Antipeptide antibodies targeted against specific regions of rat CYP2D1 and human CYP2D6

Four peptides (pep23-33, pep26-38, pep283-297, and pep409-419) corresponding to unique sequences in rat cytochrome P450 (CYP) 2D1 and/or human CYP2D6 were selected for production of antipeptide antibodies. Rat liver microsomal protein was recognized by anti-serum to all four peptides in ELISAs; however, antisera against pep23-33 and pep26-36 proved not usable for any other applications. Western blots of microsomal protein from a cell line specifically expressing human CYP2D6 revealed that antisera to pep283-297 and pep409-419 recognized a single protein band of the same molecular size as CYP2D6. Antisera to pep283-297 and pep409-419 recognized a rat microsomal protein presumed to be CYP2D1, because it comigrated with human CYP2D6 and had an apparent molecular size of 52 kDa. An unknown protein of approximately 85 kDa was also recognized by pep409-419. Recognition of microsomal protein(s) by antisera to pep283-297 and pep409-419 was blocked by pep283-297 or a bovine serum albumin-pep409-419 conjugate, respectively. Antiserum to pep283-297 was used to analyze sex and strain differences in liver microsomes prepared from Sprague-Dawley, Fischer 344, and Dark Agouti male and female rats. Sprague-Dawley and Fischer 344 rats expressed similar amounts of CYP2D1, but expression in Dark Agouti rats was significantly lower. The antiserum did not detect a sexual dimorphism in any of the strains. A significant correlation between antipeptide283-297 immunoreactivity and Vmax for dextromethorphan O-demethylation existed in female rat strains; however, this relationship did not exist in male rat strains.(ABSTRACT TRUNCATED AT 250 WORDS)