Tolbutamide and phenytoin hydroxylations by cDNA-expressed human liver cytochrome P4502C9.
A human cytochrome P4502C9 cDNA clone has been isolated from a human liver bacteriophage Lambda gt11 library using oligonucleotide probes. Expression of the 1762 base pair cDNA in COS cells demonstrated that the encoded enzyme has a molecular mass of 55 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The expressed enzyme catalysed the methylhydroxylation of tolbutamide with an apparent Km of 131.7 microM, similar to that observed in human liver microsomes. P4502C9 also catalysed the 4-hydroylation of phenytoin, and inhibition experiments demonstrated that phenytoin was a competitive inhibitor of tolbutamide hydroxylation with an apparent Ki of 19.1 microM. Sulphaphenazole was a potent inhibitor of the expressed enzyme with respect to both tolbutamide and phenytoin hydroxylations. These data demonstrate that a single isozyme can catalyse the hydroxylations of both tolbutamide and phenytoin, and suggest that both reactions are mediated by the same isozyme(s) of cytochrome P450 in human liver.
Veronese
M E
ME
Department of Clinical Pharmacology, School of Medicine, Flinders University of South Australia, Adelaide.
Mackenzie
P I
PI
Doecke
C J
CJ
McManus
M E
ME
Miners
J O
JO
Birkett
D J
DJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010672:Phenytoin / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013426:Sulfaphenazole / Q000494:pharmacology; D014044:Tolbutamide / Q000378:metabolism*; D014162:Transfection
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D013426:Sulfaphenazole; D010672:Phenytoin; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide