Production of a form-specific, inhibitory antibody against rat cytochrome P450 2B1 using a synthetic peptide antigen against a putative substrate binding site.
Rat cytochrome P450 2B1 antipeptide antibodies were produced by immunizing rabbits with a synthetic peptide antigen. The anti-CYP2B1 IgG obtained did not cross-react with CYP2B2, which has 97% identity in primary sequence of CYP2B1. This result demonstrates that a difference of 2 amino acid residues among 12 is sufficient to produce a form-specific antibody. The CYP2B1 antipeptide IgG inhibited pentoxyresorufin O-dealkylase activity of microsomes obtained from phenobarbital-treated rats in a dose-dependent manner, whereas it did not inhibit ethoxyresorufin O-deethylase activity of microsomes obtained from 3-methylcholanthrene-treated rats. These results suggest that the selected amino acid sequence, which coincides with one of the substrate binding sites of Pseudomonas putida CYP101A (P450cam) and one of the putative substrate binding sites of CYP2B2, is located on the surface of the CYP2B1 molecule, as opposed to inside the molecule or in the lipid bilayer of microsomes.
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D000918:Antibody Specificity; D000941:Antigens / Q000737:chemistry / Q000276:immunology*; D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D015153:Blotting, Western; D019362:Cytochrome P-450 CYP2B1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000276:immunology
D000906:Antibodies; D000941:Antigens; D065607:Cytochrome P-450 Enzyme Inhibitors; D007074:Immunoglobulin G; D010455:Peptides; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C025619:steroid 16-beta-hydroxylase