The structure of phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4. Production and characterization of site-specific antibodies.
Fifteen peptides corresponding in sequence to segments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the antipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demonstrated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 125I-labeled P-450 PB-4 and also in vitro-synthesized apo-P-450 PB-4, the yields of immunoprecipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibodies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5. Three antipeptide antibodies raised against hydrophilic segments located in the amino-terminal one-third of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed O-deethylation of the model substrate 7-ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 beta NF-B, the major aromatic hydrocarbon-inducible rat hepatic P-450, suggesting that certain amino acid sequences or regions of secondary structure are conserved between the major phenobarbital-induced and polycyclic-induced rat liver P-450 isoenzymes. These studies demonstrate the utility of antipeptide antibodies for evaluation of antigenic sites exposed in native P-450 PB-4, for identification of specific amino acid sequences important for the interaction of P-450 PB-4 with its substrate and/or with cytochrome P-450 reductase in a reconstituted system and for elucidation of structural and immunochemical homologies between P-450 PB-4 and other P-450 isoenzymes present in rat liver endoplasmic reticulum.
D015660:7-Alkoxycoumarin O-Dealkylase; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000276:immunology*; D004790:Enzyme Induction; D004797:Enzyme-Linked Immunosorbent Assay; D007527:Isoenzymes / Q000096:biosynthesis / Q000276:immunology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010105:Oxygenases / Q000037:antagonists & inhibitors; D010446:Peptide Fragments / Q000276:immunology; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains
D000906:Antibodies; D007527:Isoenzymes; D010446:Peptide Fragments; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D010634:Phenobarbital