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• W2017363944 abstract "Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. We investigated whether the ICDH would be a vulnerable target of peroxynitrite anion (ONOO-) as a purified enzyme, in intact cells, and in liver mitochondria from ethanol-fed rats. Synthetic peroxynitrite and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a peroxynitrite-generating compound, inactivated ICDH in a dose- and time-dependent manner. The inactivation of ICDH by peroxynitrite or SIN-1 was reversed by dithiothreitol. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. Immunoblotting analysis of peroxynitrite-modified ICDH indicates that S-nitrosylation of cysteine and nitration of tyrosine residues are the predominant modifications. Using electrospray ionization mass spectrometry (ESI-MS) with tryptic digestion of protein, we found that peroxynitrite forms S-nitrosothiol adducts on Cys305 and Cys387 of ICDH. Nitration of Tyr280 was also identified, however, this modification did not significantly affect the activity of ICDH. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by peroxynitrite. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and binding of the hydrophobic probe 8-anilino-1-napthalene sulfonic acid. When U937 cells were incubated with 100 μm SIN-1 bolus, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitation and ESI-MS, we were also able to isolate and positively identify S-nitrosylated and nitrated mitochondrial ICDH from SIN-1-treated U937 cells as well as liver from ethanol-fed rats. Inactivation of ICDH resulted in the pro-oxidant state of cells reflected by an increased level of intracellular reactive oxygen species, a decrease in the ratio of [NADPH]/[NADPH + NADP+], and a decrease in the efficiency of reduced glutathione turnover. The peroxynitrite-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. We investigated whether the ICDH would be a vulnerable target of peroxynitrite anion (ONOO-) as a purified enzyme, in intact cells, and in liver mitochondria from ethanol-fed rats. Synthetic peroxynitrite and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a peroxynitrite-generating compound, inactivated ICDH in a dose- and time-dependent manner. The inactivation of ICDH by peroxynitrite or SIN-1 was reversed by dithiothreitol. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. Immunoblotting analysis of peroxynitrite-modified ICDH indicates that S-nitrosylation of cysteine and nitration of tyrosine residues are the predominant modifications. Using electrospray ionization mass spectrometry (ESI-MS) with tryptic digestion of protein, we found that peroxynitrite forms S-nitrosothiol adducts on Cys305 and Cys387 of ICDH. Nitration of Tyr280 was also identified, however, this modification did not significantly affect the activity of ICDH. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by peroxynitrite. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and binding of the hydrophobic probe 8-anilino-1-napthalene sulfonic acid. When U937 cells were incubated with 100 μm SIN-1 bolus, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitation and ESI-MS, we were also able to isolate and positively identify S-nitrosylated and nitrated mitochondrial ICDH from SIN-1-treated U937 cells as well as liver from ethanol-fed rats. Inactivation of ICDH resulted in the pro-oxidant state of cells reflected by an increased level of intracellular reactive oxygen species, a decrease in the ratio of [NADPH]/[NADPH + NADP+], and a decrease in the efficiency of reduced glutathione turnover. The peroxynitrite-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition. Peroxynitrite anion (ONOO-) is a potent oxidant generated from the interaction of nitric oxide (NO) and superoxide (O2.¯) (1Beckman J.S. Beckman T.W. Chen J. Marshall M.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6647) Google Scholar, 2Padjama S. Huie R.E. Biochem. Biophys. Res. Commun. 1993; 195: 539-544Crossref PubMed Scopus (272) Google Scholar, 3Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722PubMed Google Scholar). At physiological concentrations, NO is the only known biological molecule that can out-compete endogenous superoxide dismutase (SOD) 1The abbreviations used are: SODsuperoxide dismutaseROSreactive oxygen speciesICDHNADP+-dependent isocitrate dehydrogenaseDTTdithiothreitolDTNB5,5′-dithiobis(2-nitrobenzoic acid)ANSA8-anilino-1-naphthalenesulfonic acidDCFH-DA2′,7′-dichlorofluorescein diacetateDCF2′,7′-dichlorofluoresceinSIN-13-morpholinosydnomine N-ethylcarbamideGSTglutathione S-transferaseIANBDN,N′-dimethyl-N(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyleneamineESI-MSelectrospray ionization mass spectrometryCDcircular dichroismiNOSinducible nitric-oxide synthase.1The abbreviations used are: SODsuperoxide dismutaseROSreactive oxygen speciesICDHNADP+-dependent isocitrate dehydrogenaseDTTdithiothreitolDTNB5,5′-dithiobis(2-nitrobenzoic acid)ANSA8-anilino-1-naphthalenesulfonic acidDCFH-DA2′,7′-dichlorofluorescein diacetateDCF2′,7′-dichlorofluoresceinSIN-13-morpholinosydnomine N-ethylcarbamideGSTglutathione S-transferaseIANBDN,N′-dimethyl-N(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyleneamineESI-MSelectrospray ionization mass spectrometryCDcircular dichroismiNOSinducible nitric-oxide synthase. for available O2.¯ (4Beckman J.S. Carson M. Smith C.D. Koppenol W.H. Nature. 1993; 364: 584Crossref PubMed Scopus (784) Google Scholar), and formation of ONOO- can count for both O2.¯ and NO-dependent toxicities (3Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722PubMed Google Scholar). The in vivo formation of this compound has recently been demonstrated in endothelial cells, Kupffer cells, neutrophils, neurons, macrophages, and other cellular systems (5Ischiropoulos H. Zhu L. Beckman J.S. Arch. Biochem. Biophys. 1992; 298: 446-451Crossref PubMed Scopus (1084) Google Scholar, 6Carreras M.C. Pargament G.A. Catz C.D. Poderosa J.J. Boveris A. FEBS Lett. 1994; 341: 65-68Crossref PubMed Scopus (334) Google Scholar, 7Kooy N.W. Royall J.A. Arch. Biochem. Biophys. 1994; 310: 352-359Crossref PubMed Scopus (241) Google Scholar, 8Wang P. Zweier J.L. J. Biol. Chem. 1996; 271: 29223-29230Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, 9Huhmer A.F. Gerber N.C. de Montellano P.R. Schoneich C. Chem. Res. Toxicol. 1996; 9: 484-491Crossref PubMed Scopus (50) Google Scholar). Peroxynitrite is a relatively stable species, but its protonated form decays with a rate constant of 1.3 s-1 at 25 °C (10Koppenol W.H. Moreno J.J. Pryor W.A. Ischiropoulos H. Beckman J.S. Chem. Res. Toxicol. 1992; 5: 834-842Crossref PubMed Scopus (1269) Google Scholar). Peroxynitrite reacts with a diverse array of other biological target molecules, including cysteine, tyrosine, methionine, and tryptophan residues of proteins (11Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar, 12Beckman J.S. Crow C.P. Biochem. Soc. Trans. 1993; 21: 330-334Crossref PubMed Scopus (630) Google Scholar, 13Beckman J.S. Chen J. Ischiropoulos H. Crow C.P. Methods Enzymol. 1994; 233: 229-240Crossref PubMed Scopus (961) Google Scholar). Peroxynitrite has been demonstrated to readily oxidize or nitrate various enzymes such as metalloproteinase-1 inhibitor, alcohol dehydrogenase, aconitase, xanthine oxidase, cytosolic glyceraldehyde-3-phosphate dehydrogenase, glutamine synthetase, creatine kinase, and succinate dehydrogenase (14Frears E.R. Zhang Z Black D.R. O'Connell J.P. Winyard P.G. FEBS Lett. 1996; 381: 21-24Crossref PubMed Scopus (148) Google Scholar, 15Crow C.P. Beckman J.S. McCord J.M. Biochemistry. 1995; 34: 3544-3552Crossref PubMed Scopus (237) Google Scholar, 16Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 17Houston M. Chumley P. Radi R. Rubbo H. Freeman B.A. Arch. Biochem. Biophys. 1998; 355: 1-8Crossref PubMed Scopus (56) Google Scholar, 18Souza J.M. Radi R. Arch. Biochem. Biophys. 1998; 360: 187-194Crossref PubMed Scopus (143) Google Scholar, 19Berlett B.S. Friguet B Yim M.B. Chock P.B. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1776-1780Crossref PubMed Scopus (164) Google Scholar, 20Konorev E.A. Hogg N. Kalyanaraman B. FEBS Lett. 1998; 427: 171-174Crossref PubMed Scopus (134) Google Scholar, 21Rubbo H. Denicola A. Radi R. Arch. Biochem. Biophys. 1994; 308: 96-102Crossref PubMed Scopus (110) Google Scholar). superoxide dismutase reactive oxygen species NADP+-dependent isocitrate dehydrogenase dithiothreitol 5,5′-dithiobis(2-nitrobenzoic acid) 8-anilino-1-naphthalenesulfonic acid 2′,7′-dichlorofluorescein diacetate 2′,7′-dichlorofluorescein 3-morpholinosydnomine N-ethylcarbamide glutathione S-transferase N,N′-dimethyl-N(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyleneamine electrospray ionization mass spectrometry circular dichroism inducible nitric-oxide synthase. superoxide dismutase reactive oxygen species NADP+-dependent isocitrate dehydrogenase dithiothreitol 5,5′-dithiobis(2-nitrobenzoic acid) 8-anilino-1-naphthalenesulfonic acid 2′,7′-dichlorofluorescein diacetate 2′,7′-dichlorofluorescein 3-morpholinosydnomine N-ethylcarbamide glutathione S-transferase N,N′-dimethyl-N(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyleneamine electrospray ionization mass spectrometry circular dichroism inducible nitric-oxide synthase. Antioxidant enzymes, which provide a substantial defense against damage induced by reactive oxygen species (ROS), could be susceptible to the damaging effect of peroxynitrite. It has been shown that glutathione peroxidase, manganese SOD (Mn-SOD), and glutathione reductase are inactivated by peroxynitrite (22Padmaja S. Squadrito G.L. Pryor W.A. Arch. Biochem. Biophys. 1998; 349: 1-6Crossref PubMed Scopus (108) Google Scholar, 23MacMillan-Crow L.A. Crow J.P. Kerby J.D. Beckman J.S. Thompson J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11853-11858Crossref PubMed Scopus (715) Google Scholar, 24Savvides S.N. Scheiwein M. Bohme C.C. Arteel G.E. Karplus P.A. Becker K. Schirmer R.H. J. Biol. Chem. 2002; 277: 2779-2784Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It is implied that the inactivation of antioxidant enzymes by peroxynitrite may lead to the perturbation of the cellular antioxidant defense system and subsequently exacerbate the harmful effect of peroxynitrite as well as ROS. The isocitrate dehydrogenases (ICDHs, EC1.1.1.41 and EC1.1.1.42) catalyze oxidative decarboxylation of isocitrate to α-ketoglutarate and require either NAD+ or NADP+, producing NADH and NADPH, respectively (25Koshland Jr., D.E. Walsh K. LaPorte D.C. Curr. Top. Cell Regul. 1985; 27: 13-22Crossref PubMed Scopus (59) Google Scholar). NADPH is an essential reducing equivalent for the regeneration of reduced glutathione (GSH) by glutathione reductase and for the activity of NADPH-dependent thioredoxin system (26Chae H.Z. Chung S.J. Rhee S.G. J. Biol. Chem. 1994; 269: 27670-27678Abstract Full Text PDF PubMed Google Scholar, 27Kwon S.J. Park J.-W. Choi W.K. Kim I.H. Kim K. Biochem. Biophys. Res. Commun. 1994; 201: 8-15Crossref PubMed Scopus (100) Google Scholar), both are important in the protection of cells from oxidative damage. Therefore, ICDH may play an antioxidant role during oxidative stress. We recently reported that ICDH is involved in the supply of NADPH needed for GSH production against cytosolic and mitochondrial oxidative damage (28Lee S.M. Koh H.J. Park D.C. Song B.J. Huh T.L. Park J.-W. Free Radic. Biol. Med. 2002; 32: 1185-1196Crossref PubMed Scopus (333) Google Scholar, 29Jo S.-H. Son M.-K. Koh H.-J. Lee S.-M. Song I.-H. Kim Y.-O. Lee Y.S. Jeong K.-S. Kim W.B. Park J.-W. Song B.J. Huh T.-L. J. Biol. Chem. 2001; 276: 16168-16176Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Hence, the damage of ICDH may result in the perturbation of the balance between oxidants and antioxidants and subsequently lead to a pro-oxidant condition. Because cysteine residues serve as an essential role in the catalytic function of ICDH (30Smyth G.E. Colman R.F. J. Biol. Chem. 1991; 266: 14918-14925Abstract Full Text PDF PubMed Google Scholar, 31Fatania H.R. Al-Nassar K.E. Thomas N. FEBS Lett. 1993; 322: 245-248Crossref PubMed Scopus (20) Google Scholar), the highly reactive sulfhydryl groups in ICDH could be potential targets of peroxynitrite. In this study we show that peroxynitrite modifies tyrosine and thiol groups of ICDH, thus forming nitrotyrosine and S-nitrosothiol adducts with a concomitant loss of its activity in vitro and in vivo. Materials—Isocitrate, β-NADP+, NADPH, mitochondrial ICDH from pig heart, GSH, cysteine, dithiothreitol (DTT), methionine, penicillamine, 2-mercaptoethanol, ebselen, selenomethionine, selenocysteine, human serum albumin, 5′-dithiobis-(2-nitrobenzoate) (DTNB), xylenol orange, o-phthaldehyde, lucigenin, bovine erythrocyte SOD, Pronase, trypsin, and 8-anilino-1-naphthalene sulfonic acid (ANSA) were purchased from Sigma Chemical Co. (St. Louis, MO). 3-Morpholinosydnomine N-ethylcarbamide (SIN-1), anti-nitrotyrosine antibody, anti-nitrosocysteine antibody, and anti-inducible nitric-oxide synthase (iNOS) antibody were purchased from Calbiochem (La Jolla, CA). 2′,7′-Dichlorofluoroscein diacetate (DCFH-DA) and N,N′-dimethyl-N(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethyleneamine (IANBD) were obtained from Molecular Probes (Eugene, OR). Electrophoreses reagents and Bio-Rad protein assay kit were purchased from Bio-Rad (Hercules, CA). Peroxynitrite was synthesized from hydrogen peroxide and isoamyl nitrite as described previously (32Uppu R.M. Pryor W.A. Anal. Biochem. 1996; 236: 242-249Crossref Scopus (224) Google Scholar). Contaminating hydrogen peroxide was eliminated with manganese dioxide, and peroxynitrite concentration was determined at 302 nm (ϵ = 1.67 mm-1 cm-1). To prepare recombinant cytosolic ICDH, Escherichia coli transformed with pGEX-2λT containing an insert of mouse cytosolic ICDH cDNA construct was grown and lysed, and the glutathione S-transferase (GST) fusion protein was purified on glutathione-agarose as described elsewhere (33Smith D.B. Johnson K.S. Gene (Amst.). 1988; 15: 31-40Crossref Scopus (5028) Google Scholar). Antibody against mitochondrial ICDH was prepared from mitochondrial ICDH-immunized rabbit, and the antibody was purified by Protein A affinity chromatography. Cell Culture—U937, a human histiocytic lymphoma cell line was purchased from the American Type Culture Collection and maintained in RPMI 1640 and Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate, respectively. These cells were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Animals and Treatment—Young adult male Sprague-Dawley rats weighing 200-250 g were randomly separated into two groups: (a) rats fed ethanol-containing liquid diets for 30 days (n = 5); (b) rats pair-fed isocaloric liquid diets without ethanol for 30 days (n = 5). They were housed in individual cages under conditions of constant temperature (22 °C) and humidity. The calorie distribution of liquid diets component is as follows: 16% as protein, 36% as fat, 13% as carbohydrate, and 33% as either ethanol or additional carbohydrate in the isocaloric liquid diets. Ethanol was incorporated into the liquid diets containing all required nutrients, and liquid diets were the only source of fluid and food provided, as recommended by the manufacturer. Rats given isocaloric liquid diets without ethanol were pair-fed daily on an isoenergetic basis with the corresponding littermates fed the ethanol-containing diets. Before sacrifice, the rats were fasted overnight but had free access to drinking water for 14-16 h. Preparation of Liver Mitochondrial Fraction—Rats were deeply anesthetized by intraperitoneal injection of pentobarbital sodium (1 mg/kg body weight), and the liver was removed and dissected for biochemical analysis. For the preparation of mitochondria sample from liver tissue, the tissue portions were homogenized with a Dounce homogenizer in sucrose buffer (0.32 m sucrose, 10 mm Tris-Cl, pH 7.4). The mitochondrial fractions from tissue homogenates were prepared as described below. Measurement of ICDH Activity—ICDH (6.5 μg) was added to 1 ml of Tris buffer, pH 7.4, containing NADP+ (2 mm), MgCl2 (2 mm), and isocitrate (5 mm). Activity of ICDH was measured by the production of NADPH at 340 nm at 25 °C (34Loverde A.W. Lehrer G.M. J. Neurochem. 1973; 20: 441-448Crossref PubMed Scopus (33) Google Scholar). One unit of ICDH activity is defined as the amount of enzyme catalyzing the production of 1 μmol of NADPH/min. For the determination of ICDH activities in mammalian cells, cells were collected at 1,000 × g for 10 min at 4 °C and were washed once with cold PBS. Briefly, cells were homogenized with a Dounce homogenizer in sucrose buffer (0.32 m sucrose, 10 mm Tris-Cl, pH 7.4). Cell homogenates were centrifuged at 1,000 × g for 5 min, and the supernatants further centrifuged at 15,000 × g for 30 min. The resulting supernatants were used as a cytosolic fraction to measure the activity of cytosolic ICDH. The precipitates were washed twice with sucrose buffer to collect mitochondria pellet. The mitochondrial pellets were resuspended in 1× PBS containing 0.1% Triton X-100, disrupted by ultrasonication (4710 Series, Cole-Palmer, Chicago, IL) twice at 40% of maximum setting for 10 s, and centrifuged at 15,000 × g for 30 min. The supernatants were used to measure the activity of mitochondrial ICDH. The protein levels were determined by the method of Bradford using reagents purchased from Bio-Rad. Reversibility—ICDH was exposed to peroxynitrite or SIN-1 and then passed through Econo-pac 10 DG gel filtration column (Bio-Rad). The enzyme was incubated with 20 mm DTT at 37 °C. The percentage of ICDH activity recovered after inactivation was calculated. Titration of Sulfhydryl Groups and Competitive Labeling—The thiol groups of ICDH were titrated in 50 mm Tris, pH 8.0/0.5, mm EDTA/1 mm DTNB. A molar coefficient of 1.36 × 104m-1cm-1 for the anion thionitrobenzoic acid was used. Peroxynitrite-, SIN-1-treated, and untreated ICDH (50 μg) were labeled with 10-fold molar excess of IANBD (150 μm) in a total volume of 100 μl of buffer (20 mm Tris-buffer, pH 7.4, containing 100 mm NaCl). The reaction was allowed to proceed for 1 h at room temperature in the dark and was quenched by addition of 1 mm cysteine. Cysteine-reacted dye was removed by extensive dialysis against labeling buffer. IANBD-labeled samples were excited at 481 nm, and emission was monitored between 490 and 625 nm. Each recorded spectrum was corrected for background fluorescence of the relevant control. Immunoblot Analysis—Proteins were separated on 10% SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and subsequently subjected to immunoblot analysis using appropriate antibodies. Immunoreactive antigen was then recognized by using horseradish peroxidase-labeled anti-rabbit IgG and an enhanced chemiluminescence detection kit (Amersham Biosciences). Immunoprecipitation—Mitochondrial fractions were cleared with Protein-A-Sepharose (Amersham Biosciences) for 1 h at 4 °C. Supernatants were then incubated with rabbit polyclonal anti-mitochondrial ICDH (5 μg) for 12 h at 4 °C followed by protein-A-Sepharose incubation for 1 h at 4 °C. Immunoprecipitated proteins were washed, separated by SDS-PAGE, and visualized by staining with Coomassie Blue. For mass spectrometry, the immunoprecipitated proteins were washed and dissolved in 20 μl of 0.5% trifluoroacetic acid (v/v). Determination of Nitrotyrosine—Nitrotyrosine content of ICDH treated with peroxynitrite or SIN-1 was continually monitored from 250 to 500 nm using a Shimadzu UV-visible spectrophotometer. Nitrotyrosine-containing proteins were detected by incubating blots with a mouse monoclonal anti-nitrotyrosine antibody as described above. Mass Spectrometry—Positive ion electrospray ionization mass spectrometry (ESI-MS) was performed on HP 1100 Series LC/MSD triple-quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA) equipped with an atmospheric pressure ion source. Control and peroxynitrite (10 μm, 10 min)-treated ICDH samples were subjected to gel filtration and mixed with 0.1% trifluoroacetic acid. Aliquots of ICDH samples (5 μg of protein) were directly infused into the ESI source of the mass spectrometer. For the tryptic digestion and peptide mapping, ICDH samples treated with peroxynitrite were denatured in the 95 °C water for 20 min and cleaved with trypsin for 24 h at 37 °C, at an enzyme/substrate ratio of 1/10 (w/w). Positive ion mass spectra were acquired for capillary LC/MS analyses. The effluent from octadecyl silica gel (C-18) reverse-phase column (5 mm; 4.6 × 250 mm) (Beckman Coulter Inc., Fullerton, CA) was introduced directly into the ionization needle of the mass spectrometer. Solvent A was 0.1% trifluoroacetic acid in ultra pure water, and solvent B was 0.1% trifluoroacetic acid in acetonitrile. Peptides were eluted using an increasing linear gradient of solvent B from 0-60% in 60 min, 60-100% in 40 min, with a flow rate of 1 ml/min. The molecular masses of fractionated peptide fragments were examined by ESI-MS, and the amino acid sequences were assigned using the data obtained. Structural Analysis—For circular dichroism (CD) spectroscopy, samples of ICDH were desalted on Econo-Pac 10 DG column (Bio-Rad) equilibrated in 20 mm Tris buffer, pH 7.4, and fractions containing the protein were pooled. CD spectra were recorded on a temperature-controlled spectropolarimeter (JASCO, J-810). Spectra were recorded at 25 °C in 0.05-cm quartz cells from 190 to 250 nm with protein concentrations of 0.05 mg/ml at a digital resolution of 0.5 nm, with scan speed of 5 nm/min for wavelength above and below 190 nm, respectively. Multiple spectra were recorded for duplicated samples. These spectra were averaged and corrected for baseline contribution from the buffer. Susceptibility to proteolysis was measured by the incubation of ICDH samples (65 μg) with 12.5 μg of Pronase in 250 μl of 25 mm Hepes (pH 8.0)/100 mm NaCl for 1 h at 37 °C. After incubation, the aliquots were removed and subjected to a 10% trichloroacetic acid treatment. After centrifugation of precipitated proteins for 10 min at maximum speed in an Eppendorf microcentrifuge, the supernatant was first neutralized with a predetermined volume of 2 m potassium borate, pH 10. The amount of small peptides in the supernatant was then determined as described by Church et al. (35Church F.C. Porter D.H. Catignani G.L. Swaisgood H.E. Anal. Biochem. 1985; 146: 343-348Crossref PubMed Scopus (146) Google Scholar). ANSA (100 μm) was incubated with the various forms of ICDH in 25 mm potassium phosphate buffer, pH 7.0/50 mm KCl. The fluorescence emission spectra (excitation, 370 nm) of the different mixtures were monitored on spectrofluorometer. Binding of ANSA to the protein was evidenced by subtracting the emission spectrum of ANSA from that of ANSA in the presence of enzyme. Cellular NADPH and GSH Levels—NADPH was measured using the enzymatic cycling method as described by Zerez et al. (36Zerez C.R. Lee S.J. Tanaka K.R. Anal. Biochem. 1987; 164: 367-373Crossref PubMed Scopus (156) Google Scholar). Briefly, the reaction mixture, which combined 100 mm Tris (pH 8.0), 5 mm EDTA, 2 mm phenazine ethosulfate, 0.5 mm 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 1.3 units of glucose-6-phosphate dehydrogenase, and appropriate amounts of the cell extracts, was preincubated for 5 min at 37 °C. The reaction was started by the addition of 1 mm glucose 6-phosphate. The absorbance at 570 nm was measured for 3 min. The concentration of total glutathione was determined by the rate of formation of 5-thio-2-nitrobenzoic acid at 412 nm (ϵ = 1.36 × 104m-1cm-1) as the method described by Akerboom and Sies (37Akerboom T.P.M. Sies H. Methods Enzymol. 1981; 77: 373-382Crossref PubMed Scopus (1442) Google Scholar), and GSSG was measured by the DTNB-GSSG reductase recycling assay after treating GSH with 2-vinylpyridine (38Anderson M.E. Methods Enzymol. 1985; 113: 548-555Crossref PubMed Scopus (2380) Google Scholar). Measurement of Intracellular ROS—Intracellular peroxide production was measured using the oxidant-sensitive fluorescent probe DCFH-DA with confocal microscopy (39Schwarz M.A. Lazo J.S. Yalowich J.C. J. Biol. Chem. 1994; 269: 15238-15243Abstract Full Text PDF PubMed Google Scholar). Cells were grown at 2 × 106 cells per 100-mm plate containing slide glass coated with poly-l-lysine and maintained in the growth medium for 24 h. Cells were exposed to 10 μm DCFH-DA for 15 min and treated with 100 μm SIN-1 for 5 min. Cells on the slide glass were washed with PBS, and a coverglass was put on the slide glass. 2′7′-Dichlorofluorescein (DCF) fluorescence (excitation, 488 nm; emission, 520 nm) was imaged on a laser confocal scanning microscope (DM/R-TCS, Leica) coupled to a microscope (Leitz DM REB). Hydrogen peroxide oxidizes ferrous (Fe2+) to ferric ion (Fe3+) selectively in dilute acid, and the resulting ferric ions can be determined using a ferric sensitive dye, xylenol orange, as an indirect measure of hydrogen peroxide concentration. Mitochondrial fractions were added to FOX solution (0.1 mm xylenol orange, 0.25 mm ammonium ferrous sulfate, 100 mm sorbitol, and 25 mm H2SO4) and incubated in a room temperature for 30 min, and absorbance was measured at 560 nm. Hydrogen peroxide was used to draw standard curve as described (40Jiang Z.Y. Hunt J.V. Wolff S.P. Anal. Biochem. 1992; 202: 384-389Crossref PubMed Scopus (1431) Google Scholar). Measurements of O2.¯/NO Formation and SOD/iNOS Activity—Superoxide was measured using lucigenin-enhanced chemiluminescence (41Li Y. Zhu H. Kuppusamy P. Rouband V. Zweier J.L. Trush M.A. J. Biol. Chem. 1998; 273: 2015-2023Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). Nitrite, a stable product of NO oxidation, was determined by the Griess reaction (42Green L.C. Wagner D.A. Glogowsk J. Skipper P.L. Wishnok J.S. Tannenbaum S.R. Anal. Biochem. 1982; 126: 131-138Crossref PubMed Scopus (10586) Google Scholar). The activity of iNOS was determined by the rate of conversion of [l-3H]arginine to [l-3H]citrulline as described (43Przedborski S. Jackson-Lewis V. Yokoyama R. Shibata T. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4565-4571Crossref PubMed Scopus (589) Google Scholar). The iNOS protein was assessed immunochemically in Western blots by using polyclonal anti-iNOS antibodies. SOD activity was assayed spectrophotometrically using a pyrogallol assay (44Marklund S. Marklund G. Eur. J. Biochem. 1974; 47: 469-474Crossref PubMed Scopus (7732) Google Scholar). Replicates—Unless otherwise indicated, each result described in the paper is representative of at least three separate experiments. Inactivation of ICDH by Peroxynitrite—Incubation of mitochondrial ICDH with synthesized peroxynitrite or SIN-1 at pH 7.4 at 37 °C resulted in a time- and concentration-dependent loss of enzyme activity as shown in Fig. 1 (A and B). Previously decomposed peroxynitrite does not inactivate the enzyme (data not shown), indicating that the decomposition products of peroxynitrite are not responsible for the inactivation of enzyme. SIN-1 is a nitric oxide and superoxide anion donor and thus considered a peroxynitrite releasing compound (45Hogg N. Darley-Usmar V.M. Wilson M.T. Moncada S. Biochem. J. 1992; 281: 419-424Crossref PubMed Scopus (619) Google Scholar). Under the conditions chosen, incubation with 0.1 mm peroxynitrite or 0.1 mm SIN-1 resulted in ∼90 or 65% inhibition. The mouse liver cytosolic ICDH was expressed as a fusion protein to GST, and it was purified as a recombinant protein, which was almost pure as estimated by SDS-PAGE (data not shown). The inhibition of recombinant cytosolic ICDH by peroxynitrite or SIN-1 demonstrated basically the same pattern, as shown in Fig. 1C. We evaluated whether the presence of product would protect the active site of ICDH from peroxynitrite- and SIN-1-mediated inactivation. The inactivation of enzyme by peroxynitrite or SIN-1 was partially prevented in the presence of NADPH in a dose-dependent manner as shown in Fig. 1D. This observation strongly suggests that the inactivation with peroxynitrite or SIN-1 takes place in the vicinity of the active site of ICDH. To gain insight into the mechanism by which peroxynitrite or SIN-1 inactivates ICDH, the protective effect of several molecules that react with either peroxynitrite or secondary oxidants with hydroxyl radical-like reactivity from peroxynitrite decomposition was evaluated. Table I shows that hydroxyl radical scavengers such as mannitol," @default.
• W2017363944 created "2016-06-24" @default.
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• W2017363944 title "Inactivation of NADP+-dependent Isocitrate Dehydrogenase by Peroxynitrite" @default.
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