Molecular cloning and characterization of mouse CYP2J6, an unstable cytochrome P450 isoform.
A cDNA encoding a new cytochrome P450 was cloned from a mouse liver library. Sequence analysis revealed that this 2046-bp cDNA encodes a 501-amino acid polypeptide that is 72-94% identical to other CYP2J subfamily P450s and is designated CYP2J6. Northern analysis demonstrated that CYP2J6 transcripts are abundant in the small intestine and present at lower levels in other mouse tissues. In situ hybridization revealed that CYP2J6 mRNAs are present in luminal epithelial cells of the gastrointestinal mucosa. The CYP2J6 cDNA was expressed in Sf9 cells using baculovirus. The heterologously expressed CYP2J6 protein displayed a typical P450 CO-difference spectrum; however, the protein was unstable as evidenced by the loss of the Soret maxima at 450nm and the appearance of a 420nm peak when CYP2J6-expressing cells were disrupted by mechanical homogenization, sonication, or freeze-thaw. Immunoblotting of mouse microsomes with the anti-human CYP2J2 IgG, which cross-reacts with rodent CYP2Js, demonstrated the presence of multiple distinct murine CYP2J immunoreactive proteins in various tissues. Immunoblotting with an antibody to a CYP2J6-specific peptide detected a prominent 55-57kDa protein in Sf9 cell extracts expressing recombinant CYP2J6 but did not detect a protein of similar molecular mass in mouse small intestinal microsomes. Mixing experiments demonstrated that recombinant CYP2J6 is degraded rapidly in the presence of small intestinal microsomes consistent with proteolysis at highly sensitive sites. Sf9 cells, which express both CYP2J6 and NADPH-P450 oxidoreductase, metabolized benzphetamine but not arachidonic acid. We conclude that CYP2J6 is an unstable P450 that is active in the metabolism of benzphetamine, but not arachidonic acid.
Ma
Jixiang
J
Division of Intramural Research, National Institute of Environmental Health Sciences, 111 T.W. Alexander Drive, Building 101, Research Triangle Park, NC 27709, USA.
Bradbury
J Alyce
JA
King
Lorraine
L
Maronpot
Robert
R
Davis
Linda S
LS
Breyer
Matthew D
MD
Zeldin
Darryl C
DC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis; D004795:Enzyme Stability; D005260:Female; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D014018:Tissue Distribution
C000717350:CYP2J2 protein, human; C000717353:Cyp2j6 protein, mouse; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D000090762:Cytochrome P-450 CYP2J2