Fatty acid metabolism by cytochrome P450 4A3 expressed in baculovirus-insect cell system

20-Hydroxyeicosatetraenoic acid (20-HETE), a potent modulator of the renal vascular and tubular functions, is the major cytochrome P450 (CYP)-arachidonic acid (AA) metabolite in the kidney. Its synthesis is believed to be catalyzed by enzymes of the CYP4A family, which, in the rat, comprises four major isoforms: 4A1, 4A2, 4A3 and 4A8. Due to a high sequence homology associated with these enzymes, the baculovirusSf9 insect cell expression system has been successfully used to characterize each CYP4A isoform. In this study, CYP4A3 was expressed and characterized with regard to its 20-HETE synthesis. A high level of expression of CYP4A3 was evident by Western and spectral analyses revealing a P450 content of 0.24 nmol/mg microsomal protein. The membrane fraction of expressed CYP4A3 was used to investigate the catalytic activities of lauric, linoleic and arachidonic acids. All three fatty acid oxidations catalyzed by CYP4A3 were surprisingly higher than that catalyzed by CYP4A2, the most abundant CYP4A isozyme in the kidney of male rats, which displays 97% sequence homology with CYP4A3. The