EXPRESSION AND CHARACTERIZATION OF CANINE CYTOCHROME-P450 2D15

CYP2D15 is the canine ortholog of human CYP2D6, the human CYP2D isofor m involved in the metabolism of drugs such as antiarhythmics, adrenoce ptor antagonists, and tricyclic antidepressants. Similar to human, can ine CYP2D15 is expressed in the liver, with detectable levels in sever al other tissues. Three different CYP2D15 cDNA clones were obtained by RT-PCR from dog liver RNA. Two clones corresponded to variant full-le ngth CYP2D15 cDNAs (termed CYP2D15 WT2 and CYP2D15 V1); the third was identified as a splicing variant missing exon 3 (termed CYP2D15 V2). R ecombinant baculoviruses were constructed containing full-length cDNAs and used to express CYP2D15 WT2 and CYP2D15 V1 in Spodoptera frugiper da (Sf9) cells with expression levels of up to 0.14 nmol/mg cell prote in. As with human CYP2D6, the recombinant CYP2D15 enzymes exhibited bu furalol 1'-hydroxylase and dextromethorphan O-demethylase activities w hen coexpressed with rabbit NADPH:P450 oxidoreductase, For bufuralol 1 '-hydroxylase, apparent K-m values were 4.9, 3.7, and 2.5 mu M and the V-max values were 0.14, 0.034, and 0.60 nmol/min/mg protein for dog l iver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively . For dextromethorphan O-demethylase, apparent K-m values were 0.6, 0. 6, and 2.0 mu M and the V-max values were 0.18, 0.034, and 0.057 nmol/ min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. The human CYP2D6-specific inhibitor quinidin e and the rat CYP2D1-specific inhibitor quinine were both shown to be inhibitors of bufuralol 1'-hydroxylase activity for dog liver microsom es, CYP2D15 WT2, and the CYP2D15 V1 variant with nearly equal potency. Thus, the dog expresses a CYP2D ortholog possessing enzymatic activit ies similar to human CYP2D6, but is affected by the inhibitors quinine and quinidine in a manner closer to that of rat CYP2D1. (C) 1998 Acad emic Press.