J. Biochem, 1998, Vol. 123, No. 1 162-168
© 1998 Japanese Biochemical Society
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Expression and Characterization of Dog CYP2D15 Using Baculovirus Expression System1
* Laboratory of Toxicology, Graduate School of Veterinary Medicine, Hokkaido University N18, W9, Kita-ku, Sapporo 060
Laboratory of Infectious Diseases, Graduate School of Veterinary Medicine, Hokkaido University N18, W9, Kita-ku, Sapporo 060
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University Kita-ku, Sapporo 060
4To whom correspondence should be addressed. Fax: +81-11-706-5105, E-mail: fujita{at}vetmed.hokudai.ac.jp
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 µM), the catalytic activity for that of debrisoquine (50 µM) 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.
1The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases with the accession number AB004268. The nomenclature number CYP2D15 was provided by Nelson et al. (1). This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan to S.F. (No. 07558236), A.K. (No. 08456155), and H.I. (No. 09306021).
2The data in this paper are taken from a thesis to be submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy at the Graduate School of Veterinary Medicine, Hokkaido University.
3Present address: Research Laboratories, Nippon Shinyaku Co., Ltd., Nishioji-Hachijo, Minami-ku, Kyoto 601.
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