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.
Highlights
From the Departments of $Medicine, llBiochemistry, and )ICell Biology, Vanderbilt University Medical School, Nashville, Tennessee 37232 and the §Department of Molecular Genetics, SouthwesternMedical Center, Dallas, Teras75235
Sequenceanalysisshowed thatwith the date, arachidonic acid epoxygenase activity has been demonexception of[11] nucleotides,this cDNA is identicalwith strated in numerous tissues, including kidney, liver, brain, the published sequence for ratliver cytochrome 2C23 pituitary,adrenal,and endothelium
Thedemonstration of a role for P-450 in the in uiuo acidblothybridizationindicatedthatthe levels of expression of the correspondinmgRNA are high in rat kidney and liver and are undetectable in brain and heart
Summary
E with RD3 (67 and 61% for nucleotide and amino acid sequence, respectively). the differences in nucleotide sequence between RD3 and other members of the 2C gene subfamily were randomly distributed along the entire length of the cDNA, indicating that thegene for P-450 2C23 or RD3. Microsomal fractions isolated from cells transfected with the sense RD3 plasmid metabolized the fatty acid to products with chromatographic properties similar to those of synthetic EETs and DHETs. as shown, the catalysis of EET and DHET formation was incubation time-dependent. Studies utilizing purified hemoproteins and/or this is the first time that the P-450 2C23 gene product has microsomal fractions, isolated from animals treated with sebeen expressed and a catalytic activity for the recombinant lected P-450 inducers, demonstrated thecoexistence in liver protein determined. Radiomatic Flow-one 6 Detector (Radiomatic InstrumentCo.).Shown are the radiochromatograms derived from a total of 1 mg of microsomal protein isolated from control, nontransfected cells (bottom) or cells transfected with the epoxygenase cDNA (top).The retention times for 11,12-DHET ( A ) , 5,6-DHET ( B ) ,1 4 , S E E T( C ) ,11,lZ-EET (D)a,nd.
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