Abstract
The cDNA of cytochrome P450 (CYP) 2C43 was cloned from cynomolgus monkey liver by RT-PCR. The deduced amino acid sequence showed 93% and 91% identity to human CYP2C9 and CYP2C19, respectively. The cDNA was expressed in Escherichia coli and purified by a series of chromatography steps, yielding a specific content of 11.5 nmol P450/mg protein. The substrate specificity of the purified CYP2C43 was examined in a reconstitution system comprising NADPH-P450 reductase, lipid, cytochrome b(5) and CYP2C marker substrates. The purified CYP2C43 showed high activity for testosterone 17-oxidation and progesterone 21-hydroxylation, which were also observed for CYP2C19 but not CYP2C9. In addition, CYP2C43 showed activity for (S)-mephenytoin 4'-hydroxylation, a marker reaction for CYP2C19. With CYP2C9 marker substrates, CYP2C43 exhibited low activity for diclofenac 4'-hydroxylation and no activity for tolbutamide p-methylhydroxylation. Therefore, in terms of substrate specificity, our results indicate that CYP2C43 is similar to CYP2C19, rather than CYP2C9.
MeSH terms
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Animals
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Aryl Hydrocarbon Hydroxylases / isolation & purification
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Aryl Hydrocarbon Hydroxylases / metabolism
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Cloning, Molecular
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Cricetinae
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Cytochrome P-450 CYP2C19
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Cytochrome P-450 CYP2C9
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Cytochrome P-450 Enzyme System / biosynthesis
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Cytochrome P-450 Enzyme System / isolation & purification*
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Cytochrome P-450 Enzyme System / metabolism*
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Diclofenac / pharmacokinetics
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Guinea Pigs
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Humans
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Kinetics
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Macaca fascicularis
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Mephenytoin / pharmacokinetics
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Mice
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Microsomes, Liver / enzymology*
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Mixed Function Oxygenases / isolation & purification
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Mixed Function Oxygenases / metabolism
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Progesterone / metabolism
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Rabbits
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Rats
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Substrate Specificity / genetics
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Testosterone / metabolism
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Tolbutamide / pharmacokinetics
Substances
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Recombinant Proteins
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cytochrome P-450 CYP2C subfamily
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Diclofenac
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Testosterone
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Progesterone
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Cytochrome P-450 Enzyme System
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Tolbutamide
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Mixed Function Oxygenases
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CYP2C9 protein, human
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Cytochrome P-450 CYP2C9
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Aryl Hydrocarbon Hydroxylases
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CYP2C19 protein, human
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Cytochrome P-450 CYP2C19
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Mephenytoin