM4. The phenolic products were eluted in two peaks, A and B, that contained primarily 9-hydroxy- and 3-hydroxybenzo[a]pyrene, respectively. The ratio of peak A to peak B phenols was 0.11 for P-450LM2 and 0.45 for P-450LM4. Thus, the relative amounts of the various phenols formed by these two cytochrome fractions differ markedly. The positional specificity of the hydroxylation is also indicated by large differences in the fluorescence spectra of the phenolic products formed by the two cytochromes. P-450LM2 and P-450LM4 did not form benzo[a]pyrene dihydrodiols, thereby showing that benzo[a]pyrene oxide hydratase activity was absent from these purified preparations. Ninety percent of the phenols formed by P-450LM1,7 were eluted in peak B; the metabolites produced by this preparation also included dihydrodiols, thus indicating the presence of hydratase activity. The positional specificities of different forms of cytochrome P-450 may channel polycyclic aromatic hydrocarbon metabolism into the various activation and detoxification pathways and thereby help determine the cytotoxic and carcinogenic activity of these compounds.

Purified liver microsomal cytochrome P-450. Separation and characterization of multiple forms.

During the purification of rabbit liver microsomal cytochrome P-450 (P-450LM), evidence was obtained for the occurrence of at least four distinct forms. These were distinguished by polyacrylamide gel electrophoresis after treatment with sodium dodecyl sulfate in the presence or absence of mercaptoethanol and were shown to have characteristic spectra as the reduced carbon monoxide complexes. They are designated by their relative electrophoretic mobilities. P-450LM2, which was purified to apparent homogeneity, is induced by phenobarbital and has a subunit molecular weight of 50,000. P-450LM4, which was also extensively purified, is induced by beta-naphthoflavone and has a molecular weight of 54,000. P-450LM1,7, which is induced neither by phenobarbital nor beta-naphthoflavone, is a mixtureMIXTURE OF ABOUT EQUAL AMOUNTS OF TWO FORMS WITH MOLECULAR WEIGHTS OF 47,000 AND 60,000 RESPECTIVELY. Some preparations were obtained containing primarily P-450LM1 or P-450LM7. Benzphetamine, ethylmorphine, and p-nitroanisole are hydroxylated preferentially by P-450LM2, and benzpyrene by P-450LM1,7. Biphenyl is hydroxylated in both positions 2 and 4 by all of the preparations, but the latter position is strongly favored by the action of P-450LM2. Testosterone is hydroxylated primarily in position 16alpha by P-450LM2 and in position 6beta by P-450LM1,7. Although the occurrence of additional forms of the cytochrome with highly similar electrophoretic behavior is not ruled out, it appears that the presence of these forms differing in subunit molecular weight may account for the variety of catalytic activities attributed to this pigment of liver microsomes.