A NEW CYTOCHROME-P450 FORM BELONGING TO THE CYP2D IN DOG LIVER-MICROSOMES - PURIFICATION, CDNA CLONING, AND ENZYME CHARACTERIZATION

A new form of cytochrome P450 (P450 DUT2) was purified from untreated male dog liver microsomes. The final preparation (a specific content o f 19.1 nmol P450/mg protein) showed a single band with an apparent mon omeric molecular weight of 50,000 on sodium dodecyl sulfate-polyacryla mide gel electrophoresis, but was further separated into two apoprotei ns (P450 DUT2(a) and P450 DUT2(b)) by reverse-phase HPLC. Both protein s had identical NH2-terminal amino acid sequences, but the first three amino acids of P450 DUT2(b) were truncated in P450 DUT2(a). Purified P450 DUT2 showed 5 to 18 times higher catalytic activities than did he patic microsomes toward desipramine, metoprolol, and dextromethorphan. These activities in dog liver microsomes were strongly inhibited by a nti-P450 DUT2-IgG. A 1.7-kilobase pair cDNA (cDUT2) encoding a male do g liver P450 of 500 amino acid residues (molecular weight 56,400) was isolated and sequenced. The first 35 NH2-terminal amino acid sequence of P450 DUT2(b) coincided with the deduced amino acid sequence of cDUT 2 at 2-36. The deduced total amino acid sequence of cDUT2 shared high similarity with the reported 2D forms (with 2D6, 74.6%; 2D14, 75.4%; 2 D1, 65.4%; and 2D9, 63.6%). Moreover, the expressed P450 DUTS in COS-7 cells had catalytic activities similar to those of purified P450 DUT2 . Therefore, this paper is the first report about dog CYP2D. Furthermo re, Northern and Western blot analyses indicated that the expressed le vels of mRNA and protein were almost equal between male and female dog s. Western blot analysis suggested that P450 DUT2 is a constitutive an d major (approximately 20% of the total P450) form, indicating that th e 2D subfamily P450 in dog Liver is quite unique from CYP2D members of other species. (C) 1995 Academic Press, Inc.