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• W1964159597 abstract "A cDNA encoding a new cytochrome P450 was isolated from a mouse liver library. Sequence analysis reveals that this 1,886-base pair cDNA encodes a 501-amino acid polypeptide that is 69–74% identical to CYP2J subfamily P450s and is designated CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 cells using a baculovirus system. Microsomal fractions of CYP2J5/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolize arachidonic acid to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 11- and 15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of product/nmol of cytochrome P450/min at 37 °C); thus CYP2J5 is enzymologically distinct. Northern analysis reveals that CYP2J5 transcripts are most abundant in mouse kidney and present at lower levels in liver. Immunoblotting using a polyclonal antibody against a CYP2J5-specific peptide detects a protein with the same electrophoretic mobility as recombinant CYP2J5 most abundantly in mouse kidney microsomes. CYP2J5 is regulated during development in a tissue-specific fashion. In the kidney, CYP2J5 is present before birth and reaches maximal levels at 2–4 weeks of age. In the liver, CYP2J5 is absent prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistochemical staining of kidney sections with anti-human CYP2J2 IgG reveals that CYP2J protein(s) are present primarily in the proximal tubules and collecting ducts, sites where the epoxyeicosatrienoic acids are known to modulate fluid/electrolyte transport and mediate hormonal action.In situ hybridization confirms abundant CYP2J5 mRNA within tubules of the renal cortex and outer medulla. Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney thus providing direct evidence for the in vivo metabolism of arachidonic acid by the mouse renal epoxygenase(s). Based on these data, we conclude that CYP2J5 is an enzymologically distinct, developmentally regulated, protein that is localized to specific nephron segments and contributes to the oxidation of endogenous renal arachidonic acid pools. In light of the well documented effects of epoxyeicosatrienoic acids in modulating renal tubular transport processes, we postulate that CYP2J5 products play important functional roles in the kidney. A cDNA encoding a new cytochrome P450 was isolated from a mouse liver library. Sequence analysis reveals that this 1,886-base pair cDNA encodes a 501-amino acid polypeptide that is 69–74% identical to CYP2J subfamily P450s and is designated CYP2J5. Recombinant CYP2J5 was co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 cells using a baculovirus system. Microsomal fractions of CYP2J5/NADPH-cytochrome P450 oxidoreductase-transfected cells metabolize arachidonic acid to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 11- and 15-hydroxyeicosatetraenoic acids (catalytic turnover, 4.5 nmol of product/nmol of cytochrome P450/min at 37 °C); thus CYP2J5 is enzymologically distinct. Northern analysis reveals that CYP2J5 transcripts are most abundant in mouse kidney and present at lower levels in liver. Immunoblotting using a polyclonal antibody against a CYP2J5-specific peptide detects a protein with the same electrophoretic mobility as recombinant CYP2J5 most abundantly in mouse kidney microsomes. CYP2J5 is regulated during development in a tissue-specific fashion. In the kidney, CYP2J5 is present before birth and reaches maximal levels at 2–4 weeks of age. In the liver, CYP2J5 is absent prenatally and during the early postnatal period, first appears at 1 week, and then remains relatively constant. Immunohistochemical staining of kidney sections with anti-human CYP2J2 IgG reveals that CYP2J protein(s) are present primarily in the proximal tubules and collecting ducts, sites where the epoxyeicosatrienoic acids are known to modulate fluid/electrolyte transport and mediate hormonal action.In situ hybridization confirms abundant CYP2J5 mRNA within tubules of the renal cortex and outer medulla. Epoxyeicosatrienoic acids are endogenous constituents of mouse kidney thus providing direct evidence for the in vivo metabolism of arachidonic acid by the mouse renal epoxygenase(s). Based on these data, we conclude that CYP2J5 is an enzymologically distinct, developmentally regulated, protein that is localized to specific nephron segments and contributes to the oxidation of endogenous renal arachidonic acid pools. In light of the well documented effects of epoxyeicosatrienoic acids in modulating renal tubular transport processes, we postulate that CYP2J5 products play important functional roles in the kidney. Cytochrome P450s have been the subject of intense investigation by toxicologists and pharmacologists over the past 4 decades because they catalyze the oxidative, peroxidative, and reductive metabolism of a wide variety of drugs, industrial chemicals, environmental pollutants, and carcinogens (1Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2619) Google Scholar, 2Guengerich F.P. J. Biol. Chem. 1991; 266: 10019-10022Abstract Full Text PDF PubMed Google Scholar). Some of these enzymes also catalyze the NADPH-dependent oxidation of arachidonic acid tocis-epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and 14,15-EET), 1The abbreviations used are: EET, cis-epoxyeicosatrienoic acid; DHET, vic-dihydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; HPLC, high performance liquid chromatography; P450, cytochrome P450; CYPOR, NADPH-cytochrome P450 oxidoreductase; kb, kilobase; PCR, polymerase chain reaction; GC/MS, gas chromatography/mass spectrometry. 1The abbreviations used are: EET, cis-epoxyeicosatrienoic acid; DHET, vic-dihydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; HPLC, high performance liquid chromatography; P450, cytochrome P450; CYPOR, NADPH-cytochrome P450 oxidoreductase; kb, kilobase; PCR, polymerase chain reaction; GC/MS, gas chromatography/mass spectrometry. mid-chaincis-trans-conjugated dienols (5-, 8-, 9-, 11-, 12-, and 15-HETE), and/or ω-terminal alcohols of arachidonic acid (16-, 17-, 18, 19-, and 20-HETE) (3Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6 (and references therein): 731-736Crossref PubMed Scopus (275) Google Scholar, 4Oliw E.H. Prog. Lipid Res. 1994; 33 (and references therein): 329-354Crossref PubMed Scopus (135) Google Scholar, 5Capdevila J.H. Zeldin D.C. Karara A. Falck J.R. Adv. Cell Mol. Biol. 1996; 14 (and references therein): 317-339Crossref Scopus (9) Google Scholar). A particular interest in the epoxygenase reaction has developed because the EETs are endogenous constituents of numerous tissues and because they possess a myriad of potent biological activities (3Capdevila J.H. Falck J.R. Estabrook R.W. FASEB J. 1992; 6 (and references therein): 731-736Crossref PubMed Scopus (275) Google Scholar, 4Oliw E.H. Prog. Lipid Res. 1994; 33 (and references therein): 329-354Crossref PubMed Scopus (135) Google Scholar, 5Capdevila J.H. Zeldin D.C. Karara A. Falck J.R. Adv. Cell Mol. Biol. 1996; 14 (and references therein): 317-339Crossref Scopus (9) Google Scholar, 6McGiff J.C. Annu. Rev. Pharmacol. Toxicol. 1991; 31 (and references therein): 339-369Crossref PubMed Scopus (286) Google Scholar, 7McGiff J.C. Quilley C.P. Carroll M.A. Steroids. 1993; 58: 573-579Crossref PubMed Scopus (23) Google Scholar). For example, the EETs have been shown to control peptide hormone secretion in the pancreas, pituitary, and hypothalamus (8Falck J.R. Manna S. Moltz J. Chacos N. Capdevila J. Biochem. Biophys. Res. Commun. 1983; 114: 743-749Crossref PubMed Scopus (144) Google Scholar, 9Capdevila J. Chacos N. Falck J.R. Manna S. Negro-Vilar A. Ojeda S.R. Endocrinology. 1983; 113: 421-423Crossref PubMed Scopus (108) Google Scholar), regulate vascular tone in the intestine and brain (10Proctor K.G. Falck J.R. Capdevila J. Circ. Res. 1986; 60: 50-59Crossref Scopus (150) Google Scholar, 11Gebremedhin D. Ma Y.-H. Falck J.R. Roman R.J. VanRollins M. Harder D.R. Am. J. Physiol. 1992; 263: H519-H525PubMed Google Scholar), affect ion transport and airway smooth muscle tone in the lung (12Zeldin D.C. Plitman J.D. Kobayashi J. Miller R.F. Snapper J.R. Falck J.R. Szarek J.L. Philpot R.M. Capdevila J.H. J. Clin. Invest. 1995; 95: 2150-2160Crossref PubMed Scopus (105) Google Scholar,13Pascual M.M.S. McKenzie A. Yankaskas J.R. Falck J.R. Zeldin D.C. J. Pharmacol. Exp. Ther. 1998; 286: 772-779PubMed Google Scholar), and improve functional recovery following global ischemia in the heart (14Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). P450-derived eicosanoids have been extensively studied in the kidney and have been shown to substantially contribute to integrated renal function (6McGiff J.C. Annu. Rev. Pharmacol. Toxicol. 1991; 31 (and references therein): 339-369Crossref PubMed Scopus (286) Google Scholar, 7McGiff J.C. Quilley C.P. Carroll M.A. Steroids. 1993; 58: 573-579Crossref PubMed Scopus (23) Google Scholar). Thus, the EETs and/or their hydration products (the DHETs) inhibit Na+ transport and mediate angiotensin II-induced rises in cytosolic Ca2+ in the proximal tubule (15Madhun Z.T. Goldthwait D.A. McKay D. Hopfer U. Douglas J.G. J. Clin. Invest. 1991; 88: 456-461Crossref PubMed Scopus (115) Google Scholar, 16Romero M.F. Hopfer U. Madhun Z.T. Zhou W. Douglas J.G. Renal Physiol. Biochem. 1991; 14: 199-207PubMed Google Scholar), stimulate prostaglandin synthesis, and inhibit Na+ reabsorption, K+ secretion, and vasopressin-stimulated water reabsorption in the collecting duct (17Jacobson H.R. Corona S. Capdevila J. Chacos N. Manna S. Womack A. Falck J.R. Braquet P. Prostaglandins and Membrane Ion Transport. Raven Press, Ltd., New York1984: 311-318Google Scholar, 18Hirt D.L. Capdevila J. Falck J.R. Breyer M.D. Jacobson H.R. J. Clin. Invest. 1989; 84: 1805-1812Crossref PubMed Scopus (61) Google Scholar, 19Sakairi Y. Jacobson H.R. Noland T.D. Capdevila J.H. Falck J.R. Breyer M.D. Am. J. Physiol. 1995; 268: F931-F939PubMed Google Scholar), activate Na+/H+ exchange, amplify vasopressin-induced increases in cytosolic Ca2+, and stimulate cellular proliferation in renal glomerular mesangial cells (20Harris R.C. Homma T. Jacobson H.R. Capdevila J. J. Cell. Physiol. 1990; 144: 429-437Crossref PubMed Scopus (91) Google Scholar, 21Force T. Hyman G. Hajjar R. Sellmayer A. Bonventre J.V. J. Biol. Chem. 1991; 266: 4295-4302Abstract Full Text PDF PubMed Google Scholar), modulate renal Na+/K+-ATPase (22Ominato M. Satoh T. Katz A.I. J. Membr. Biol. 1996; 152: 235-243Crossref PubMed Scopus (117) Google Scholar), affect intrarenal vascular tone, and renal vascular smooth muscle cell K+ channel activity (23Katoh T. Takahashi K. Capdevila J. Karara A. Falck J.R. Jacobson H.R. Badr K.F. Am. J. Physiol. 1991; 261: F578-F586PubMed Google Scholar, 24Zou A. Fleming J.T. Falck J.R. Jacobs E.R. Gebremedhin D. Harder D.R. Roman R.J. Am. J. Physiol. 1996; 270: F822-F832PubMed Google Scholar, 25Carroll M.A. Balazy M. Margiotta P. Falck J.R. McGiff J.C. J. Biol. Chem. 1993; 268: 12260-12266Abstract Full Text PDF PubMed Google Scholar), and inhibit renal cortical renin release (26Henrich W.L. Falck J.R. Campbell W.B. Am. J. Physiol. 1990; 258: E269-E274PubMed Google Scholar). Recent studies demonstrating that (a) the rat renal epoxygenase(s) are under regulatory control by dietary salt, (b) clotrimazole inhibition of the rat renal epoxygenase(s) leads to the development of salt-dependent hypertension, (c) the salt-sensitive phenotype in the Dahl rat model of genetic hypertension is associated with decreased renal microsomal arachidonic acid epoxygenase activity and an inability to up-regulate this activity in response to salt loading, (d) the developmental phase of hypertension in spontaneously hypertensive rats is associated with alterations in kidney P450 arachidonic acid metabolism, and (e) the urinary excretion of epoxygenase metabolites is increased during pregnancy-induced hypertension in humans have supported the hypothesis that P450-derived arachidonic acid metabolites may be involved in the pathophysiology of hypertension (6McGiff J.C. Annu. Rev. Pharmacol. Toxicol. 1991; 31 (and references therein): 339-369Crossref PubMed Scopus (286) Google Scholar,7McGiff J.C. Quilley C.P. Carroll M.A. Steroids. 1993; 58: 573-579Crossref PubMed Scopus (23) Google Scholar, 27Capdevila J.H. Wei S. Yan J. Karara A. Jacobson H.R. Falck J.R. Guengerich F.P. Dubois R.N. J. Biol. Chem. 1992; 267: 21720-21726Abstract Full Text PDF PubMed Google Scholar, 28Makita K. Takahashi K. Karara A. Jacobson H.R. Falck J.R. Capdevila J.H. J. Clin. Invest. 1994; 94: 2414-2420Crossref PubMed Scopus (175) Google Scholar, 29Catella F. Lawson J.A. Fitzgerald D.J. Fitzgerald G.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5893-5897Crossref PubMed Scopus (152) Google Scholar, 30Omata K. Abraham N.G. Escalante B. Schwartzman M.L. Am. J. Physiol. 1992; 262: F8-F16PubMed Google Scholar, 31Imig J.D. Falck J.R. Gebremedhin D. Harder D.R. Roman R.J. Hypertension. 1993; 22: 357-364Crossref PubMed Scopus (108) Google Scholar, 32Ma Y.-H. Schwartzman M.L. Roman R.J. Am. J. Physiol. 1994; 36: R579-R589Google Scholar). Together, these findings suggest that the renal P450 arachidonic acid monooxygenases may be important in controlling blood pressure and body fluid volume/composition and may be part of an adaptive response of the kidney to excess dietary salt intake (6McGiff J.C. Annu. Rev. Pharmacol. Toxicol. 1991; 31 (and references therein): 339-369Crossref PubMed Scopus (286) Google Scholar,7McGiff J.C. Quilley C.P. Carroll M.A. Steroids. 1993; 58: 573-579Crossref PubMed Scopus (23) Google Scholar). Despite considerable investigations into the physiological aspects of the renal epoxygenase pathway, little is known with regard to the biochemical and molecular properties of the renal P450 enzyme(s) active in the biosynthesis of the EETs. A member of the CYP2C subfamily was purified from rabbit kidney and shown to catalyze arachidonic acid epoxidation and ω/ω-1 hydroxylation (33Laethem R.M. Laethem C.L. Koop D.R. J. Biol. Chem. 1992; 267: 5552Abstract Full Text PDF PubMed Google Scholar). Immunochemical studies suggested that CYP2C isoforms were responsible for some of the arachidonic acid epoxygenase activity present in rat and rabbit kidney microsomal fractions (27Capdevila J.H. Wei S. Yan J. Karara A. Jacobson H.R. Falck J.R. Guengerich F.P. Dubois R.N. J. Biol. Chem. 1992; 267: 21720-21726Abstract Full Text PDF PubMed Google Scholar, 33Laethem R.M. Laethem C.L. Koop D.R. J. Biol. Chem. 1992; 267: 5552Abstract Full Text PDF PubMed Google Scholar). Work by Karara et al. (34Karara A. Makita K. Jacobson H.R. Falck J.R. Guengerich F.P. DuBois R.N. Capdevila J.H. J. Biol. Chem. 1993; 268: 13565-13570Abstract Full Text PDF PubMed Google Scholar) and Imaoka et al. (35Imaoka S. Wedlund P.J. Ogawa H. Kimura S. Gonzalez F.J. Kim H.Y. J. Pharmacol. Exp. Ther. 1993; 267: 1012-1016PubMed Google Scholar) demonstrated that CYP2C23 was highly expressed in rat kidney and active in the regio- and stereoselective epoxidation of arachidonic acid (34Karara A. Makita K. Jacobson H.R. Falck J.R. Guengerich F.P. DuBois R.N. Capdevila J.H. J. Biol. Chem. 1993; 268: 13565-13570Abstract Full Text PDF PubMed Google Scholar, 35Imaoka S. Wedlund P.J. Ogawa H. Kimura S. Gonzalez F.J. Kim H.Y. J. Pharmacol. Exp. Ther. 1993; 267: 1012-1016PubMed Google Scholar). CYP2C8, cloned from a human kidney cDNA library, was also an active arachidonic acid epoxygenase; however, this P450 was expressed at relatively low levels in human kidney (36Zeldin D.C. DuBois R.N. Falck J.R. Capdevila J.H. Arch. Biochem. Biophys. 1995; 322: 76-86Crossref PubMed Scopus (151) Google Scholar, 37Zeldin D.C. Moomaw C.R. Jesse N. Tomer K.B. Beetham J. Hammock B.D. Wu S. Arch. Biochem. Biophys. 1996; 330: 87-96Crossref PubMed Scopus (128) Google Scholar). More recently, our laboratory identified a human P450 of the CYP2J subfamily that was highly expressed in a number of extrahepatic tissues including the kidney and active in the metabolism of arachidonic acid to EETs (38Wu S. Moomaw C.R. Tomer K.B. Falck J.R. Zeldin D.C. J. Biol. Chem. 1996; 271: 3460-3468Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). However, studies on the regulation, importance, and functional role of this new human P450 epoxygenase in the kidney have not been possible because of the limited availability of fresh, histologically normal and abnormal human kidney tissues. Herein, we report the cDNA cloning and heterologous expression of CYP2J5, a new mouse P450 that is abundant in the kidney and active in the epoxidation and midchain hydroxylation of arachidonic acid. We show that this heme-thiolate protein is regulated during development and in a tissue-specific fashion. We further demonstrate, using immunohistochemical techniques and in situ hybridization, that CYP2J5 mRNA and protein are localized to sites within the nephron where the EETs are known to affect fluid/electrolyte transport and mediate hormonal action. [α-32P]dATP and [1-14C]arachidonic acid were purchased from NEN Life Science Products. Restriction enzymes were purchased from New England Biolabs (Beverly, MA). PCR reagents including AmpliTaq® DNA polymerase were purchased from Perkin-Elmer. Triphenylphosphine, α-bromo-2,3,4,5,6-pentafluorotoluene,N,N-diisopropylethylamine, N,N-dimethylformamide, and diazald were purchased from Aldrich. All other chemicals and reagents were purchased from Sigma unless otherwise specified. An oligo(dT)-primed Lambda ZAP B6/CBAF1J male mouse liver cDNA library (Stratagene, La Jolla, CA) was screened with the 1.8-kb CYP2J3 cDNA probe under conditions identical to those described previously (14Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Approximately 100 duplicate positive clones were identified, of which 21, selected at random, were plaque-purified and rescued into pBluescript SK(+) (Stratagene). Plasmid DNAs were isolated using a Qiagen Plasmid Purification Kit (Qiagen Inc., Chatsworth, CA), and insert sizes were determined by agarose gel electrophoresis after EcoRI digestion. The pBluescript cDNA inserts were partially sequenced by the dideoxy chain termination method using Sequenase version 2.0 (U. S. Biochemical Corp.) and T3/T7 oligonucleotide primers. Nucleotide sequences were analyzed by searching GenBankTMand EMBL data bases utilizing GCG software (Genetics Computer Group, Inc., Madison, WI). Sixteen of the duplicate positive clones contained sequences that were identical and shared homology with several human and rodent CYP2 family P450s (1Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2619) Google Scholar). One of these clones (clone JM-6) 2The new sequence that is reported in this paper was submitted to the Committee on Standardized Cytochrome P450 Nomenclature and has been designated CYP2J5. was completely sequenced utilizing a total of 16 oligonucleotide primers (21–25 nucleotides, each) that spanned the entire length of the sense and antisense cDNA strands. Oligonucleotides were synthesized and purified as described (14Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Co-expression of the protein encoded by the cloned 1.886-kb JM-6 cDNA insert (CYP2J5) with CYPOR in Sf9 insect cells was accomplished with the pAcUW51-CYPOR shuttle vector (kindly provided by Dr. Cosette Serabjit-Singh, Glaxo Wellcome, Research Triangle Park, NC) and the BaculoGold Baculovirus Expression System (PharMingen, San Diego, CA) using methods similar to those previously described (14Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 39Lee C. Kadwell S.H. Kost T.A. Serabjit-Singh C.J. Arch. Biochem. Biophys. 1995; 319: 157-167Crossref PubMed Scopus (92) Google Scholar). The CYP2J5 cDNA (nucleotides 42–1886) including the entire coding region was ligated into a slightly modified pAcUW51-CYPOR vector, and the orientation and identity of the resulting expression vector (pAcUW51-CYPOR-CYP2J5) were confirmed by sequence analysis. In this construct, the expression of CYPOR was controlled by the p10 promoter, whereas the expression of CYP2J5 was independently controlled by the polyhedrin promoter. Cultured Sf9 cells were co-transfected with the pAcUw51-CYPOR-CYP2J5 vector and linear wild-type BaculoGold viral DNA, and recombinant viruses were purified as described (14Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Cultured Sf9 cells grown in spinner flasks at a density of 1.5–2 × 106 cells/ml were then infected with high titer CYP2J5-CYPOR recombinant baculovirus stock in the presence of δ-aminolevulinic acid and iron citrate (100 μm each). Cells co-expressing recombinant CYP2J5 and CYPOR were harvested 72 h after infection, washed twice with phosphate-buffered saline, and were used to prepare microsomal fractions by differential centrifugation at 4 °C as described previously (12Zeldin D.C. Plitman J.D. Kobayashi J. Miller R.F. Snapper J.R. Falck J.R. Szarek J.L. Philpot R.M. Capdevila J.H. J. Clin. Invest. 1995; 95: 2150-2160Crossref PubMed Scopus (105) Google Scholar). P450 content was determined spectrally according to the method of Omura and Sato (40Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar) using a Shimadzu UV-3000 dual-wavelength/double-beam spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD). CYP2J5 protein was also expressed without CYPOR in Sf9 cells using the pBlueBacIV expression vector (Invitrogen, San Diego, CA). Sf9 cells expressing recombinant CYPOR but not P450 were prepared as described (39Lee C. Kadwell S.H. Kost T.A. Serabjit-Singh C.J. Arch. Biochem. Biophys. 1995; 319: 157-167Crossref PubMed Scopus (92) Google Scholar). Reaction mixtures containing 0.05 mTris-Cl buffer (pH 7.5), 0.15 m KCl, 0.01 mMgCl2, 8 mm sodium isocitrate, 0.5 IU/ml isocitrate dehydrogenase, 0.1–0.2 mg of CYP2J5-CYPOR-transfected Sf9 cell microsomal protein/ml, and [1-14C]arachidonic acid (25–55 μCi/μmol; 100 μm, final concentration) were constantly stirred at 37 °C. After temperature equilibration, NADPH (1 mm, final concentration) was added to initiate the reaction. At different time points, aliquots were withdrawn, and the reaction products were extracted and analyzed by reverse-phase HPLC as described (41Zeldin D.C. Foley J. Goldsworthy S.M. Cook M.E. Boyle J.E. Ma J. Moomaw C.R. Tomer K.B. Steenbergen C. Wu S. Mol. Pharmacol. 1997; 51: 931-943Crossref PubMed Scopus (119) Google Scholar). Products were identified by comparing their reverse- and normal-phase HPLC properties with those of authentic standards and by GC/MS (42Capdevila J.H. Falck J.R. Dishman E. Karara A. Methods Enzymol. 1990; 187: 385-394Crossref PubMed Scopus (89) Google Scholar,43Capdevila J. Yadagiri P. Manna S. Falck J.R. Biochem. Biophys. Res. Commun. 1986; 141: 1007-1011Crossref PubMed Scopus (105) Google Scholar). For kinetic experiments, arachidonic acid concentrations were varied from 5 to 100 μm, and reactions were only allowed to proceed for 2–3 min to ensure that the quantitative assessment of the rates of product formation reflected initial rates. For chiral analysis, the EETs were collected from the HPLC eluent, derivatized, and resolved into the corresponding antipodes by chiral-phase HPLC as described previously (44Capdevila J.H. Dishman E. Karara A. Falck J.R. Methods Enzymol. 1991; 206: 441-453Crossref PubMed Scopus (72) Google Scholar, 45Hammonds T.D. Blair I.A. Falck J.R. Capdevila J.H. Anal. Biochem. 1989; 182: 300-303Crossref PubMed Scopus (47) Google Scholar). Control studies were performed by incubating uninfected Sf9 cell microsomes and baculovirus-infected Sf9 cell microsomes expressing recombinant CYPOR but containing no spectrally evident P450 with arachidonic acid under identical conditions. Methods used to quantify endogenous EETs present in mouse kidney were similar to those used to quantify EETs in rat liver and heart (14Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 46Karara A. Dishman E. Blair I. Falck J.R. Capdevila J.H. J. Biol. Chem. 1989; 264: 19822-19827Abstract Full Text PDF PubMed Google Scholar). Briefly, kidneys were frozen in liquid nitrogen and homogenized in phosphate-buffered saline containing triphenylphosphine. The homogenate was extracted as described (14Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), and the combined organic phases were evaporated in tubes containing mixtures of [1-14C]8,9-EET, [1-14C]11,12-EET, and [1-14C]14,15-EET internal standards (56–57 μCi/μmol, 80 ng each). Saponification, silica column purification, HPLC resolution, GC/MS analysis, and quantification were done as described elsewhere (14Wu S. Chen W. Murphy E. Gabel S. Tomer K.B. Foley J. Steenbergen C. Falck J.R. Moomaw C.R. Zeldin D.C. J. Biol. Chem. 1997; 272: 12551-12559Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 45Hammonds T.D. Blair I.A. Falck J.R. Capdevila J.H. Anal. Biochem. 1989; 182: 300-303Crossref PubMed Scopus (47) Google Scholar, 46Karara A. Dishman E. Blair I. Falck J.R. Capdevila J.H. J. Biol. Chem. 1989; 264: 19822-19827Abstract Full Text PDF PubMed Google Scholar). Normal mouse tissues were obtained from adult male C57BL/6J mice fed NIH 31 rodent chow (Agway, St. Mary, OH) ad libitum and sacrificed by lethal CO2 inhalation. RNA was prepared by the guanidinium thiocyanate/cesium chloride density gradient centrifugation method as described previously (47Maniatis T. Sambrook J. Fritsch E.F. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Total RNA (25 μg) prepared from mouse testes, small intestine, muscle, lung, liver, kidney, heart, colon, and brain was denatured and electrophoresed in 1.2% agarose gels containing 2.2 m formaldehyde. After capillary pressure transfer to Hybond-N+ membranes (Amersham Pharmacia Biotech), the blots were hybridized with either the cloned 1.886-kb JM-6 cDNA insert or with the following JM-6 sequence-specific oligonucleotide probe (5′-TCAAAAACATAATCATATTTGGGATCCTTGCACAC-3′, complementary to nucleotides 23–57 of the JM-6 cDNA). With the cDNA probe, hybridizations were performed at 42 °C in 50% formamide containing 0.7 m NaCl, 70 mm sodium citrate, 5× Denhardt's solution, 0.5% (w/v) SDS, and 0.1 mg of heatdenatured salmon sperm DNA/ml. With the oligonucleotide probe, hybridizations were performed at 50 °C in 10% formamide containing 0.9m NaCl, 90 mm sodium citrate, 2× Denhardt's solution, 0.5% (w/v) SDS, and 0.1 mg of heat-denatured salmon sperm DNA/ml. The double-stranded cDNA probe was labeled with [α-32P]dATP by nick translation usingEscherichia coli polymerase I. The oligonucleotide probe was end-labeled using T4 polynucleotide kinase and [γ-32P]ATP. Northern blot results were confirmed by PCR amplification of reversetranscribed mouse RNAs using the GeneAmp® RNA PCR Kit (Perkin-Elmer). The following JM-6 sequence-specific oligonucleotides were used: forward primer, 5′-ATCAGAGAAGCGAAAAGAATGTAG-3′, corresponding to nucleotides 1549–1572 of the JM-6 cDNA; reverse primer, 5′-CCATTTCCTCTGATTCTGACTCAT-3′, complementary to nucleotides 1810–1833 of the JM-6 cDNA. Reverse transcription was performed with 1 μg of total RNA in a buffer containing 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 5 mm MgCl2, 2.5 μm random hexamers, 1 mm each of dGTP, dATP, dTTP, and dCTP, and 50 units of Moloney murine leukemia virus reverse transcriptase incubated at 42 °C for 1 h. The PCR amplifications were performed in the presence of 2 mm MgCl2, 0.15 μmforward and reverse primers, and 2.5 units of AmpliTaq® DNA polymerase. Following an initial incubation for 105 s at 95 °C, samples were subjected to 35 cycles of 15 s at 95 °C and 30 s at 62 °C. The PCR products were electrophoresed on 1.2% agarose gels containing ethidium bromide. The following β-actin sequence-specific primers were used to control for the quality and amount of RNA: forward primer, 5′-GAGCTATGAGCTGCCTGACG-3′, corresponding to nucleotides 776–795 of the mouse β-actin cDNA; reverse primer, 5′-AGCACTTGCGGTGCACGATG-3′, complementary to nucleotides 1166–1185 of the mouse β-actin cDNA. Microsomal fractions were prepared from adult male C57BL/6J mouse tissues by differential centrifugation at 4 °C as described previously (12Zeldin D.C. Plitman J.D. Kobayashi J. Miller R.F. Snapper J.R. Falck J.R. Szarek J.L. Philpot R.M. Capdevila J.H. J. Clin. Invest. 1995; 95: 2150-2160Crossref PubMed Scopus (105) G" @default.
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• W1964159597 title "Molecular Cloning, Enzymatic Characterization, Developmental Expression, and Cellular Localization of a Mouse Cytochrome P450 Highly Expressed in Kidney" @default.
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