title: CDNA cloning, heterologous expression, and characterization of rat intestinal CYP2J4.
abstract: The small intestine is the major portal of entry of ingested xenobiotics. Previous studies from this and other laboratories indicated that at least 6 of the 33 xenobiotic metabolizing forms of P450 currently identified are expressed in rat small intestinal epithelial cells. In the present study, a previously unidentified rat P450, designated CYP2J4, was identified in rat small intestine using PCR. The full-length CYP2J4 cDNA contains an open reading frame for a protein of 501 residues and is 72.5 and 75.8% identical to rabbit CYP2J1 and human CYP2J2, respectively, in deduced amino acid sequences. The coding region of CYP2J4 cDNA has been cloned into a baculoviral expression vector (pVL1392) and expressed in cultured Spodoptera frugiperta (SF9) cells. The heterologously expressed CYP2J4 protein displayed a typical p450 CO-difference spectrum, with maximum absorbance at 449 nm. When purified to near electrophoretic homogeneity, it was active toward arachidonic acid in a reconstituted system with NADPH-P450 reductase and phospholipid, producing both hydroxyeicosatetraenoic and epoxyeicosatrienoic acids. RNA blot analysis with CYP2J4 cDNA as a probe detected two mRNA species, about 2.0 and 2.4 kb, respectively, in RNA preparations from liver, intestine, olfactory mucosa, kidney, heart, and lung. The 2.0-kb mRNA species was abundant in liver, small intestine, and olfactory mucosa, whereas the 2.4-kb mRNA species was predominant only in the olfactory mucosa. Immunoblot analysis of microsomal fractions from different rat tissues with a polyclonal anti-peptide antibody to CYP2J4 detected a protein with the same electrophoretic mobility as purified CYP2J4 most abundantly in small intestine and to a lesser extent in liver and other immunoreactive proteins with slightly higher electrophoretic mobility than purified CYP2J4 in a number of tissues, including small intestine, liver, kidney, lung, and olfactory mucosa. The predominant distribution of CYP2J4, which has activity toward arachidonic acid, is provocative, but its physiological function is as yet unknown.
authors.affiliation: Wadsworth Center, New York State Department of Health 12201-0509, USA.
authors.forename: Q Y
authors.initials: QY
authors.lastname: Zhang
authors.forename: X
authors.initials: X
authors.lastname: Ding
authors.forename: L S
authors.initials: LS
authors.lastname: Kaminsky
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007422:Intestines / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D005810:Multigene Family; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
chemical_list: D018076:DNA, Complementary; D003577:Cytochrome P-450 Enzyme System; C112496:Cyp2j4 protein, rat; D000072467:Cytochrome P450 Family 2