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 immuni zing rabbits with a synthetic peptide antigen. The anti-CYP2B1 IgG obt ained did not cross-react with CYP2B2, which has 97% identity in prima ry 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-dea lkylase activity of microsomes obtained from phenobarbital-treated rat s in a dose-dependent manner, whereas it did not inhibit ethoxyresoruf in O-deethylase activity of microsomes obtained from 3-methylcholanthr ene-treated rats. These results suggest that the selected amino acid s equence, which coincides with one of the substrate binding sites of Ps eudomonas putida CYP101A (P450cam) and one of the putative substrate b inding sites of CYP2B2, is located on the surface of the CYP2B1 molecu le, as opposed to inside the molecule or in the lipid bilayer of micro somes. (C) 1995 Academic Press, Inc.