Tolbutamide and phenytoin hydroxylations by cDNA-expressed human liver cytochrome P4502C9

Biochem Biophys Res Commun. 1991 Mar 29;175(3):1112-8. doi: 10.1016/0006-291x(91)91680-b.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Line
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism*
  • DNA / genetics
  • DNA / isolation & purification
  • Gene Library
  • Humans
  • Hydroxylation
  • Kinetics
  • Liver / enzymology*
  • Microsomes, Liver / enzymology
  • Molecular Sequence Data
  • Oligonucleotide Probes
  • Phenytoin / metabolism*
  • Phenytoin / pharmacology
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Sulfaphenazole / pharmacology
  • Tolbutamide / metabolism*
  • Transfection


  • Oligonucleotide Probes
  • Recombinant Proteins
  • Sulfaphenazole
  • Phenytoin
  • DNA
  • Cytochrome P-450 Enzyme System
  • Tolbutamide