ANTIPEPTIDE ANTIBODIES TARGETED AGAINST SPECIFIC REGIONS OF RAT CYP2D1 AND HUMAN CYP2D6

Four peptides (pep(23-33), pep(26-36), pep(283-297) and pep(409-419)) corresponding to unique sequences in rat cytochrome P450 (CYP) 2D1 and /or human CYP2D6 were selected for production of antipeptide antibodie s. Rat liver microsomal protein was recognized by antiserum to all fou r peptides in ELISAs; however, antisera against pep(23-33) and pep(26- 36) proved not usable for any other applications. Western blots of mic rosomal protein from a cell line specifically expressing human CYP2D6 revealed that antisera to pep(283-297) and pep(409-419) recognized a s ingle protein band of the same molecular size as CYP2D6. Antisera to p ep(283-297) and pep(409-419) recognized a rat microsomal protein presu med to be CYP2D1, because it comigrated with human CYP2D6 and had an a pparent molecular size of 52 kDa. An unknown protein of similar to 85 kDa was also recognized by pep(409-419). Recognition of microsomal pro tein(s) by antisera to pep(283-297) and pep(409-419) was blocked by pe p(283-297) or a bovine serum albumin-pep(409-419) conjugate, respectiv ely. Antiserum to pep(283-297) was used to analyze sex and strain diff erences 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 Agout i rats was significantly lower. The antiserum did not detect a sexual dimorphism in any of the strains. A significant correlation between an tipeptide(283-297) immunoreactivity and V-max for dextromethorphan O-d emethylation existed in female rat strains; however, this relationship did not exist in male rat strains. These data suggest antisera agains t pep(283-297) and pep(409-419) are useful in studying expression of r at CYP2D1 and human CYP2D6.