title: Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2.
abstract: Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.
authors.affiliation: Institute of Toxicology, University of Mainz, West Germany.
authors.forename: F
authors.initials: F
authors.lastname: Oesch
authors.forename: D J
authors.initials: DJ
authors.lastname: Waxman
authors.forename: J J
authors.initials: JJ
authors.lastname: Morrissey
authors.forename: W
authors.initials: W
authors.lastname: Honscha
authors.forename: W
authors.initials: W
authors.lastname: Kissel
authors.forename: T
authors.initials: T
authors.lastname: Friedberg
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D015151:Immunoblotting; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D010446:Peptide Fragments / Q000276:immunology; D010634:Phenobarbital / Q000494:pharmacology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology*
chemical_list: D000906:Antibodies; D010446:Peptide Fragments; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D004262:DNA Restriction Enzymes; D010634:Phenobarbital