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

X. N. Nguyen, Mong-Heng Wang, M. L. Schwartzman

Research output: Contribution to journalArticlepeer-review

1 Scopus citations


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 <o-hydroxylation of AA (50 uM) to 20-HETE by CYP4A3 was higher than that of CYP4A2 (0.53 and 0.36 nmol/min/nmol P450, respectively). The AA w-hydroxylation activity of the expressed CYP4A3 was dependent on the concentrations of NADPH-cytochrome P450 oxidoreductase and cytochrome bs, with maximal activity being achieved at a 14-fold excess of reductase and a 4-fold excess of b,. The results show that the baculovirus-insect cell system provides high levels of P450 expression necessary for catalytic and metabolic studies of the rat CYP4A isoforms. Our data suggest that in addition to CYP4A2, CYP4A3, which is highly expressed in rat proximal tubules, may contribute to the generation of endogenous 20-HETE. Most importantly, CYP4A3 may be an essential source of 20HETE synthesis in female rats, where expression of CYP4A2 is barely detectable.

Original languageEnglish (US)
JournalFASEB Journal
Issue number9
StatePublished - Dec 1 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


Dive into the research topics of 'Fatty acid metabolism by cytochrome P450 4A3 expressed in baculovirus-insect cell system'. Together they form a unique fingerprint.

Cite this