Multiplicity of steroid-inducible cytochromes P-450 in rat liver microsomes

Highly purified preparations of steroid-inducible cytochromes P-450 have been isolated from liver microsomes of adult male Sprague-Dawley rats treated with pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), or triacetyloleandomycin (TAO). The interrelationships among these preparations as well as their relationship to the major forms of cytochrome P-450 of this gene family identified in other laboratories have been evaluated by amino-terminal sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, chromatography on DEAE-Sephacel, and Ouchterlony double-immunodiffusion. The results indicate that a cytochrome P-450 previously isolated in this laboratory and referred to as PCNa (P.E. Graves, L.S. Kaminsky, and J. Halpert (1987) Biochemistry 26, 3887-3894) is the major cytochrome P-450 induced in rats by TAO, and corresponds to P-450p (K.A. Hostetler, S.A. Wrighton, P. Kremers, and P.S. Guzelian (1987) Biochem. J. 245, 27-33) and to P450PCN1 (F.J. Gonzalez, B.J. Song, and J.P. Hardwick (1986) Mol. Cell. Biol. 6, 2969-2976). A second previously isolated cytochrome P-450 termed PCNb in this laboratory appears to be identical to PB/PCN-E (F.P. Guengerich, G.A. Dannan, S.T. Wright, M.V. Martin, and L.S. Kaminsky (1982) Biochemistry 21, 6019-6030). PCNb as well as a third cytochrome P-450 termed PCNc isolated from PB-treated rats both correspond in amino-terminal sequence to the putative protein product of the pP450PCN2/cDNA clone of Gonzalez et al. These results document at the protein level the multiplicity of steroid-inducible rat liver cytochromes P-450.