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Cloning and characterization of three new mouse P450 enzymes
Author(s) -
Graves Joan P.,
Wang Hong,
Bradbury J. Alyce,
Zeldin Darryl C.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a458-b
Subject(s) - biology , recombinant dna , gene , microbiology and biotechnology , cloning (programming) , complementary dna , open reading frame , molecular cloning , pseudogene , sf9 , polyclonal antibodies , biochemistry , antibody , genetics , peptide sequence , genome , spodoptera , computer science , programming language
We report the physical map of the murine CYP2J subfamily which contains 8 genes and 2 pseudogenes, located on a 0.58 Mb region of chromosome 4. Three novel Cyp2j genes were identified and designated Cyp2j11 , Cyp2j12 and Cyp2j13 . The cDNAs were cloned by PCR from reverse‐transcribed mouse kidney or brain RNA. The cDNAs contain open reading frames that encode polypeptides which have conserved regions present in all P450s. RT‐PCR analysis showed that the CYP2J11 transcript is most abundant in the kidney, the CYP2J12 transcript is most abundant in the brain, and the CYP2J13 transcript is present in multiple tissues including kidney. Northern analysis with full‐length cDNAs confirm the results for CYP2J11 and CYP2J13. The recombinant CYP2J proteins were expressed in Sf9 or Sf21 insect cells using the baculovirus system. Spectral analysis of microsomal fractions of infected insect cells show typical CO‐difference spectra for all three P450s with Soret maxima at ~450 nm. Each of the three recombinant proteins metabolized arachidonic acid (AA) to epoxyeicosatrienoic and hydroxyeicosatrienoic acids, albeit with different rates and profiles. Polyclonal antibodies were produced to specific CYP2J peptides in order to examine the expression of these new P450s at the protein level. Immunoblotting studies confirmed that these antibodies reacted with their respective CYP2J recombinant proteins. We propose that these three new CYP2J enzymes may be involved in the metabolism of AA in mouse extrahepatic tissues. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.