Purification and characterization of cytochrome P450 isozymes from beta-naphthoflavone-induced adult hen liver.
Cytochromes P450 beta NF-A, beta NF-B, and beta NF-C were purified from beta-naphthoflavone-treated adult hens. Cytochrome P450 beta NF-A, however, appeared at two places in the purification scheme. They were designated as cytochromes P450 beta NF-A1 and beta NF-A2 for property comparison. The cytochromes beta NF-A1 and beta NF-A2 were induced by both phenobarbital and beta-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 NH2-terminal amino acid sequence. However, P450 PB-A differed from beta NF-A1/beta NF-A2 in peptide pattern after partial proteolysis by alpha-chymotrypsin and Staphylococcus aureus V8 protease, and complete digestion of 125I-labeled cytochromes by trypsin. The cytochrome P450 PB-A also differed from beta NF-A1/beta NF-A2, in that its antibodies cross-reacted with P-450 of normal, PB-, and beta-NF-induced rabbit liver microsomes. The cytochromes beta NF-B and beta NF-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 NH2-terminal amino acid sequence. The most notable difference between beta NF-B and beta NF-C was that the anti-beta NF-C IgG completely inhibited O-dealkylation of 7-methoxyresorufin and 7-ethoxyresorufin by beta-NF-induced microsomes. These activities increased 40- to 50-fold in beta-NF-induced microsomes as compared to only 2- to 4-fold in PB-treated hens. The amino-terminal sequences of beta NF-B and beta NF-C were different from those of mammalian and other nonmammalian species.
Gupta
R P
RP
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina.
Lapadula
D M
DM
Abou-Donia
M B
MB
D000595:Amino Acid Sequence; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology*; D002645:Chickens; D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D017886:Durapatite; D004790:Enzyme Induction; D005260:Female; D006882:Hydroxyapatites; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010634:Phenobarbital / Q000494:pharmacology; D013379:Substrate Specificity; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D006882:Hydroxyapatites; D007527:Isoenzymes; D010446:Peptide Fragments; D019324:beta-Naphthoflavone; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D010634:Phenobarbital