Molecular Pharmacology, Vol 12, 746-758, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Immunochemical Evidence for Six Forms of Rat Liver Cytochrome P450 Obtained Using Antibodies against Purified Rat Liver Cytochromes P450 and P448

¹ Department of Biochemistry and Drug Metabolism, Research Division, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110

The immune globulin G fraction (anti-PB-P450) isolated from rabbits immunized against purified hepatic cytochrome P450 from phenobarbital-treated rats is quite specific. In Ouchterlony double-diffusion plates, anti-PB-P450 reacts well with its homologous antigen, but poorly and incompletely with purified rat liver cytochrome P448 from 3-methylcholanthrene-treated rats. Purified rabbit cytochrome P450 LM₂ reacts poorly while rabbit cytochrome P448, beef adrenal mitochondrial P450, and Pseudomonas putida P450 show no reaction with anti-PB-P450. Using this antibody preparation in conjunction with our previously reported antibody [Thomas, P. E., Lu, A. Y. H., Ryan, D., West, S. B., Kawalek, J. & Levin, W. (1976) J. Biol. Chem., 251, 1385-1391] to purified hepatic cytochrome P448 from 3-methylcholanthrene-treated rats (anti-MC-P448), we have detected a total of six different but immunochemically related forms of rat liver cytochrome P450. Anti-MC-P448 cross-reacts with cytochrome P450 in Ouchterlony immunodiffusion plates and gives three heme-staining precipitin bands, but the intensity of these bands suggests that only a portion of the total hemeprotein is precipitated by this antibody. When both antibodies are used in the same immunodiffusion plate, anti-PB-P450 precipitates the hemeprotein which does not cross-react with anti-MC-P448. The presence of four heme-staining precipitin bands is evidence for at least four cytochromes P450 in the purified rat PB-P450 preparation. When anti-PB-P450 cross-reacts with cytochrome P448, less of the hemeprotein is precipitated than with the homologous antigen, which also suggests that one or more forms in the purified rat MC-P448 preparation are not recognized by the heterologous antibody. Again, using both antibodies in the same immunodiffusion plate, there is at least one hemeprotein in the cytochrome P448 preparation that is precipitated by anti-MC-P448 but not by anti-PB-P450. The differential effects of the antibodies on the catalytic activity of the reconstituted system also suggest a multiplicity of cytochromes P450 in the purified preparations obtained from phenobarbital- and 3-methylcholanthrene-treated rats. Thus the immunological and catalytic properties of both purified hemeprotein preparations indicate that the rat is capable of producing at least six immunochemically distinguishable forms of cytochrome P450.

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ACKNOWLEDGMENTS We thank Dr. Minor Coon of the Department of Biological Chemistry, University of Michigan, for the rabbit cytochrome P450 LM₂, Dr. David Copper of the Harrison Department of Surgical Research, University of Pennsylvania School of Medicine, for the beef adrenal mitochondrial cytochrome P450, and Dr. Julian Peterson of the Department of Biochemistry, University of Texas Health Science Center, for Pseudomonas putida cytochrome P450. Thanks are also due to Dr. Jim Primus of the Department of Immunology, Hoffmann-La Roche, Inc., for his helpful discussions, and to Mrs. Cathy Chvasta for help in preparing this manuscript.

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