Purification and characterization of cytochrome P450 isozymes from β-naphthoflavone-induced adult hen liver

Abstract

Cytochromes P450 βNF-A, βNF-B, and βNF-C were purified from β-naphthoflavone-treated adult hens. Cytochrome P450 βNF-A, however, appeared at two places in the purification scheme. They were designated as cytochromes P450 βNF-A 1 and βNF-A 2 for property comparison. The cytochromes βNF-A 1 and βNF-A 2 were induced by both phenobarbital and β-naphthoflavone treatment and were similar to P450 PB-A (previously purified from phenobarbital-induced hen livers) in molecular weights, isoelectric pH, spectral properties, behavior on chromatography columns, catalysis of substrates, immunological cross-reactivity on Ouchterlony plates and by immunoblotting, and NH 2-terminal amino acid sequence. However, P450 PB-A differed from βNF-A 1/βNF-A 2 in peptide pattern after partial proteolysis by α-chymotrypsin and Staphylococcus aureus V 8 protease, and complete digestion of 125I-labeled cytochromes by trypsin. The cytochrome P450 PB-A also differed from βNF-A 1/βNF-A 2, in that its antibodies cross-reacted with P-450 of normal, PB-, and β-NF-induced rabbit liver microsomes. The cytochromes βNF-B and βNF-C, although immunochemically cross-reactive with each other, were distinct enzymes on the basis of molecular weights, spectral characteristics, isoelectric pH, peptide pattern on partial proteolysis, tryptic peptide pattern, cross-reactivity of their antibodies with other species, and NH 2-terminal amino acid sequence. The most notable difference between βNF-B and βNF-C was that the anti-βNF-C IgG completely inhibited O-dealkylation of 7-methoxyresorufin and 7-ethoxyresorufin by β-NF-induced microsomes. These activities increased 40- to 50-fold in β-NF-induced microsomes as compared to only 2- to 4-fold in PB-treated hens. The amino-terminal sequences of βNF-B and βNF-C were different from those of mammalian and other nonmammalian species.