Purification and characterization of a dog cytochrome P450 isozyme belonging to the CYP2D subfamily and development of its antipeptide antibody.
Species differences in the metabolism of bunitrolol (BTL) and propranolol (PL) in liver microsomes from rats and dogs were investigated. Hepatic microsomes from dogs lacked the ability to catalyze PL 7-hydroxylation, which is mediated by the CYP2D subfamily in rats. This suggested that dogs might lack the CYP2D subfamily; however, the antibody against cytochrome P450 (P450) BTL (CYP2D2) recognized a protein of approximately 49 kDa in hepatic microsomes from dogs, indicative of the presence of the CYP2D subfamily in dogs. The P450 purified from dog hepatic microsomes was designated P450 Canis familiaris (CF)1. It cross-reacted with the antibody against P450 BTL. The apparent molecular weight of the purified P450 CF1 was estimated to be 49 kDa. Its N-terminal amino acid sequence resembled the sequences of the members of the rat CYP2D subfamily and was the same as the sequences of the dog CYP2D subfamily, as deduced from the cDNA, except for the lack of four residues at the N-terminal. P450 CF1 could mediate metabolism of BTL and PL. P450 CF1, however, could not mediate PL 7-hydroxylation, which is almost exclusively mediated by CYP2D in rats. These findings indicate that P450 CF1 belongs to the CYP2D subfamily and that it differs functionally from the rat CYP2D subfamily. An antipeptide antibody against the synthetic peptide (DPTQPPRH), the sequence of which occurs in dog CYP2D at position 266-273 (S. Kirita et al., unpublished data), inhibited BTL 4-hydroxylase activity by 71% in dog hepatic microsomes at the substrate concentration of 0.01 M. This is further evidence that the CYP2D subfamily, in particular P450 CF1, is largely responsible for the oxidation of beta-blockers in dog hepatic microsomes.
Nakamura
A
A
Department of Toxicology, Faculty of Veterinary Medicine, Hokkaido University, Japan.
Yamamoto
Y
Y
Tasaki
T
T
Sugimoto
C
C
Masuda
M
M
Kazusaka
A
A
Fujita
S
S
D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004285:Dogs; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D011803:Quinine / Q000494:pharmacology; D051381:Rats; D013045:Species Specificity
D000319:Adrenergic beta-Antagonists; D000939:Epitopes; D007527:Isoenzymes; D010455:Peptides; D003577:Cytochrome P-450 Enzyme System; D011803:Quinine; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6