Identification of a Novel Rat Microsomal Vitamin D3 25-Hydroxylase
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m311346200
ISSN1083-351X
AutoresTomoaki Yamasaki, Shunsuke Izumi, Hiroshi Ide, Yoshihiko Ohyama,
Tópico(s)Diet and metabolism studies
ResumoVitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1α-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1α-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50–60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1α-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver. Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1α-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1α-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50–60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1α-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver. Vitamin D3 25-hydroxylation, which mainly occurs in the liver, is the initial step of vitamin D3 activation (1DeLuca H.F. FASEB J. 1988; 2: 224-236Crossref PubMed Scopus (491) Google Scholar). The 25-hydroxylated vitamin D3 product circulates in blood as a complex with the vitamin D-binding protein. The complex is filtered once through the glomerulus and reabsorbed by the endocytic receptor megalin into the proximal tubular cell in the kidney, where 25-hydroxyvitaimin D3 (25-OH-D3) 1The abbreviations used are: 25-OH-D3, 25-hydroxyvitaimin D3 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; 1α-OH-D3, 1α-hydroxyvitamin D3; CYP, cytochrome P450; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MEGA-10, n-decanoyl-N-methylglucamide; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; δ-ALA, δ-aminolevulinic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PEG, polyethylene glycol. is 1α-hydroxylated to yield 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) (2Jones G. Strugnell S.A. DeLuca H.F. Physiol. Rev. 1998; 78: 1193-1231Crossref PubMed Scopus (1054) Google Scholar, 3Brown A.J. Dusso A. Slatopolsky E. Am. J. Physiol. 1999; 277: F157-F175Crossref PubMed Google Scholar, 4Omdahl J.L. Morris H.A. May B.K. 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Suda T. Yamamoto O. Noshiro M. Kato Y. J. Biol. Chem. 1994; 269: 10545-10550Abstract Full Text PDF PubMed Google Scholar). These two enzymes are apparently regulated in a reciprocal manner depending on physiological factors concerning calcium homeostasis, by which the biological activity of vitamin D3 is maintained at appropriate levels in the body (1DeLuca H.F. FASEB J. 1988; 2: 224-236Crossref PubMed Scopus (491) Google Scholar). In contrast to the 1α-hydroxylase and 24-hydroxylase in kidney, the 25-hydroxylase of vitamin D3 in liver remains not fully understood, although 25-OH-D3 is the major circulation form of vitamin D3 in mammals. It has been established that 25-hydroxylation is catalyzed by cytochrome P450 enzymes present in microsomes and mitochondria of liver (15Björkhem I. Holmberg I. J. Biol. Chem. 1978; 253: 842-849Abstract Full Text PDF PubMed Google Scholar, 16Madhok T.C. DeLuca H.F. Biochem. J. 1979; 184: 491-499Crossref PubMed Scopus (89) Google Scholar). Until now, one mitochondrial and three microsomal cytochrome P450 enzymes have been reported as vitamin D3 25-hydroxylases: CYP2C11 (17Andersson S. Jörnvall H. J. Biol. Chem. 1986; 261: 16932-16936Abstract Full Text PDF PubMed Google Scholar, 18Hayashi S. Noshiro M. Okuda K. J. Biochem. (Tokyo). 1986; 99: 1753-1763Crossref PubMed Scopus (65) Google Scholar), CYP27A1 (19Masumoto O. Ohyama Y. Okuda K. J. Biol. Chem. 1988; 263: 14256-14260Abstract Full Text PDF PubMed Google Scholar, 20Usui E. Noshiro M. Okuda K. FEBS Lett. 1990; 262: 135-138Crossref PubMed Scopus (113) Google Scholar, 21Su P. Rennert H. Shayiq R.M. Yamamoto R. Zheng Y.M. Addya S. Strauss III, J.F. Avadhani N.G. DNA Cell Biol. 1990; 9: 657-667Crossref PubMed Scopus (109) Google Scholar, 22Guo Y.D. Strugnell S. Back D.W. Jones G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8668-8672Crossref PubMed Scopus (151) Google Scholar), CYP2D25 (23Axén E. Bergman T. Wikvall K. Biochem. J. 1992; 287: 725-731Crossref PubMed Scopus (22) Google Scholar, 24Postlind H. Axén E. Bergman T. Wikvall K. Biochem. Biophys. Res. Commun. 1997; 241: 491-497Crossref PubMed Scopus (37) Google Scholar), and CYP2R1 (25Cheng J.B. Motola D.L. Mangelsdorf D.J. Russell D.W. J. Biol. Chem. 2003; 278: 38084-38093Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). They show broad substrate specificities and low catalytic activities toward vitamin D3 relative to other substrates. CYP2C11 is a rat male-specific P450 (26Yoshioka H. Morohashi K. Sogawa K. Miyata T. Kawajiri K. Hirose T. Inayama S. Fujii-Kuriyama Y. Omura T. J. Biol. Chem. 1987; 262: 1706-1711Abstract Full Text PDF PubMed Google Scholar) and catalyzes the 2α- and 16α-hydroxylation of testosterone 40-fold more efficiently than vitamin D3 25-hydroxylation (18Hayashi S. Noshiro M. Okuda K. J. Biochem. (Tokyo). 1986; 99: 1753-1763Crossref PubMed Scopus (65) Google Scholar). CYP27A1 is a mitochondrial P450 catalyzing not only vitamin D3 25-hydroxylation but also sterol 27-hydroxylation. The latter reaction is 100-fold more efficient than vitamin D3 25-hydroxylation and is essential for the catabolism of cholesterol to bile acid (27Ohyama Y. Masumoto O. Usui E. Okuda K. J. Biochem. (Tokyo). 1991; 109: 389-393Crossref PubMed Scopus (27) Google Scholar). Mutation of this gene causes cerebrotendinous xanthomatosis, leading to accumulation of cholestanol and cholesterol in most tissues (28Björkhem I. Boberg K.M. Leitersdorf E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 2961-2988Google Scholar). The disruption of the Cyp27a1 gene in mice severely compromises cholesterol metabolism but not vitamin D3 metabolism (29Rosen H. Reshef A. Maeda N. Lippoldt A. Shpizen S. Triger L. Eggertsen G. Björkhem I. Leitersdorf E. J. Biol. Chem. 1998; 273: 14805-14812Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). CYP2D25 was purified from pig liver and kidney microsomes, and its cDNA was isolated (23Axén E. Bergman T. Wikvall K. Biochem. J. 1992; 287: 725-731Crossref PubMed Scopus (22) Google Scholar, 24Postlind H. Axén E. Bergman T. Wikvall K. Biochem. Biophys. Res. Commun. 1997; 241: 491-497Crossref PubMed Scopus (37) Google Scholar). A series of reports by Wikvall and co-workers have shown that (i) this enzyme hydroxylates not only at the C-25 of vitamin D3 but also at the C-1α and C-26 of 25-OH-D3 (30Araya Z. Hosseinpour F. Bodin K. Wikvall K. Biochim. Biophys. Acta. 2003; 1632: 40-47Crossref PubMed Scopus (32) Google Scholar); (ii) it is expressed in kidney as well as in liver (31Hosseinpour F. Norlin M. Wikvall K. Biochim. Biophys. Acta. 2002; 1580: 133-144Crossref PubMed Scopus (14) Google Scholar); and (iii) a possible human ortholog, CYP2D6, exhibits no 25-hydroxylation activity (32Hosseinpour F. Wikvall K. J. Biol. Chem. 2000; 275: 34650-34655Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Very recently, Cheng et al. (25Cheng J.B. Motola D.L. Mangelsdorf D.J. Russell D.W. J. Biol. Chem. 2003; 278: 38084-38093Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) have reported a microsomal P450 (CYP2R1) in mice and humans as a 25-hydroxylase. They successfully isolated this P450 by expression cloning from a CYP27A1-deficient mouse cDNA library, and the 25-hydroxylase activity of the recombinant enzyme was demonstrated. However, detailed analysis of the enzyme activity including substrate specificity and kinetic parameters has not yet been reported. The aim of the present study is to investigate whether other P450 enzymes with a 25-hydroxylation activity higher than those identified exist in mammals. Previous studies showed that CYP2C11 is a male-specific P450 expressed in rat liver and scarcely expressed in female rat liver (26Yoshioka H. Morohashi K. Sogawa K. Miyata T. Kawajiri K. Hirose T. Inayama S. Fujii-Kuriyama Y. Omura T. J. Biol. Chem. 1987; 262: 1706-1711Abstract Full Text PDF PubMed Google Scholar). However, female rat liver microsomes exhibit substantial vitamin D3 25-hydroxylation activity (17Andersson S. Jörnvall H. J. Biol. Chem. 1986; 261: 16932-16936Abstract Full Text PDF PubMed Google Scholar, 33Hayashi S. Usui E. Okuda K. J. Biochem. (Tokyo). 1988; 103: 863-866Crossref PubMed Scopus (25) Google Scholar), suggesting the existence of a yet unidentified vitamin D3 25-hydroxylase. In the present study, we have identified the P450 enzyme responsible for this 25-hydroxylation activity from female rat liver microsomes. Here we report CYP2J3 as a principal microsomal vitamin D3 25-hydroxylase in rat. Materials—Vitamin D3 and testosterone were obtained from Katayama Chemical (Osaka, Japan), and 25-OH-D3, 1,25-(OH)2D3, 1α-OH-D3, cholic acid, and isopropyl-1-thio-β-d-galactoside were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CHAPS and MEGA-10 were from Dojindo Laboratories (Kumamoto, Japan). Emulgen 911 was supplied by Kao-Atlas Co. (Tokyo, Japan). β-NADPH was the product of Oriental Co. (Tokyo, Japan). Restriction enzymes were purchased from New England Biolabs. δ-Aminolevulinic acid (δ-ALA) was from Sigma. The chaperone plasmid, pGro7, was purchased from Takara Bio Inc. (Otsu, Japan). Male and female rats (Wistar strain, 8–10 weeks old) were obtained from Hiroshima Jikken Doubutsu (Hiroshima, Japan). All other chemicals were of the highest quality commercially available. Purification of Female Rat Microsomal 25-Hydroxylase—Livers (∼100 g) were excised from 15 female rats that had been starved for 20 h before sacrifice and perfused with 0.9% NaCl solution. All subsequent operations were performed at 4 °C or on ice, and all buffers contained 1 μg/ml of leupeptin and 1 μg/ml pepstatin except for the hydroxyapatite II step. Livers were minced and homogenized with 5 volumes of 20 mm Tris-HCl (pH 7.5) containing 0.25 m sucrose, 1 mm EDTA, and 1 mm phenylmethanesulfonyl fluoride. The homogenate was centrifuged at 500 × g for 10 min and subsequently at 9300 × g for 20 min. The supernatant was centrifuged at 187,000 × g for 1 h. The precipitated pellet of microsomes was suspended in 20 mm Tris-HCl (pH 7.5) containing 0.15 m KCl, 1 mm EDTA, and 1 mm phenylmethanesulfonyl fluoride and centrifuged again. All buffers used in the following purification procedures contained 20% glycerol and 0.5 mm dithiothreitol. The pellet of microsomes was suspended in buffer A (100 mm potassium phosphate buffer (pH 7.4) containing 0.1 m KCl, 1 mm EDTA) containing 1 mm phenylmethanesulfonyl fluoride to the final protein concentration of 10 mg/ml. To solubilize microsomes, 10% cholate was slowly added to a final concentration of 1.0%, and then the mixture was slowly stirred on ice for 1 h. The solubilized solution was fractionated with polyethylene glycol (PEG) 6000 (Katayama Chemical) as described (34Coon M.J. van der Hoeven T.A. Dahl S.B. Haugen D.A. Methods Enzymol. 1978; 52: 109-117Crossref PubMed Scopus (150) Google Scholar). The precipitate with 4–16% PEG was suspended in buffer A. The suspension was dialyzed overnight against buffer A. The turbid dialysate was resolubilized by adding 10% cholate and the same volume of 200 mm potassium phosphate buffer (pH 7.4) containing 0.2 m KCl and 2mm EDTA, resulting in the final concentration of 1.0% cholate and 9.2 mg of protein/ml in buffer A. The mixture was stirred on ice for 45 min and centrifuged at 187,000 × g for 1 h. The supernatant was then diluted with the same volume of buffer A to reduce the cholate concentration. The diluted sample (80 ml) was applied to an ω-aminohexyl-Sepharose column (3.0 × 12 cm) equilibrated with buffer B (100 mm potassium phosphate buffer (pH 7.4) containing 1 mm EDTA and 0.5% cholate). The column was washed with buffer B and then eluted with buffer B containing 0.05% Emulgen 911. The fractions with relatively high 1α-OH-D3 25-hydroxylation activity were pooled and dialyzed against buffer C (25 mm potassium phosphate buffer (pH 7.4) containing 0.5 mm EDTA, 0.3% cholate, and 0.05% Emulgen 911). The dialyzed fraction (70 ml) was applied to a Bio-Gel HTP hydroxyapatite (Bio-Rad) column (2.5 × 3.0 cm) (the hydroxyapatite I step). The column was washed with buffer C and then eluted stepwise with 60, 100, and 300 mm potassium phosphate buffer (pH 7.4) containing 0.5 mm EDTA, 0.3% cholate, and 0.05% Emulgen 911. The fractions eluted with 60 mm potassium phosphate were pooled and dialyzed against buffer D (20 mm Tris-HCl (pH 7.8), 1 mm EDTA, 0.1% cholate, and 0.2% CHAPS). The dialyzed fraction (14 ml) was applied to a DEAE-Sepharose Fast Flow (Amersham Biosciences) column (1.6 × 2.5 cm) (the DEAE-Sepharose I step). The column was washed with buffer D and then eluted stepwise with the same buffer containing 100, 200, and 300 mm NaCl. The fractions (100 mm NaCl eluate) containing relatively high activity were pooled and dialyzed against 50 mm Tris-HCl (pH 7.5), 1 mm EDTA, and 0.1% cholate. The dialyzed fraction (4.2 ml) was applied to a DEAE-Sepharose Fast Flow column (1.0 × 2.5 cm) equilibrated with buffer E, consisting of 50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 0.1% cholate, and 0.1% MEGA-10 (the DEAE-Sepharose II step). The column was washed with buffer E and eluted stepwise with the same buffer containing 40, 80, and 300 mm NaCl. The fractions (40 mm NaCl) containing high activity were pooled and dialyzed against buffer F (20 mm potassium phosphate buffer (pH 7.4) containing 0.5 mm EDTA and 0.05% cholate). The dialysate (2.5 ml) was applied to a Bio-Gel HTP hydroxyapatite column (1.0 × 2.0 cm) (the hydroxyapatite II step). The column was washed with buffer F and then eluted with 400 mm potassium buffer (pH 7.4) containing 0.5 mm EDTA and 0.05% cholate. The fractions with relatively high activity were stored at –80 °C and used for enzyme characterization. Enzyme Assay—25-Hydroxylase activity was assayed with a reconstituted method (35Ohyama Y. Hayashi S. Usui E. Noshiro M. Okuda K. Methods Enzymol. 1997; 282: 186-199Crossref PubMed Scopus (5) Google Scholar). Typically, the assay mixture (0.49 ml) consisted of 10–300 μg of protein, NADPH-P450 reductase (0.5 units based on cytochrome c reducing activity (36Yasukochi Y. Masters B.S.S. J. Biol. Chem. 1976; 251: 5337-5344Abstract Full Text PDF PubMed Google Scholar)), 100 mm Tris-HCl (pH 7.7), 1 μm EDTA, and 10 nmol of 1α-OH-D3 dissolved in 5 μl of ethanol. The assay mixture was preincubated for 2 min at 37 °C, and then the reaction was started by adding 10 μl of 50mm β-NADPH. The incubation was carried out for 10 min at 37 °C, and the reaction was terminated by the addition of 100 μl of 1 n NaOH. The reaction products were extracted with 4 ml of benzene. The organic phase (3 ml) was evaporated under reduced pressure. The residue was dissolved in chloroform and ethyl acetate (4:1, v/v). An aliquot was subjected to HPLC analysis (PU-980 pump, UV-975 detector, JASCO Co. Ltd., Tokyo, Japan) using a Finepak SIL-5 column (4.6 × 250 mm; JASCO). The sample was eluted with isopropyl alcohol/methanol/hexane (7:7:86, v/v/v) at a flow rate of 1.4 ml/min, and effluents were monitored by absorbance at 265 nm (λmax of vitamin D3 derivatives). The amount of the product was calculated according to the standard curve based on the peak height. In the assay of microsomal activity, the NADPH-P450 reductase was omitted from the assay mixture. The hydroxylation activity toward vitamin D3 was assayed as follows. 0.98 ml of a typical assay mixture consisting of P450 fraction, 1.0 unit of NADPH-P450 reductase, 100 μm Tris-HCl (pH 7.7), 1.0 μm EDTA, and 100 nmol of vitamin D3 dissolved in 10 μl of ethanol was preincubated for 2 min at 37 °C, and then the reaction was started by adding 20 μl of 50mm β-NADPH. The incubation was carried out for 10 min at 37 °C, and the reaction was terminated by adding 1 ml of ethanol. Subsequent procedures were similar to those for the 1α-OH-D3 assays. HPLC analysis were performed with isopropyl alcohol/methanol/hexane (5:2:93, v/v/v) at a flow rate of 1.2 ml/min. The testosterone hydroxylation activity was measured using 50 nmol of testosterone as a substrate by the method similar to that used for 25-hydroxylase activity. HPLC analysis was performed with isopropyl alcohol/methanol/hexane (5:15:85, v/v/v) at a flow rate of 1.2 ml/min, and effluents were monitored by absorbance at 238 nm (18Hayashi S. Noshiro M. Okuda K. J. Biochem. (Tokyo). 1986; 99: 1753-1763Crossref PubMed Scopus (65) Google Scholar). To confirm the product identities, the reversed phase HPLC analysis (Finepak SIL C18T-5 column, 4.6 × 250 mm; JASCO) was carried out in parallel. HPLC analysis for 25-OH-D3 was performed with water/methanol (5:95, v/v) at a flow rate of 0.7 ml/min, and that for 1,25-(OH)2D3 was with water/methanol (10:90, v/v) at a flow rate of 0.8 ml/min (35Ohyama Y. Hayashi S. Usui E. Noshiro M. Okuda K. Methods Enzymol. 1997; 282: 186-199Crossref PubMed Scopus (5) Google Scholar). Kinetic parameters Km and Vmax were determined by measuring the activities under varying substrate concentrations. Vitamin D3 concentrations ranging from 0.5 to 5.0 μm were used for 4.8 pmol of dN2J3 and 1α-OH-D3 from 0.25 to 2.0 μm were employed for 0.95 pmol of dN2J3. Experiments were carried out at least three times, and data are presented as the averages. Hydroxylation activities of dN2J3 for 25-OH-D3 were also examined by HPLC analysis of metabolites (35Ohyama Y. Hayashi S. Usui E. Noshiro M. Okuda K. Methods Enzymol. 1997; 282: 186-199Crossref PubMed Scopus (5) Google Scholar). In-gel Digestion with Trypsin—The in-gel digestion procedure was performed as previously described (37Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7878) Google Scholar, 38Taniguchi H. Kikuchi M. Saibo Kogaku. 2002; 21: 524-534Google Scholar). Briefly major protein bands with apparent molecular mass between 50 and 60 kDa of the purified fraction were excised from SDS-polyacrylamide gel and cut into pieces. The gel pieces were destained by washes with either 100 μl of 50% acetonitrile and 25 mm ammonium bicarbonate repeatedly (in the case of Coomassie Brilliant Blue staining) or with 100 μl of 15mm potassium ferricyanide and 50 mm sodium thiosulfate once and water repeatedly (for silver staining) (39Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Electrophoresis. 1999; 20: 601-605Crossref PubMed Scopus (843) Google Scholar). After shaking with 100 μl of acetonitrile for 5 min, acetonitrile was removed, and the gel pieces were dried in a vacuum centrifuge for 20 min at 30 °C. The gel pieces were shaken with 100 μl of reducing solution (10 mm dithiothreitol and 25 mm ammonium bicarbonate) for 60 min at 56 °C and then washed with 100 μl of 25mm ammonium bicarbonate for 10 min. Subsequently, the gel pieces were shaken under dark conditions with 100 μl of alkylating solution (55 mm iodoacetamide and 25 mm ammonium bicarbonate) for 45 min and washed with 100 μl of 25 mm ammonium bicarbonate. The gel pieces were shaken with 50% acetonitrile and 25 mm ammonium bicarbonate for 10 min twice. The solvent was removed, and the gel pieces were dried again. The gel pieces were immersed in 90 μl of digestion solution containing 10 μg/ml sequencing grade modified trypsin (Promega) and 50 mm ammonium bicarbonate and incubated on ice for 30 min. After removing unabsorbed solution, the gel pieces were incubated for 18 h at 37 °C. Digested peptides were extracted twice from gel pieces with 50 μl of 50% acetonitrile and 5% trifluoroacetic acid. The extracts were combined, and the solvent was evaporated to a volume of ∼20 μl. The concentrated samples were subjected to MALDI-TOF mass analysis as below. Peptide Mass Fingerprinting—One μl of matrix solution (10 mg/ml α-cyano-4-hydroxy-trans-cinnamic acid (Sigma) in 0.1% trifluoroacetic acid, 50% acetonitrile (v/v)) was added to 1 μl of sample in the Eppendorf tube, and then 1 μl of the sample-matrix solution was spotted onto a stainless steel probe tip and allowed to air-dry for 10 min at room temperature. Samples were measured on a Bruker Biflex II MALDI-TOF mass spectrometer (Bruker) equipped with an ion source with visualization optics and an N2 laser (337 nm). Mass spectra were recorded in the reflector positive mode at a 28.5-kV acceleration voltage and 1.5 kV in the detector by accumulating 200 spectra of single laser shots under threshold irradiance. The fragments in the range from m/z 800 to 4000 were detected. Highly intense, well resolved mass signals arising from 3–5 selected target spots were considered. If the signal/noise ratio of the profile was low, the sample was desalted with ZipTip (Millipore Corp.) before mixing with matrix solution, and then desalted sample was remeasured as described. All MALDI spectra were calibrated externally using a standard peptide mixture (angiotensin II (Mr = 1047.2), adrenocorticotropic hormone fragment 18–39 (Mr = 2466.7), and insulin (Mr = 5734.6)). Construction of Expression Plasmids—Standard methods for construction of plasmids were as described by Sambrook et al. (40Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Rat liver cDNA libraries (for male and female, separately) were prepared by a reverse transcription reaction with total RNA, oligo(dT)12–18 primer and SuperScript II (Invitrogen). CYP2J3 cDNA (41Wu 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 (217) Google Scholar) (GenBank™ accession number U39943) was isolated by PCR from the cDNA library (female) using a GeneAmp High Fidelity PCR System (Applied Biosystems) as follows: at 94 °C for 2 min, 40 cycles at 94 °C for 15 s, 60 °C for 30 s, 72 °C for 1 min, and finally 72 °C for 7 min. To prepare a cDNA for an N-terminal modified CYP2J3 protein (mN2J3), TY-1 (forward primer, 5′-TCA CAT ATG GCT GTC ACA GCT GGT TCC CTA CTA G-3′) and TY-2 (reverse primer, 5′-CTC AAG CTT AGC AGA CAC CAG TGT CCT CCA TG-3′) were used as a PCR primer set. TY-1 and TY-2 primers include a NdeI site that coincides with the initiation codon (ATG) and a HindIII site after the stop codon, respectively (indicated with underlines) to facilitate the subsequent cloning into the NdeI/HindIII sites of an expression vector. Moreover, TY-1 primer was designed to alter the second codon to GCT (Ala) and one guanine in the fifth codon (GCG) to thymine to achieve the efficient expression as described by Barnes et al. (42Barnes H.J. Arlotto M.P. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5597-5601Crossref PubMed Scopus (551) Google Scholar) (see Fig. A in Supplemental Materials). A PCR product (1.6 kb) was purified by agarose gel electrophoresis and ligated into the pGEM-T Easy vector (Promega). After transformation of Escherichia coli XL1-Blue, plasmid was purified and verified to be free of mutations by DNA sequence analysis using an ABI PRISM 310 sequencer (Applied Biosystems). The plasmid was completely digested by NdeI and then partially with HindIII because an additional HindIII site is present in the coding region. The 1.6-kb NdeI-HindIII fragment containing the mN2J3 open reading frame was purified from agarose gel and ligated into the NdeI/HindIII site of the pKSN2 vector (43Akiyoshi-Shibata M. Sakaki T. Ohyama Y. Noshiro M. Okuda K. Yabusaki Y. Eur. J. Biochem. 1994; 224: 335-343Crossref PubMed Scopus (148) Google Scholar) modified from pKK223-3, and the plasmid was named pK-mN2J3. To prepare an expression plasmid for an N-terminal truncated CYP2J3 (dN2J3), pK-mN2J3 was amplified by the primer set of TY-5 (forward primer, 5′-CAT ATG GCT AAA CAC AGA CGT CCC AAG AAC TAC-3′) and TY-2 (see Fig. A in Supplemental Materials). TY-5 primer was designed to remove 33 amino acid residues corresponding to positions 3–35 of mN2J3 and to alter the fifth and sixth codons to an AT-rich sequence without amino acid changes (see Fig. A in Supplemental Materials). The PCR product was finally cloned into the pKSN2 vector with a similar manner for mN2J3. The plasmid was named pK-dN2J3. Heterologous Expression of Recombinant P450s in E. coli—E. coli JM109 was used as a host for the expression. The strains harboring various expression vectors were grown in 18 ml of LB medium containing 50 μg/ml ampicill
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