Differences in the binding of 3-methylcholanthrene and phenobarbital to rat liver cytosolic and nuclear protein fractions

Abstract

The inducers of cytochrome P-450c and P-450b, 3-methylcholanthrene and phenobarbital, respectively, have been studied in their interaction with subcellular fractions from rat liver. 3-Methylcholanthrene bound to both nuclear and cytoplasmic components as demonstrated by DNA-cellulose chromatography. The binding of 3-methylcholanthrene to cytosolic proteins, on DNA-cellulose, was approximately 27 fmol/mg of applied protein, whereas the binding to nuclear proteins was 250–570 fmol/mg applied protein. Phenobarbital did not bind to proteins of rat serum, rat liver cytosol, or rat liver nuclei which could bind to DNA-cellulose. Further examination of the potential interaction of phenobarbital to rat liver cytosolic proteins was carried out using either DEAE A-50 Sephadex chromatography, charcoal dextran analysis, or sucrose density gradients. No binding of phenobarbital to rat liver cytosolic proteins was observed under these experimental conditions. In contrast, the binding of 3-methylcholanthrene to cytosolic proteins showed four peaks of radioactivity after DEAE A-50 Sephadex chromatography, two peaks by sucrose density gradient analysis, and specific binding (0.13 pmol/mg protein) was observed using the charcoal dextran technique. One of the peaks on sucrose gradients was labile in the presence of salt. The uptake and intranuclear distribution of 3-methylcholanthrene and phenobarbital were markedly different after incubation with whole nuclei: 64% of the available 3-methylcholanthrene but only 3% of the available phenobarbital radioactivity became associated with nuclei. Of this radioactivity, the highest specific activity of the 3-methylcholanthrene radioactivity was associated with the 2 m KCl-resistant nuclear pellet and the highest specific activity of the phenobarbital radioactivity was associated with the nuclear fraction soluble in the absence of salt. These results are interpreted in regard to the induction of cytochrome P-450c.