MOLECULAR-CLONING, EXPRESSION, AND ENZYMATIC CHARACTERIZATION OF THE RAT-KIDNEY CYTOCHROME-P-450 ARACHIDONIC-ACID EPOXYGENASE

A cDNA containing an open reading frame coding for the rat kidney cyto chrome 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 ex pression of the corresponding mRNA are high in rat kidney and liver an d are undetectable in brain and heart. The cDNA coding region was clon ed into a pCMV2 vector and expressed in COS-1 cells. The recombinant m icrosomal protein catalyzed the NADPH-dependent metabolism of arachido nic acid to a mixture of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrie noic acids as the only oxygenation products. The enantiofacial selecti vity of the recombinant protein was nearly identical with that reporte d 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%, respect ively. On the basis of mRNA abundance and the close similarities betwe en the regio- and stereochemical selectivity of the recombinant and ki dney microsomal proteins, we concluded that cytochrome P-450 2C23 is t he predominant enzyme isoform responsible for arachidonic acid epoxida tion in the rat kidney.