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-16α-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 pP450 PCN2 cDNA clone of Gonzalez et al. These results document at the protein level the multiplicity of steroid-inducible rat liver cytochromes P-450.