Expression and characterization of dog CYP2D15 using baculovirus expression system.
Dog CYP2D15 was expressed in Sf9 cells with a recombinant baculovirus. Infection of Sf9 insect cells with a recombinant dog CYP2D15-virus resulted in the expression of a protein which cross-reacted with a polyclonal antibody against a dog CYP2D15-specific peptide. The difference spectrum of CO-complex of reduced P450 of the infected cell microsomes had a maximal absorbance at 449 nm. The specific content of P450 was calculated to be 0.56 nmol/mg of Sf9 cell microsomal protein. Although the expressed dog CYP2D15 showed high catalytic activity for the hydroxylations of bunitrolol and imipramine at low substrate concentration (10 microM), the catalytic activity for that of debrisoquine (50 microM) was extremely low as compared with that of CYP2D from other species. Dog liver microsomes also showed bunitrolol and imipramine hydroxylase activities, but not debrisoquine hydroxylase activity at the same substrate concentrations. In addition, the expressed CYP2D showed high catalytic activity for imipramine N-demethylation. Thus, our study reveals that the expressed dog CYP2D15 engages in high catalytic activity and has a unique substrate specificity from other CYP2D subfamilies. Western blot analysis suggested that the dog CYP2D15 contents were less than 4% of the total liver P450 content, assuming that 100% of expressed CYP2D15 incorporated heme.
Tasaki
T
T
Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo.
Nakamura
A
A
Itoh
S
S
Ohashi
K
K
Yamamoto
Y
Y
Masuda
M
M
Iwata
H
H
Kazusaka
A
A
Kamataki
T
T
Fujita
S
S
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D003647:Debrisoquin / Q000378:metabolism; D004285:Dogs; D004791:Enzyme Inhibitors / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D004364:Pharmaceutical Preparations / Q000378:metabolism; D011803:Quinine / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D011803:Quinine; D001189:Aryl Hydrocarbon Hydroxylases; C110942:cytochrome P-450 CYP2D15 (dog); D003647:Debrisoquin