Molecular cloning, expression, and enzymatic characterization of the rat kidney cytochrome P-450 arachidonic acid epoxygenase

Armando Karara, Keiko Makita, Harry R. Jacobson, John R. Falck, F. Peter Guengerich, Raymond N. DuBois, Jorge H. Capdevila

Research output: Contribution to journalArticle

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Abstract

A cDNA containing an open reading frame coding for the rat kidney cytochrome P-450 arachidonic acid epoxygenase was isolated from a male rat kidney cDNA library. Sequence analysis showed that with the exception of 11 nucleotides, this cDNA is identical with the published sequence for rat liver cytochrome 2C23 and encodes a polypeptide of 494 amino acids. Nucleic acid blot hybridization indicated that the levels of expression of the corresponding mRNA are high in rat kidney and liver and are undetectable in brain and heart. The cDNA coding region was cloned into a pCMV2 vector and expressed in COS-1 cells. The recombinant microsomal protein catalyzed the NADPH-dependent metabolism of arachidonic acid to a mixture of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids as the only oxygenation products. The enantiofacial selectivity of the recombinant protein was nearly identical with that reported for the kidney microsomal enzyme and generated 8(R),9(S)- , 11(R),12(S)-, and 14(S),15(R) with optical purities of 95, 85, and 75%, respectively. On the basis of mRNA abundance and the close similarities between the regio- and stereochemical selectivity of the recombinant and kidney microsomal proteins, we concluded that cytochrome P-450 2C23 is the predominant enzyme isoform responsible for arachidonic acid epoxidation in the rat kidney.

Original languageEnglish (US)
Pages (from-to)13565-13570
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number18
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Karara, A., Makita, K., Jacobson, H. R., Falck, J. R., Guengerich, F. P., DuBois, R. N., & Capdevila, J. H. (1993). Molecular cloning, expression, and enzymatic characterization of the rat kidney cytochrome P-450 arachidonic acid epoxygenase. Journal of Biological Chemistry, 268(18), 13565-13570.