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• W2131960328 abstract "Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.AF212319 Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP+-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.AF212319 Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase.Journal of Biological ChemistryVol. 276Issue 28PreviewDr. Huh's name was misspelled. The correct spelling is shown above. Full-Text PDF Open Access reactive oxygen species glucose-6-phosphate dehydrogenase isocitrate dehydrogenase mitochondrial NAD+-dependent isocitrate dehydrogenase mitochondrial NADP+-dependent isocitrate dehydrogenase cytosolic NADP+-dependent isocitrate dehydrogenase 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 2′,7′-dichlorofluorescein fluorescence-activated cell sorter malondialdehyde Cell damage induced by oxidative stress and reactive oxygen species (ROS)1 has been implicated in several human diseases including aging, alcohol-mediated organ damage, neurodegenerative diseases, many types of cancers, cardiovascular diseases, and UV-mediated skin disorders (1Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7913-7922Crossref Scopus (5418) Google Scholar). As one of the major sources of ROS (2Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4830) Google Scholar), mitochondria are highly susceptible to oxidative damage. ROS can damage mitochondrial enzymes directly (3Lenaz G. Biochim. Biophys. Acta. 1998; 1366: 53-67Crossref PubMed Scopus (569) Google Scholar), and they can cause mutation in mitochondrial DNAs (4Esposito L.A. Melov S. Panov A. Cottrell B.A. Wallace D.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4820-4825Crossref PubMed Scopus (537) Google Scholar). At the same time, ROS can change the mitochondrial transmembrane potential (Δψm), which is indicative of mitochondrial membrane integrity (5Huang P. Feng L. Oldham E.A. Keating M.J. Plunkett W. Nature. 2000; 407: 390-395Crossref PubMed Scopus (749) Google Scholar) and precedes cell death induced by various toxic compounds and cytokines (6Lemasters J.J. Nieminen A.L. Qian T. Trost L.C. Elmore S.P. Nishimura Y. Crowe R.A. Cascio W.E. Bradham C.A. Brenner D.A. Herman B. Biochim. Biophys. Acta. 1998; 1366: 177-196Crossref PubMed Scopus (1227) Google Scholar). Recent reports indicate that mitochondrial ROS cause apoptosis (7Hockenbery D.M. Oltvai Z.N. Yin X.-M. Milliman C.L. Korsmeyer S.J. Cell. 1993; 75: 241-251Abstract Full Text PDF PubMed Scopus (3294) Google Scholar,8Nomura K. Imai H. Koumura T. Arai M. Nakagawa Y. J. Biol. Chem. 1999; 274: 29294-29302Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) by activating various apoptotic effectors such as cytochromec release, procaspase-2, procaspase-9, procaspase-3, and latent apoptosis-inducing factor, which is released from the mitochondria during apoptosis (9Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 10Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Brenner C. Larochette N. Prevost M.C. Alzari P.M. Kroemer G. J. Exp. Med. 1999; 189: 381-394Crossref PubMed Scopus (637) Google Scholar, 11Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3452) Google Scholar). Another report also suggested that mitochondrial ROS directly caused apoptosis of T cells (12Hildeman D.A. Mitchell T. Teague T.K. Henson P. Day B.J. Kappler J. Marrack P.C. Immunity. 1999; 10: 735-744Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). It was also reported that tumor necrosis factor α causes a rapid production of mitochondrial ROS (13Goossens V. Grooten J. De Vos K. Fiers W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8115-8119Crossref PubMed Scopus (556) Google Scholar) and that ceramide, an apoptotic stimulus, also plays a crucial role in tumor necrosis factor α-induced mitochondrial ROS generation (14Garcia-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J.C. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (715) Google Scholar). Furthermore, several other investigators demonstrated that ROS are involved in the signaling pathway of certain growth factors (15Sundaresan M., Yu, Z.-X. Ferrans C.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2314) Google Scholar) and cytokines (16Lo Y.Y.C. Cruz T.F. J. Biol. Chem. 1995; 270: 11727-11730Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar). In addition, mitochondrial ROS, under hypoxic conditions, activate the transcription of the genes for glycolytic enzymes as well as erythropoietin and vascular endothelial growth factor by up-regulating a transcriptional factor, hypoxia-inducible factor 1 (17Semenza G.L. Cell. 1999; 98: 281-284Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), suggesting that mitochondrial ROS mediate cross-talk between the nucleus and the mitochondria. These reports suggest an important role of ROS in the regulation of cellular homeostasis including cell death and signal transduction pathway after treatments with various agents or growth factors. During aerobic respiration to generate ATP in mitochondria, leakage of electrons frequently produces mitochondrial superoxide anions that are rapidly reduced to H2O2 by manganese superoxide dismutase. Because catalase, which metabolizes H2O2, is absent in the mitochondria of most animal cells (18Esworthy R, S. Ho Y.S. Chu F.F. Arch. Biochem. Biophys. 1997; 340: 59-63Crossref PubMed Scopus (134) Google Scholar), mitochondrial glutathione peroxidase plays a key role in metabolizing H2O2. Therefore, reduced glutathione (GSH), an efficient antioxidant and free radical scavenger by itself and required for the activity of mitochondrial glutathione peroxidase, becomes the best defense available against the potential toxicity of H2O2 in the mitochondria. Nevertheless, GSH is known to be synthesized in the cytosol and transported into the mitochondria (19Griffith O.W. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4668-4672Crossref PubMed Scopus (445) Google Scholar) through rapid exchange of GSH between the cytosol and mitochondria (20Martensson J. Lai J.C.K. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7185-7189Crossref PubMed Scopus (263) Google Scholar). In contrast, oxidized glutathione disulfide (GSSG) in the mitochondria cannot be exported into the cytosol (21Olafsdottier K. Reed D.J. Biochim. Biophys. Acta. 1988; 964: 377-382Crossref PubMed Scopus (160) Google Scholar) for reconversion into GSH. These facts underscore the importance of mitochondrial NADPH as a necessary reducing equivalent for the regeneration of GSH from GSSG by the activity of mitochondrial glutathione reductase. Until now, glucose-6-phosphate dehydrogenase (Glu-6-P dehydrogenase) was regarded as the major source of cellular NADPH because it reduces cellular oxidative stress by increasing the GSH concentration (22Salvemini F. Franze A. Iervolino A. Filosa S. Salzano S. Ursini M.V. J. Biol. Chem. 1999; 274: 2750-2757Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Because Glu-6-P dehydrogenase is absent in the mitochondria, the mechanism for maintaining the mitochondrial NADPH pool, crucial to the control of mitochondrial redox balance, remains to be elucidated. In mammals, three classes of isocitrate dehydrogenase (ICDH) isoenzymes exist: mitochondrial NAD+-dependent ICDH (IDH), mitochondrial NADP+-dependent ICDH (IDPm) and cytosolic NADP+-dependent ICDH (IDPc) (23Plaut G.W.E. Gabriel J.L. Biochemistry of Metabolic Process. Elsevier Science Publishing Co., Inc., New York1983: 285-301Google Scholar). Among the eukaryotic ICDH isoenzymes, IDH has been assumed to play a major role in the oxidative decarboxylation of isocitrate in the tricarboxylic acid cycle (24Cupp J.R. McAlister-Henn L. J. Biol. Chem. 1991; 266: 22199-22205Abstract Full Text PDF PubMed Google Scholar). However, the exact roles of IDPm and IDPc, which catalyze decarboxylation of isocitrate into α-ketoglutarate with concurrent production of NADPH in the mitochondria and cytosol, respectively, have not been elucidated. We have reported previously the isolation and molecular characterization of cDNA clones for bovine IDPm (25Huh T.-L. Ryu J.H. Huh J.W. Sung H.C. Oh I.-U. Song B.J. Veech R.L. Biochem. J. 1993; 292: 705-710Crossref PubMed Scopus (35) Google Scholar) and other IDH subunits (26Kim Y.-O. Koh H.-J. Kim S.-H. Jo S.-H. Huh J.-W. Jeong K.-S. Lee J.I. Song B.J. Huh T.-L. J. Biol. Chem. 1999; 274: 36866-36875Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In this study, we investigated the potential role of IDPm in the defense against ROS-induced oxidative damage and cell death. Our study was performed by overproducing the coding region of a cDNA for mouse IDPm followed by measurement of cell death and various indicators of oxidative stress. Reduced expression of IDPm by transfecting the antisense cDNA increased spontaneous production of ROS and lipid peroxidation accompanied by significantly more mitochondrial injury compared with the control cells transfected with vector alone. In contrast, increased expression of IDPm derived from the sense cDNA effectively prevented or reduced ROS-related damage. Our results further provide evidence that ROS-inducible IDPm is a major producer of mitochondrial NADPH, subsequently leading to an increased mitochondrial GSH pool needed for the defense against ROS-mediated oxidative injury. Bovine IDPm cDNA (25Huh T.-L. Ryu J.H. Huh J.W. Sung H.C. Oh I.-U. Song B.J. Veech R.L. Biochem. J. 1993; 292: 705-710Crossref PubMed Scopus (35) Google Scholar) was used as a probe to screen mouse IDPm cDNA from a λ-ZAP II cDNA library of NIH3T3 cells (Stratagene). The largest IDPm cDNA was initially subcloned into the EcoRI site of pGEM7 (Promega). The resultant DNA was digested byApaI, blunt-ended, and then digested further by eitherHindIII or ClaI before the IDPm cDNA was ligated into a LNCX-retroviral vector (27Miller A.D. Rosman G.T. BioTechniques. 1989; 7: 980-990PubMed Google Scholar) in a sense or antisense orientation, respectively. In the LNCX-retroviral vector, expression of sense or antisense IDPm cDNA was directed by the cytomegalovirus promoter. The respective two recombinant IDPm DNA constructs or LNCX-vector alone was transfected into the BOSC23 retroviral packaging cells (28Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2301) Google Scholar) by the calcium phosphate method. The retrovirus particles were separated from the packaging cells by filtration through a sterile filter (0.4-μm diameter) and used to transfect into NIH3T3 cells. Stable NIH3T3 transformants were identified in the presence of G418. NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (Hyclone Laboratories) and 10 μg/ml gentamycin at 37 °C in an incubator under 5% CO2. To prepare IDPm polyclonal antibody, a peptide representing the N-terminal 16 amino acids of mouse IDPm (ADKRIKVAKPVVEMDG) was synthesized with a peptide synthesizer (Excell, Milligene Bioresearch) and purified according to the protocol suggested by the manufacturer. The purified peptide (5 mg) was conjugated by rabbit serum albumin (1 mg) using a kit (Imject, Pierce Chemical Co.) and used to prepare polyclonal anti-peptide antibodies in rabbit. The mitochondrial homogenates from cultured cells were separated on 10% SDS-polyacrylamide gel, transferred to nitrocellulose membranes (Schleicher & Shuell), and subsequently subjected to immunoblot analysis using anti-peptide antibodies. Immunoreactive antigen was then recognized by using horseradish peroxidase-labeled anti-rabbit IgG and an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech). Total RNAs from cultured cells were prepared using RNAzol (Tel-Test Inc., Friendswood, TX) according to the manufacturer's protocol. Total RNA from cultured cells was separated by electrophoresis on 0.66 m formamide, 1% agarose gels, transferred to GeneScreen membranes, and hybridized with32P-labeled mouse IDPm cDNA as a probe. A membrane for human or mouse multiple tissue Northern blot (CLONTECH) was hybridized with32P-labeled DNA probe. Hybridization and subsequent procedures were the same as those described previously (26Kim Y.-O. Koh H.-J. Kim S.-H. Jo S.-H. Huh J.-W. Jeong K.-S. Lee J.I. Song B.J. Huh T.-L. J. Biol. Chem. 1999; 274: 36866-36875Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Mitochondrial pellets (25Huh T.-L. Ryu J.H. Huh J.W. Sung H.C. Oh I.-U. Song B.J. Veech R.L. Biochem. J. 1993; 292: 705-710Crossref PubMed Scopus (35) Google Scholar) prepared from cultured cells were resuspended in 1 × phosphate-buffered saline containing 0.1% Triton X-100, disrupted by sonication (4710 series, Cole-Palmer) twice at 40% of the maximum setting for 10 s, and centrifuged at 15,000 × gfor 30 min. The supernatants were used to measure the activities of several mitochondrial enzymes. Activities of IDH and IDPm were measured by the production of NADH (26Kim Y.-O. Koh H.-J. Kim S.-H. Jo S.-H. Huh J.-W. Jeong K.-S. Lee J.I. Song B.J. Huh T.-L. J. Biol. Chem. 1999; 274: 36866-36875Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and NADPH (29Loverde A.W. Lehrer G.M. J. Neurochem. 1973; 20: 441-448Crossref PubMed Scopus (33) Google Scholar), respectively, at 340 nm at 25 °C. 1 unit of IDPm activity is defined as the amount of enzyme catalyzing the production of 1 μmol of NADPH/min. Activities for manganese superoxide dismutase, mitochondrial glutathione reductase, and mitochondrial glutathione peroxidase were determined by published methods (30Marklund S.L. Marklund G. Eur. J. Biochem. 1974; 47: 469-474Crossref PubMed Scopus (8071) Google Scholar, 31Pinto R.E. Bartley W. Biochem. J. 1969; 112: 109-115Crossref PubMed Scopus (350) Google Scholar). Activities for Glu-6-P dehydrogenase and catalase were analyzed by the methods described (22Salvemini F. Franze A. Iervolino A. Filosa S. Salzano S. Ursini M.V. J. Biol. Chem. 1999; 274: 2750-2757Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 32Aebi H. Method. Enzymol. 1984; 105: 121-126Crossref PubMed Scopus (18681) Google Scholar). Cells (2 × 104) were grown until 80% confluence in 96-well plates, and cell viability after treatment with H2O2 was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (33Matteo M.A. Loweth A.C. Thomas S. Mabley J.G. Apoptosis. 1997; 2: 164-177Crossref PubMed Scopus (53) Google Scholar). After the cells were treated for 48 h with various concentrations of H2O2, 50 μl of MTT (2 mg/ml, Sigma) solution was added and incubated for another 4 h at 37 °C. The MTT solution was discarded by aspiration, and the resulting formazan product converted by the viable cells was dissolved in 150 μl of dimethyl sulfoxide. The absorbance at 540 nm with a 620 nm reference was read with an enzyme-linked immunosorbent assay plate reader. Cell viability was expressed as a percentage of untreated control cells. For analyses of DNA fragmentation, cells exposed to different concentrations of H2O2 for 1 h were lysed in NTE buffer, pH 8.0 (100 mm NaCl, 10 mm Tris, 1 mm EDTA) containing 1% SDS and proteinase K (0.2 mg/ml). DNA extraction and purification were performed by the method described by Bernhard et al. (34Bernhard C.T. Klas C. Peters A.M.J. Matzku S. Moller P. Falk W. Debatin K.-M. Krammer P.H. Science. 1989; 245: 301-304Crossref PubMed Scopus (1667) Google Scholar). To analyze the degree of DNA fragmentation, 5 μg of each DNA sample was resolved on 1% agarose gel and visualized under UV illumination. Total peroxide concentrations were calculated by the rate of oxidation of ferrous (Fe2+) to ferric ion (Fe3+) (35Jiang Z.Y. Hunt J.V. Wolff S.P. Anal. Biochem. 1992; 202: 384-389Crossref PubMed Scopus (1462) Google Scholar). Cells (2 × 106) were either untreated or pretreated with 0.1 mm H2O2 for 1 h. Cell extracts were incubated with the reaction mixture (0.1 mm xylenol orange, 0.25 mm ammonium ferrous sulfate, 100 mm sorbitol, and 25 mmH2SO4) at 22 °C for 30 min prior to measurement of the absorbance at 560 nm. H2O2(0–5 μm) was used to produce a standard curve. Cells (1 × 106) were grown on poly-l-lysine-coated slide glasses and untreated or treated with 1.0 mm H2O2 for 5 min. Intracellular ROS generation was monitored by the fluorescence produced from 2′,7′-dichlorofluorescein (DCF) after oxidation of 10 μm dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR) (36Bass D.A. Parce J.W. Dechatelet L.R. Szejda P. Seeds M.C. Thomas M. J. Immunol. 1983; 130: 1910-1917PubMed Google Scholar). Images of DCF fluorescence (excitation, 488 nm; emission, 520 nm) were acquired using a laser confocal scanning microscope (DM/R-TCS, Leica) coupled to a microscope (Leitz DM RBE). To measure the fluorescence intensity, 20 cells from each image were picked randomly, and their averages of fluorescence intensity were calculated as described (15Sundaresan M., Yu, Z.-X. Ferrans C.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2314) Google Scholar). For FACS analyses, cells (2 × 106) were pretreated with 5 μmdichlorodihydrofluorescein diacetate and followed by exposure to 30 μm C2-ceramide (N-acetyl-d-sphingosine, Sigma) for 15 min. Measurements of DCF fluorescence in trypsin-treated cells were made at least 10,000 events/test using a FACS Calibar flow cytometer (Becton Dickinson) with a fluorescein isothiocyanate filter. For measuring lipid peroxidation, cells (2 × 106) were either untreated or pretreated with 0.1 mm H2O2 for 1 h and analyzed by measuring of the concentration of malondialdehyde (MDA). The concentration of MDA in different cells was measured by a spectrophotometric assay (37Buege J.A. Aust S.D. Methods Enzymol. 1978; 52: 302-310Crossref PubMed Scopus (10824) Google Scholar). Cells (2 × 106) were either untreated or pretreated with 0.1 mm H2O2 for 1 h. Then cell extracts (500 μl) were mixed with 1 ml of thiobarbituric acid-trichloroacetic acid-HCl solution (0.375% thiobarbituric acid, trichloroacetic acid in 0.25 n HCl, pH 2.0) and heated at 100 °C for 15 min. The absorbance of thiobarbituric acid-reactive substance was determined at 535 nm. Cells grown to 80% confluence were either untreated or pretreated with 0.1 mmH2O2 for 2 h, rinsed twice with phosphate-buffered saline, pH 7.3, and centrifuged at 50 ×g for 5 min. Cell pellets were fixed immediately in 2.5% (v/v) glutaraldehyde in 0.1 m phosphate buffer for 2 h at 4 °C. Cells were postfixed in 1% osmium tetroxide for 30 min, washed with water, and then subjected to a dehydration procedure using graded ethanol series. For preparing the specimen, cells were embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, PA), and two random areas were cut and processed. The sections (60–70 nm) were cut with an ultramicrotome (Soya MT-7000), transferred to copper grids, and stained with uranyl acetate and lead citrate. At least 40 cells of each sample were examined and photographed using Hitachi H-7100 transmission electron microscope (Hitachi Co., Japan) at 75 kV. Intracellular ATP levels were determined by using luciferin-luciferase (38Spragg R.G. Hinshaw D.B. Hyslop P.A. Schraufstätter I.U. Cochrane C.G. J. Clin. Invest. 1985; 76: 1471-1476Crossref PubMed Scopus (204) Google Scholar). Cells (5 × 106) either untreated or treated with 0.1 mmH2O2 for 2 h were collected by centrifugation, resuspended in 250 μl of extraction solution (10 mm KH2PO4, 4 mmMgSO4, pH 7.4), heated at 98 °C for 4 min, and placed on ice. For ATP measurement, an aliquot of a 50-μl sample was added to 100 μl of reaction solution (50 mm NaAsO2, 20 mm MgSO4, pH 7.4) containing 800 μg of luciferin/luciferase (Sigma). Light emission was quantified in a Turner Designs TD 20/20 luminometer (Stratec Biomedical Systems, Germany). For all experiments, ATP standard curves were run and were linear in the range of 5–2500 nm. Concentrations of ATP stock solution were calculated from spectrophotometric absorbance at 259 nm using an extinction coefficient of 15,400. NADPH values were determined by the method of Zerez et al. (39Zerez C.R. Lee S.J. Tanaka K.R. Anal. Biochem. 1987; 1964: 367-373Crossref Scopus (157) Google Scholar) and expressed as the ratio of NADPH to the total NADP pool [NADPH]/[NADP+ + NADPH]. The GSH level was analyzed by producing of 5-thio-2-nitrobenzoate at 412 nm (ε= 1.36 × 104m−1 cm−1) by the method described previously (40Akerboom T.P.M. Sies H. Methods Enzymol. 1981; 77: 373-382Crossref PubMed Scopus (1464) Google Scholar). Total GSH level was measured in 0.1 m potassium phosphate buffer (pH 7.0) containing 1 mm EDTA, 0.2 mg of NADPH, 30 μg of 5,5′-dithio-bis(2-nitrobenzoic acid), and 0.12 unit of glutathione reductase (Sigma). The GSSG level was measured as the same as total the GSH level after treatment with 1 μl of 2-vinylpyridine and 3 μl of triethanolamine for 1 h (41Anderson M.E. Methods Enzymol. 1985; 113: 548-555Crossref PubMed Scopus (2421) Google Scholar). To isolate cDNAs for mouse IDPm, a cDNA library of NIH3T3 cells (Stratagene) was screened with the cDNA for bovine IDPm (25Huh T.-L. Ryu J.H. Huh J.W. Sung H.C. Oh I.-U. Song B.J. Veech R.L. Biochem. J. 1993; 292: 705-710Crossref PubMed Scopus (35) Google Scholar) as a probe. 11 positive cDNA clones for mouse IDPm were isolated from about two million phage plaques screened. From these clones, one clone with the largest DNA insert (1.7 kilobase pairs) was purified, subcloned into plasmid pGEM7(+), and its nucleotide sequence was determined. Mouse IDPm cDNA was 1,679 base pairs long (data not shown) with an open reading frame (1,356 base pairs) for the entire protein coding region of IDPm (Fig. 1). Structural analysis of the pig (42Haselbeck R.J. Colman R.F. McAlister-Henn L. Biochemistry. 1992; 31: 6219-6223Crossref PubMed Scopus (45) Google Scholar), bovine, and mouse IDPm (25Huh T.-L. Ryu J.H. Huh J.W. Sung H.C. Oh I.-U. Song B.J. Veech R.L. Biochem. J. 1993; 292: 705-710Crossref PubMed Scopus (35) Google Scholar) revealed that the precursor mouse IDPm protein contains 452 amino acids (50,934 Da), and the complete protein consists of 413 amino acids (46,575 Da) with the first 39 amino acids as the mitochondrial signal peptide. The deduced protein sequence of mouse IDPm showed 94.5 and 95% identity to that of bovine and porcine IDPm, respectively. However, the mitochondrial leader sequence of the mouse IDPm was quite different from the previously reported mouse IDPm (mNADP-IDH) (43Yang L. Luo H. Vinay P. Wu J. J. Cell. Biochem. 1996; 60: 400-410Crossref PubMed Scopus (15) Google Scholar). The mNADP-IDH contained an extremely long mitochondrial leader peptide (111 amino acids), and its mature protein sequence (412 amino acids) was 1 amino acid shorter than that of our clone for mouse IDPm. In addition, 11 amino acids in its mature protein sequence are different from that of our mouse IDPm (Fig. 1). To investigate the expression pattern of IDPm in different human and mouse tissues, Northern analyses were performed. One major IDPm transcript (2.2 kilobase pairs) was observed in both human and mouse tissues and expressed in a tissue-specific manner (Fig.2 A). The levels of IDPm expressed in human and mouse tissues were highest in heart, one of the most O2-consuming tissues, whereas liver and kidney contained considerable levels of IDPm transcript but significantly less than that in heart. In contrast, other tissues including brain and lung, which are vulnerable to oxidative injury, contained very low levels of IDPm message. Interestingly, the levels of IDPm expression in human and mouse skeletal muscles were strikingly different. To investigate the role of IDPm directly, two different transformants for each recombinant IDPm construct were isolated after stable transfection of the sense IDPm (S1 and S2) and antisense IDPm (AS1 and AS2) or LNCX-vector alone (control) (Fig. 2 B). Chromosomal integration of the transfected IDPm constructs was confirmed by polymerase chain reaction (data not shown). The level of IDPm transcript (2.2 kilobase pairs) in control cells was very low (Fig. 2 C). S1 cells contained much less viral IDPm transcript (2.8 kilobase pairs) than S2 cells. Both AS1 and AS2 cells contained substantially less IDPm transcript than S1 cells (Fig. 2 C). S1 and S2 cells exhibited 52.6 ± 5.1 and 66.7 ± 5.5 units of IDPm activities, respectively. These values are 3.5- and 4.5-fold higher, respectively, than that of control cells with the vector alone. In contrast, AS1 and AS2 cells exhibited 39 and 47% less IDPm activities, respectively, compared with that of control (Table I). To demonstrate any differences in ROS-mediated damage between cells with sense or antisense IDPm, we intentionally chose to use S1 and AS1 cells as a comparison pair because of less difference in IDPm activity in this pair than in the paired S2 and AS2 cells. Immunoblot analysis using anti-IDPm antibody further confirmed the increased expression of IDPm in S1 cells compared with the control cells and AS1 cells that contained significantly less expression of IDPm protein (Fig.2 D). However, immunoreactive IDPm was not detected in the cytosol of S1, AS1, or control cells (data not shown). Activities of mitochondrial IDH and other major antioxidant enzymes such as mitochondrial glutathione peroxidase, mitochondrial glutathione reductase, and manganese superoxide dismutase, were all similar in each group comparable to the control (Table I). In addition, there was less difference in Glu-6-P dehydrogenase and catalase activities in the cell lysates of S1, AS1, and control cells, suggesting that transfection of IDPm cDNAs did not affect the activities of other enzymes involved in antioxidation.Table IAntioxidant enzyme activities in NIH3T3 transfectant cellsCell linesIDPm 1-aEnzyme activities measured from mitochondrial fractions.1-bEnzyme activity represents units/g protein.mGPx 1-aEnzyme activities measured from mitochondrial fractions.1-bEnzyme activity represents units/g protein.mGRd 1-aEnzyme activities measured from mitochondrial fractions.1-cEnzyme activity represents units/mg protein.Mn-SOD 1-aEnzyme activities measured from mitochondrial fractions.1-cEnzyme activity represents units/mg protein.IDH 1-aEnzyme activities measured from mitochondrial fractions.1-bEnzyme activity represents units/g protein.G6PD 1-bEnzyme activity represents units/g protein.1-dEnzyme activities measured from total cell lysates.Catalase 1-cEnzyme activity represents units/mg protein.1-dEnzyme activities measured from total cell lysates.Vector14.9 ± 1.141.5 ± 1.37.11 ± 0.41.50 ± 0.16.07 ± 1.445.7 ± 2.43.5 ± 0.2S1 (sense IDPm)52.6 ± 5.138.6 ± 0.17.30 ± 0.11.48 ± 0.16.16 ± 0.845.2 ± 1.53.4 ± 0.2(66.7 ± 5.5) 1-eIDPm activities for S2 and AS2 cells are indicated in parentheses.AS1 (antisense IDPm)9.1 ± 1.539.1 ± 2.07.12 ± 0.11.50 ± 0.16.03 ± 0.144.8 ± 1.53.6 ± 0.3(7.9 ± 0.6) 1-eIDPm activities for S2 and AS2 cells are indicated in parentheses.Values represent means ± S.D. of three independent" @default.
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