Expression of a Human Liver Cytochrome P-450 Protein with Tolbutamide Hydroxylase Activity in Saccharomyces cerevisiae

William R. Brian, Pramod K. Srivastava, Diane R. Umbenhauer, R. Stephen Lloyd, F. Peter Guengerich

Research output: Contribution to journalArticle

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Abstract

The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this “P-450MP” family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 μM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P-450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage λgt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (~48 000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 × 104 molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity. Thus, the hydroxylation of mephenytoin and tolbutamide is catalyzed by similar but distinct P-450s. P-450MP-1 and P-450MP-2 contain mephenytoin hydroxylase activity. The MP-8 cDNA clone encodes a protein with tolbutamide hydroxylase activity, now termed P-450TB.

Original languageEnglish (US)
Pages (from-to)4993-4999
Number of pages7
JournalBiochemistry
Volume28
Issue number12
DOIs
StatePublished - Jun 1 1989

ASJC Scopus subject areas

  • Biochemistry

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