Isolation and characterization of human liver cytochrome P450 2C19: correlation between 2C19 and S-mephenytoin 4'-hydroxylation.
In an effort to identify and characterize minor forms of human liver cytochrome P450, immunoblot analyses of microsome samples were developed with antibodies to various P450s that recognized multiple human P450s. Four P450s were recognized in immunoblot analyses of human liver microsome samples developed with an antibody previously demonstrated to specifically recognize rat 2B1/2. Three of these P450s were identified as 2A6, 2C9/10, and 2E1 and the fourth was termed P450UK. A monoclonal antibody to 2C9/10 recognized P450UK in addition to 2C9/10. In order to identify P450UK, it was purified and subjected to amino-terminal amino acid analysis. The amino-terminal sequence obtained for P450UK was identical to the sequence deduced from a cDNA encoding CYP2C19, thus identifying P450UK as 2C19. The relative levels of 2C19 were determined in 14 human liver microsome samples by quantitative immunoblot analyses developed with the anti-2C9/10 antibody. These analyses demonstrated that 2C19 was not detected in one sample and its levels varied 10.5-fold in the remaining samples. The levels of 2C19 were compared to the relative levels and catalytic activities of multiple human liver P450s. The levels of 2C19 and the ability of the samples to 4'-hydroxylate S-mephenytoin were found to strongly correlate (r2 = 0.79). In summary, this is the first demonstration of the expression of 2C19 at the enzyme level, and the correlation studies suggest that 2C19 plays a role in the 4'-hydroxylation of S-mephenytoin.
Wrighton
S A
SA
Department of Drug Metabolism and Disposition, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285.
Stevens
J C
JC
Becker
G W
GW
VandenBranden
M
M
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid
D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19