Mephenytoin-type polymorphism of drug oxidation: purification and characterization of a human liver cytochrome P-450 isozyme catalyzing microsomal mephenytoin hydroxylation

Josef Gut, U. Thomas Meier, Therese Catin, Urs A. Meyer

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

A genetic polymorphism causing deficient metabolism of the anticonvulsant drug mephenytoin occurs in 5% of the Caucasian and 23% of the Japanese population. By monitoring the activities of the two major oxidative pathways of mephynotoin metabolism in the column eluates, we have purified from human livers a cytochrome P-450 isozyme, P-450 meph, which exclusively and stereoselectively catalyzes the 4-hydroxylation of (S)-mephenytoin, the major pathway affected by the polymorphism, whereas P-450 meph was virtually devoid of catalytic activity for N-demethylation of mephenytoin, the pathway remaining unaffected by the genetic deficiency. P-450 meph has an apparent Mr of 55 000 and a λmax in the reduced CO-binding spectrum of 450 nm. Polyclonal rabbit antibodies against purified human P-450 mpeh almost completely inhibited the 4-hydroxylation of mephenytoin but has little effect on N-demethylation in human liver microsomes. In microsomes of liver biopsies of two subjects characterized in vivo as 'poor metabolizers' of mephenytoin. immunocrossreactive and immunoinhibitable material was observed with similar or identical properties to those of P-450 meph. There was no difference in the extent of the immunochemical reaction between microsome of in vivo phenotypes poor metabolizers and extensive metabolizers of mephenytoin. These data suggest tat P-450 meph is the target of the genetic deficiency and support the concept that a functionally altered variant form of P-450 meph causes thus polymorphism.

Original languageEnglish (US)
Pages (from-to)435-447
Number of pages13
JournalBBA - General Subjects
Volume884
Issue number3
DOIs
StatePublished - Dec 10 1986
Externally publishedYes

Fingerprint

Mephenytoin
Hydroxylation
Polymorphism
Liver
Cytochrome P-450 Enzyme System
Isoenzymes
Purification
Oxidation
Pharmaceutical Preparations
Liver Microsomes
Metabolism
Biopsy
Genetic Polymorphisms
Carbon Monoxide
Microsomes
Anticonvulsants
Catalyst activity
Rabbits
Phenotype
Antibodies

Keywords

  • (Human liver)
  • Cytochrome P-450
  • Drug oxidation
  • Mephenytoin
  • Microsomal hydroxilation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Mephenytoin-type polymorphism of drug oxidation : purification and characterization of a human liver cytochrome P-450 isozyme catalyzing microsomal mephenytoin hydroxylation. / Gut, Josef; Meier, U. Thomas; Catin, Therese; Meyer, Urs A.

In: BBA - General Subjects, Vol. 884, No. 3, 10.12.1986, p. 435-447.

Research output: Contribution to journalArticle

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abstract = "A genetic polymorphism causing deficient metabolism of the anticonvulsant drug mephenytoin occurs in 5{\%} of the Caucasian and 23{\%} of the Japanese population. By monitoring the activities of the two major oxidative pathways of mephynotoin metabolism in the column eluates, we have purified from human livers a cytochrome P-450 isozyme, P-450 meph, which exclusively and stereoselectively catalyzes the 4-hydroxylation of (S)-mephenytoin, the major pathway affected by the polymorphism, whereas P-450 meph was virtually devoid of catalytic activity for N-demethylation of mephenytoin, the pathway remaining unaffected by the genetic deficiency. P-450 meph has an apparent Mr of 55 000 and a λmax in the reduced CO-binding spectrum of 450 nm. Polyclonal rabbit antibodies against purified human P-450 mpeh almost completely inhibited the 4-hydroxylation of mephenytoin but has little effect on N-demethylation in human liver microsomes. In microsomes of liver biopsies of two subjects characterized in vivo as 'poor metabolizers' of mephenytoin. immunocrossreactive and immunoinhibitable material was observed with similar or identical properties to those of P-450 meph. There was no difference in the extent of the immunochemical reaction between microsome of in vivo phenotypes poor metabolizers and extensive metabolizers of mephenytoin. These data suggest tat P-450 meph is the target of the genetic deficiency and support the concept that a functionally altered variant form of P-450 meph causes thus polymorphism.",
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N2 - A genetic polymorphism causing deficient metabolism of the anticonvulsant drug mephenytoin occurs in 5% of the Caucasian and 23% of the Japanese population. By monitoring the activities of the two major oxidative pathways of mephynotoin metabolism in the column eluates, we have purified from human livers a cytochrome P-450 isozyme, P-450 meph, which exclusively and stereoselectively catalyzes the 4-hydroxylation of (S)-mephenytoin, the major pathway affected by the polymorphism, whereas P-450 meph was virtually devoid of catalytic activity for N-demethylation of mephenytoin, the pathway remaining unaffected by the genetic deficiency. P-450 meph has an apparent Mr of 55 000 and a λmax in the reduced CO-binding spectrum of 450 nm. Polyclonal rabbit antibodies against purified human P-450 mpeh almost completely inhibited the 4-hydroxylation of mephenytoin but has little effect on N-demethylation in human liver microsomes. In microsomes of liver biopsies of two subjects characterized in vivo as 'poor metabolizers' of mephenytoin. immunocrossreactive and immunoinhibitable material was observed with similar or identical properties to those of P-450 meph. There was no difference in the extent of the immunochemical reaction between microsome of in vivo phenotypes poor metabolizers and extensive metabolizers of mephenytoin. These data suggest tat P-450 meph is the target of the genetic deficiency and support the concept that a functionally altered variant form of P-450 meph causes thus polymorphism.

AB - A genetic polymorphism causing deficient metabolism of the anticonvulsant drug mephenytoin occurs in 5% of the Caucasian and 23% of the Japanese population. By monitoring the activities of the two major oxidative pathways of mephynotoin metabolism in the column eluates, we have purified from human livers a cytochrome P-450 isozyme, P-450 meph, which exclusively and stereoselectively catalyzes the 4-hydroxylation of (S)-mephenytoin, the major pathway affected by the polymorphism, whereas P-450 meph was virtually devoid of catalytic activity for N-demethylation of mephenytoin, the pathway remaining unaffected by the genetic deficiency. P-450 meph has an apparent Mr of 55 000 and a λmax in the reduced CO-binding spectrum of 450 nm. Polyclonal rabbit antibodies against purified human P-450 mpeh almost completely inhibited the 4-hydroxylation of mephenytoin but has little effect on N-demethylation in human liver microsomes. In microsomes of liver biopsies of two subjects characterized in vivo as 'poor metabolizers' of mephenytoin. immunocrossreactive and immunoinhibitable material was observed with similar or identical properties to those of P-450 meph. There was no difference in the extent of the immunochemical reaction between microsome of in vivo phenotypes poor metabolizers and extensive metabolizers of mephenytoin. These data suggest tat P-450 meph is the target of the genetic deficiency and support the concept that a functionally altered variant form of P-450 meph causes thus polymorphism.

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