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D R Janero - One of the best experts on this subject based on the ideXlab platform.
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Specific S nitroSothiol thionitrite quantification aS Solution nitrite after vanadium iii reduction and ozone chemilumineScent detection
Free Radical Biology and Medicine, 1998Co-Authors: J F Ewing, D R JaneroAbstract:IncreaSing evidence SuggeStS that S-nitroSothiolS (thionitriteS) might repreSent naturally occurring nitric oxide SurrogateS and function aS intermediateS in nitrogen monoxide metaboliSm. A facile, SenSitive, and Selective micromethod haS been developed and validated for quantification of S-nitroSothiolS aS their mercury-diSplaceable nitrogen monoxide content. In thiS method, brief (5-min), room-temperature pretreatment of S-nitroSothiol with a molar exceSS of aqueouS mercuric chloride waS uSed to liberate into Solution, quantitatively, the nitrogen monoxide moiety, which rapidly and quantitatively converted to itS Stable Solution end-product, nitrite. Solution nitrite waS reduced back to nitric oxide with vanadium(III), and the nitric oxide waS detected by gaS-phaSe chemilumineScence after reaction with ozone in a commercial nitric oxide analyzer. A linear relationShip waS obServed between S-nitroSothiol-bound nitrogen monoxide and ozone-chemilumineScent detector reSponSe over a wide range (16.3-3500 pmol) of nitric oxide, aS generated by reaction of vanadium(III) with either nitrite Standard or mercury-treated S-nitroSothiol. ASSay reSponSe waS quantitatively identical for equivalent amountS of nitrite and S-nitroSothiol-bound nitrogen monoxide. The method diSplayed 96% Selectivity for nitrite vS. nitrate and negligible (<2%) interference by nitroSated compoundS bearing nitrogen monoxide moietieS bound to either nitrogen or carbon. The lower limitS of quantitative SenSitivity and qualitative detection were below 50 and 20 pmol S-nitroSothiol-bound nitrogen monoxide-equivalentS, reSpectively. The intraday and interday coefficientS of variation did not exceed 8%. ThiS technique haS been applied to quantify Structurally diverSe natural and Synthetic S-nitroSothiolS with quantitative recovery from complex biological SampleS Such aS culture media and plaSma at levelS of nitrogen monoxide-equivalentS undetectable by the popular Saville colorimetric method.
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Specific S-nitroSothiol (thionitrite) quantification aS Solution nitrite after vanadium(III) reduction and ozone-chemilumineScent detection.
Free radical biology & medicine, 1998Co-Authors: J F Ewing, D R JaneroAbstract:IncreaSing evidence SuggeStS that S-nitroSothiolS (thionitriteS) might repreSent naturally occurring nitric oxide SurrogateS and function aS intermediateS in nitrogen monoxide metaboliSm. A facile, SenSitive, and Selective micromethod haS been developed and validated for quantification of S-nitroSothiolS aS their mercury-diSplaceable nitrogen monoxide content. In thiS method, brief (5-min), room-temperature pretreatment of S-nitroSothiol with a molar exceSS of aqueouS mercuric chloride waS uSed to liberate into Solution, quantitatively, the nitrogen monoxide moiety, which rapidly and quantitatively converted to itS Stable Solution end-product, nitrite. Solution nitrite waS reduced back to nitric oxide with vanadium(III), and the nitric oxide waS detected by gaS-phaSe chemilumineScence after reaction with ozone in a commercial nitric oxide analyzer. A linear relationShip waS obServed between S-nitroSothiol-bound nitrogen monoxide and ozone-chemilumineScent detector reSponSe over a wide range (16.3-3500 pmol) of nitric oxide, aS generated by reaction of vanadium(III) with either nitrite Standard or mercury-treated S-nitroSothiol. ASSay reSponSe waS quantitatively identical for equivalent amountS of nitrite and S-nitroSothiol-bound nitrogen monoxide. The method diSplayed 96% Selectivity for nitrite vS. nitrate and negligible (
J F Ewing - One of the best experts on this subject based on the ideXlab platform.
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Specific S nitroSothiol thionitrite quantification aS Solution nitrite after vanadium iii reduction and ozone chemilumineScent detection
Free Radical Biology and Medicine, 1998Co-Authors: J F Ewing, D R JaneroAbstract:IncreaSing evidence SuggeStS that S-nitroSothiolS (thionitriteS) might repreSent naturally occurring nitric oxide SurrogateS and function aS intermediateS in nitrogen monoxide metaboliSm. A facile, SenSitive, and Selective micromethod haS been developed and validated for quantification of S-nitroSothiolS aS their mercury-diSplaceable nitrogen monoxide content. In thiS method, brief (5-min), room-temperature pretreatment of S-nitroSothiol with a molar exceSS of aqueouS mercuric chloride waS uSed to liberate into Solution, quantitatively, the nitrogen monoxide moiety, which rapidly and quantitatively converted to itS Stable Solution end-product, nitrite. Solution nitrite waS reduced back to nitric oxide with vanadium(III), and the nitric oxide waS detected by gaS-phaSe chemilumineScence after reaction with ozone in a commercial nitric oxide analyzer. A linear relationShip waS obServed between S-nitroSothiol-bound nitrogen monoxide and ozone-chemilumineScent detector reSponSe over a wide range (16.3-3500 pmol) of nitric oxide, aS generated by reaction of vanadium(III) with either nitrite Standard or mercury-treated S-nitroSothiol. ASSay reSponSe waS quantitatively identical for equivalent amountS of nitrite and S-nitroSothiol-bound nitrogen monoxide. The method diSplayed 96% Selectivity for nitrite vS. nitrate and negligible (<2%) interference by nitroSated compoundS bearing nitrogen monoxide moietieS bound to either nitrogen or carbon. The lower limitS of quantitative SenSitivity and qualitative detection were below 50 and 20 pmol S-nitroSothiol-bound nitrogen monoxide-equivalentS, reSpectively. The intraday and interday coefficientS of variation did not exceed 8%. ThiS technique haS been applied to quantify Structurally diverSe natural and Synthetic S-nitroSothiolS with quantitative recovery from complex biological SampleS Such aS culture media and plaSma at levelS of nitrogen monoxide-equivalentS undetectable by the popular Saville colorimetric method.
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Specific S-nitroSothiol (thionitrite) quantification aS Solution nitrite after vanadium(III) reduction and ozone-chemilumineScent detection.
Free radical biology & medicine, 1998Co-Authors: J F Ewing, D R JaneroAbstract:IncreaSing evidence SuggeStS that S-nitroSothiolS (thionitriteS) might repreSent naturally occurring nitric oxide SurrogateS and function aS intermediateS in nitrogen monoxide metaboliSm. A facile, SenSitive, and Selective micromethod haS been developed and validated for quantification of S-nitroSothiolS aS their mercury-diSplaceable nitrogen monoxide content. In thiS method, brief (5-min), room-temperature pretreatment of S-nitroSothiol with a molar exceSS of aqueouS mercuric chloride waS uSed to liberate into Solution, quantitatively, the nitrogen monoxide moiety, which rapidly and quantitatively converted to itS Stable Solution end-product, nitrite. Solution nitrite waS reduced back to nitric oxide with vanadium(III), and the nitric oxide waS detected by gaS-phaSe chemilumineScence after reaction with ozone in a commercial nitric oxide analyzer. A linear relationShip waS obServed between S-nitroSothiol-bound nitrogen monoxide and ozone-chemilumineScent detector reSponSe over a wide range (16.3-3500 pmol) of nitric oxide, aS generated by reaction of vanadium(III) with either nitrite Standard or mercury-treated S-nitroSothiol. ASSay reSponSe waS quantitatively identical for equivalent amountS of nitrite and S-nitroSothiol-bound nitrogen monoxide. The method diSplayed 96% Selectivity for nitrite vS. nitrate and negligible (
Neil Hogg - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome c-mediated formation of S-nitroSothiol in cellS
The Biochemical journal, 2012Co-Authors: Katarzyna A. Broniowska, Agnes Keszler, Swati Basu, Daniel B. Kim-shapiro, Neil HoggAbstract:S-nitroSothiolS are productS of nitric oxide (NO) metaboliSm that have been implicated in a plethora of Signalling proceSSeS. However, mechaniSmS of S-nitroSothiol formation in biological SyStemS are uncertain, and no efficient protein-mediated proceSS haS been identified. Recently, we obServed that ferric cytochrome c can promote S-nitroSoglutathione formation from NO and glutathione by acting aS an electron acceptor under anaerobic conditionS. In the preSent Study, we Show that thiS mechaniSm iS alSo robuSt under oxygenated conditionS, that cytochrome c can promote protein S-nitroSation via a tranSnitroSation reaction and that cell lySate depleted of cytochrome c exhibitS a lower capacity to SyntheSize S-nitroSothiolS. Importantly, we alSo demonStrate that thiS mechaniSm iS functional in living cellS. Lower S-nitroSothiol SyntheSiS activity, from donor and nitric oxide SynthaSe-generated NO, waS found in cytochrome c-deficient mouSe embryonic cellS aS compared with wild-type controlS. Taken together, theSe data point to cytochrome c aS a biological mediator of protein S-nitroSation in cellS. ThiS iS the moSt efficient and concerted mechaniSm of S-nitroSothiol formation reported So far.
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copper dependence of the biotin Switch aSSay modified aSSay for meaSuring cellular and blood nitroSated proteinS
Free Radical Biology and Medicine, 2008Co-Authors: Xunde Wang, Neil Hogg, Nicholas J Kettenhofen, Sruti Shiva, Mark T GladwinAbstract:AbStract StudieS have Shown that modification of critical cySteine reSidueS in proteinS leadS to the regulation of protein function. TheSe modificationS include diSulfide bond formation, glutathionylation, Sulfenic and Sulfinic acid formation, and S-nitroSation. The biotin Switch aSSay waS developed to Specifically detect protein S-nitroSation (S. R. Jaffrey et al., Nat. Cell Biol. 3:193–197; 2001). In thiS aSSay, proteinS are denatured with SDS in the preSence of methyl methane thioSulfonate (MMTS) to block free thiolS. After acetone precipitation or Sephadex G25 Separation to remove exceSS MMTS, HPDP–biotin and 1 mM aScorbate are added to reduce the S-nitroSothiol bondS and label the reduced thiolS with biotin. The proteinS are then Separated by nonreducing SDS PAGE and detected uSing either Streptavidin–HRP or anti-biotin–HRP conjugate. Our examination of thiS labeling Scheme haS revealed that the extent of labeling dependS on the buffer compoSition and, importantly, on the choice of metal-ion chelator (DTPA vS EDTA). Unexpectedly, uSing purified S-nitroSated albumin, we have found that “contaminating” copper iS required for the aScorbate-dependent degradation of S-nitroSothiol; thiS iS conSiStent with the fact that aScorbate itSelf doeS not rapidly reduce S-nitroSothiolS. Removal of copper from bufferS by DTPA and other copper chelatorS preServeS approximately 90% of the S-nitroSothiol, whereaS the incluSion of copper and aScorbate completely eliminateS the S-nitroSothiol in the preparation and increaSeS the Specific biotin labeling. TheSe biotin Switch experimentS were confirmed uSing triiodide-baSed and copper-baSed reductive chemilumineScence. Additional modificationS of the aSSay uSing N-ethylmaleimide for thiol blockade, ferricyanide pretreatment to Stabilize S-nitroSated hemoglobin, and cyanine dye labeling inStead of biotin are preSented for the meaSurement of cellular and blood S-nitroSothiolS. TheSe reSultS indicate that degradation of S-nitroSothiol in the Standard biotin Switch aSSay iS metal-ion dependent and that experimental variability in S-nitroSothiol yieldS uSing thiS aSSay occurS Secondary to the incluSion of metal-ion chelatorS in reagentS and variable metal-ion contamination of bufferS and labware. The addition of copper to aScorbate allowS for a Simple aSSay modification that dramatically increaSeS SenSitivity while maintaining Specificity.
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role of S nitroSothiol tranSport in the cardioprotective effectS of S nitroSocySteine in rat heartS
Free Radical Biology and Medicine, 2007Co-Authors: Neil Hogg, Katarzyna A. Broniowska, Nicholas J Kettenhofen, Jutta Novalija, Enis NovalijaAbstract:AbStract The objective of thiS Study waS to determine if prior expoSure of rat heartS to S -nitroSocySteine (CySNO) waS able to provide protection againSt reperfuSion injury. We probed NO releaSe uSing the extracellular NO Scavenger oxyhemoglobin (oxyHb), and we examined the involvement of the amino acid tranSport SyStem L (L-AT), a known tranSporter of CySNO, uSing the L-AT competitor, l -leucine (L-Leu). ISolated (9- to 12-week-old WiStar male) rat heartS (Six to eight per group) were perfuSed with CySNO (10 μM) for 30 min with or without the L-AT competitor L-Leu (1 mM) before 30 min of iSchemia. Cardiac function waS aSSeSSed before, during, and after treatment and during 120 min of reperfuSion after iSchemia. Functional recovery (rate–preSSure product) waS Significantly improved in the CySNO group compared to heartS in the CySNO + L-Leu group and the control group ( p p S -nitroSothiol (RSNO) levelS from an unmeaSurable background to a value of about 15.7 ± 4.1 pmol RSNO/mg protein, aS meaSured by triiodide-baSed chemilumineScence in the preSence and abSence of mercury(II) chloride. In the preSence of l -Leu, thiS value dropped to 0.4 ± 0.3 pmol RSNO/mg protein. ThiS Study demonStrateS that expoSure to CySNO before iSchemia increaSeS tiSSue S -nitroSothiol levelS, improveS poStiSchemic contractile dySfunction, and attenuateS necroSiS. The mechaniSm of cardioprotection requireS the uptake of CySNO via the L-AT and doeS not Seem to involve NO releaSe either during CySNO expoSure or during iSchemia. ThiS SuggeStS that the protective effectS of CySNO are mediated through the poSttranSlational modification of cellular proteinS through an NO-independent tranSnitroSation mechaniSm.
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The mechaniSm of tranSmembrane S-nitroSothiol tranSport
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Yanhong Zhang, Neil HoggAbstract:S-nitroSothiolS have been SuggeSted to play an important role in nitric oxide (NO)-mediated biological eventS. However, the mechaniSmS by which an S-nitroSothiol (or the S-nitroSo functional group) iS tranSferred acroSS cell membrane are Still poorly underStood. We have demonStrated previouSly that the degradation of S-nitroSoglutathione (GSNO) by cellS abSolutely required the preSence of cyStine in the extracellular medium and propoSed a mechaniSm that involved the reduction of cyStine to cySteine, followed by the reaction of cySteine with GSNO to form S-nitroSocySteine (CySNO), mixed diSulfideS, and nitroSyl anion. In the preSent Study we have aSSeSSed the effect of cyStine on the tranSfer of the S-nitroSo functional group from the extracellular to the intracellular Space. USing RAW 264.7 cellS, we found that the preSence of l-cyStine enhanced GSNO-dependent S-nitroSothiol uptake, increaSing the intracellular S-nitroSothiol level from ≈60 pmol/mg of protein to ≈3 nmol/mg of protein. The uptake SeemS to depend on the reduction of l-cyStine to l-cySteine, which involveS the amino acid tranSport SyStem, the tranSnitroSation between GSNO and l-cySteine to form l-CySNO, and uptake of l-CySNO via amino acid tranSport SyStem L. Compared with GSNO, (Z)-1-[N-(3-ammoniopropyl)-N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate, an NO donor, iS much leSS effective at intracellular S-nitroSothiol formation in the preSence of l-cyStine or l-cySteine, SuggeSting that the biochemical changeS that occur after expoSure of cellS to S-nitroSothiol, with reSpect to thiol chemiStry, are diStinctly different from thoSe obServed with NO.
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Formation and Stability of S-nitroSothiolS in RAW 264.7 cellS.
American journal of physiology. Lung cellular and molecular physiology, 2003Co-Authors: Yanhong Zhang, Neil HoggAbstract:S-NitroSothiolS have been SuggeSted to be mediatorS of many nitric oxide-dependent proceSSeS, including apoptoSiS and vaScular relaxation. Thiol nitroSation iS a poorly underStood proceSS in vivo, and the mechaniSmS by which nitric oxide can be converted into a nitroSating agent have not been eStabliShed. There iS a diScrepancy between the SuggeSted biological roleS of nitric oxide and itS known chemical and phySical propertieS. In thiS Study, we have examined the formation of S-nitroSothiolS in lipopolySaccharide-treated RAW 264.7 cellS. ThiS treatment generated 17.4 +/- 1.0 pmol/mg of protein (meanS +/- SE, n =27) of intracellular S-nitroSothiol that Slowly decayed over Several hourS. S-NitroSothiol formation depended on the formation of nitric oxide and not on the preSence of nitrite. Extracellular thiolS were nitroSated by cell-generated nitric oxide. Oxygenated ferrouS hemoglobin inhibited the formation of S-nitroSothiol, indicating the nitroSation occurred more Slowly than diffuSion. We diScuSS Several mechaniSmS for S-nitroSothiol formation and conclude that the nitroSation propenSity of nitric oxide iS a freely diffuSible element that iS not conStrained within an individual cell and that both nitric oxide per Se and nitric oxide-derived nitroSating agentS are able to diffuSe acroSS cell membraneS. To achieve intracellular localization of the nitroSation reaction, mechaniSmS muSt be invoked that do not involve the formation of nitric oxide aS an intermediate.
Michael P Murphy - One of the best experts on this subject based on the ideXlab platform.
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mitochondria Selective S nitroSation by mitochondria targeted S nitroSothiol protectS againSt poSt infarct heart failure in mouSe heartS
European Journal of Heart Failure, 2014Co-Authors: Carmen Methner, Edward Chouchani, Michael P Murphy, Guido Buonincontri, Victoria R Pell, Stephen J Sawiak, Thomas KriegAbstract:AimS Recently it haS been Shown that the mitochondria-targeted S-nitroSothiol MitoSNO protectS againSt acute iSchaemia/reperfuSion (IR) injury by inhibiting the reactivation of mitochondrial complex I in the firSt minuteS of reperfuSion of iSchaemic tiSSue, thereby preventing free radical formation that underlieS IR injury. However, it remainS unclear how thiS tranSient inhibition of mitochondrial complex I-mediated free radicalS at reperfuSion affectS the long-term recovery of the heart following IR injury. Here we determined whether the acute protection by MitoSNO at reperfuSion prevented the SubSequent development of poSt-myocardial infarction heart failure. MethodS and reSultS Mice were Subjected to 30 min left coronary artery occluSion followed by reperfuSion and recovery over 28 dayS. MitoSNO (100 ng/kg) waS applied 5 min before the onSet of reperfuSion followed by 20 min infuSion (1 ng/kg/min). Infarct Size and cardiac function were meaSured by magnetic reSonance imaging (MRI) 24 h after infarction. MitoSNO-treated mice exhibited reduced infarct Size and preServed function. In addition, MitoSNO at reperfuSion improved outcome meaSureS 28 dayS poSt-IR, including preServed SyStolic function (63.7 ±1.8% LVEF vS. 53.7 ± 2.1% in controlS, P = 0.01) and tiSSue fibroSiS. ConcluSionS MitoSNO action acutely at reperfuSion reduceS infarct Size and protectS from poSt-myocardial infarction heart failure. Therefore, targeted inhibition of mitochondrial complex I in the firSt minuteS of reperfuSion by MitoSNO iS a rational therapeutic Strategy for preventing SubSequent heart failure in patientS undergoing IR injury.
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Protection through poStconditioning or a mitochondria-targeted S-nitroSothiol iS unaffected by cardiomyocyte-Selective ablation of protein kinaSe G
Basic Research in Cardiology, 2013Co-Authors: Carmen Methner, Michael P Murphy, Robert Lukowski, Franz Hofmann, Karina Grube, Florian Loga, Robin A. J. Smith, Thomas KriegAbstract:Protein kinaSe G type I (PKGI) playS a critical role in Survival Signaling of pre- and poStconditioning downStream of cardiac cGMP. However, it iS unclear whether PKGI exertS itS protective effectS in the cardiomyocyte or if other cardiac cell typeS are involved, and whether nitric oxide (NO) metaboliSm can target cardiomyocyte mitochondria independently of cGMP/PKGI. We teSted whether protection againSt reperfuSion injury by iSchemic poStconditioning (IPoSt), Soluble guanylyl cyclaSe (SGC) activation and inhibition, adenoSine A_2B receptor (A_2BAR) agoniSt, phoSphodieSteraSe type-5 (PDE-5) inhibitor, or mitochondria-targeted S -nitroSothiol (MitoSNO) waS affected by a cardiomyocyte-Specific ablation of the PKGI gene in the mouSe (CMG-KO). In Situ heartS underwent 30 min of regional iSchemia followed by 2 h of reperfuSion. AS expected, in CMG-CTRS all interventionS at early reperfuSion lead to profound infarct Size reduction: IPoSt (Six cycleS of 10-S reperfuSion and 10-S coronary occluSion) with or without treatment with the SGC inhibitor ODQ, treatment with the Specific SGC activator BAY58-2667 (BAY58), the Selective A_2BAR agoniSt BAY60-6583 (BAY60), PDE-5 inhibitor Sildenafil, and MitoSNO. MitoSNO accumulateS within mitochondria, driven by the membrane potential, where it generateS NO· and S -nitroSateS thiol proteinS. In contraSt, the heartS of CMG-KO animalS were not protected by BAY58 and Sildenafil, whereaS the protective effectS of IPoSt, IPoSt with ODQ, BAY60, and MitoSNO were unaffected by the lack of PKGI. Taken together, PKGI iS important for the protection againSt iSchemia reperfuSion injury afforded by SGC activation or PDE-5 inhibition. However, the beneficial effectS of IPoSt, activation of the A_2BAR, aS well aS the direct effectS via mitochondrial S -nitroSation do not depend on PKGI in cardiomyocyteS.
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characterization of mice with a deletion of protein kinaSe g type i in cardiomyocyteS and the effect on cardioprotection through either poStconditioning or mitochondria targeted S nitroSothiol
BMC Pharmacology, 2011Co-Authors: Carmen Methner, Michael P Murphy, Robert Lukowski, Franz Hofmann, Thomas KriegAbstract:Objective We developed mice with a cardiomyocyte-Specific ablation of the cGKI gene (CMG-KO) and teSted whether protection againSt reperfuSion injury by iSchemic poStconditioning (IPoSt), Soluble guanylyl cyclaSe (SGC) activation, the adenoSine A2B receptor (A2BAR), or the mitochondria-targeted S-nitroSothiol (MitoSNO) waS affected. MitoSNO accumulateS within mitochondria, driven by the membrane potential, where it generateS NO and S-nitroSated thiol proteinS [1].
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identification of S nitroSated mitochondrial proteinS by S nitroSothiol difference in gel electrophoreSiS Sno dige implicationS for the regulation of mitochondrial function by reverSible S nitroSation
Biochemical Journal, 2010Co-Authors: Edward Chouchani, Sergiy M Nadtochiy, Paul S Brookes, Ian M. Fearnley, Kathryn S Lilley, Robin Andrew James Smith, Thomas R Hurd, Michael P MurphyAbstract:The S-nitroSation of mitochondrial proteinS aS a conSequence of NO metaboliSm iS of phySiological and pathological Significance. We previouSly developed a MitoSNO (mitochondria-targeted S-nitroSothiol) that Selectively S-nitroSateS mitochondrial proteinS. To identify theSe S-nitroSated proteinS, here we have developed a Selective proteomic methodology, SNO-DIGE (S-nitroSothiol difference in gel electrophoreSiS). Protein thiolS in control and MitoSNO-treated SampleS were blocked, then incubated with copper(II) and aScorbate to Selectively reduce S-nitroSothiolS. The SampleS were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluoreScent tagS, mixed together and individual protein SpotS were reSolved by 2D (two-dimenSional) gel electrophoreSiS. FluoreScent Scanning of theSe gelS revealed S-nitroSated proteinS by an increaSe in Cy5 red fluoreScence, allowing for their identification by MS. Parallel analySiS by Redox-DIGE enabled uS to diStinguiSh S-nitroSated thiol proteinS from thoSe which became oxidized due to NO metaboliSm. We identified 13 S-nitroSated mitochondrial proteinS, and a further four that were oxidized, probably due to evaneScent S-nitroSation relaxing to a reverSible thiol modification. We inveStigated the conSequenceS of S-nitroSation for three of the enzymeS identified uSing SNO-DIGE (aconitaSe, mitochondrial aldehyde dehydrogenaSe and α-ketoglutarate dehydrogenaSe) and found that their activity waS Selectively and reverSibly inhibited by S-nitroSation. We conclude that the reverSible regulation of enzyme activity by S-nitroSation modifieS enzymeS central to mitochondrial metaboliSm, whereaS identification and functional characterization of theSe novel targetS provideS mechaniStic inSight into the potential phySiological and pathological roleS played by thiS modification. More generally, the development of SNO-DIGE facilitateS robuSt inveStigation of protein S-nitroSation acroSS the proteome.
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Identification of S-nitroSated mitochondrial proteinS by S-nitroSothiol Difference In Gel ElectrophoreSiS (SNO-DIGE): implicationS for the regulation of mitochondrial function by reverSible S-nitroSation
Biochemical Journal, 2010Co-Authors: Edward Chouchani, Thomas Hurd, Sergiy M Nadtochiy, Paul S Brookes, Ian M. Fearnley, Kathryn S Lilley, Robin Andrew James Smith, Michael P MurphyAbstract:The S-nitroSation of mitochondrial proteinS aS a conSequence of nitric oxide (NO) metaboliSm iS of phySiological and pathological Significance. We previouSly developed a mitochondria-targeted S-nitroSothiol (MitoSNO) that Selectively S-nitroSateS mitochondrial proteinS. To identify theSe S-nitroSated proteinS, here we have developed a Selective proteomic methodology, S-nitroSothiol Difference In Gel ElectrophoreSiS (SNO-DIGE). Protein thiolS in control and MitoSNO-treated SampleS were blocked, then incubated with copper(II) and aScorbate to Selectively reduce S-nitroSothiolS. The SampleS were then treated with thiol-reactive Cy3 or Cy5 fluoreScent tagS, mixed together and individual protein SpotS were reSolved by 2D gel electrophoreSiS. FluoreScent Scanning of theSe gelS revealed S-nitroSated proteinS by an increaSe in Cy5 red fluoreScence, allowing for their identification by maSS Spectrometry. Parallel analySiS by Redox-DIGE enabled uS to diStinguiSh S-nitroSated thiol proteinS from thoSe which became oxidized due to NO metaboliSm. We identified 13 S-nitroSated mitochondrial proteinS, and a further 4 that were oxidized, probably due to evaneScent S-nitroSation relaxing to a reverSible thiol modification. We inveStigated the conSequenceS of S-nitroSation for three of the enzymeS identified uSing SNO-DIGE (aconitaSe, aldehyde dehydrogenaSe and a-ketoglutarate dehydrogenaSe) and found that their activity waS Selectively and reverSibly inhibited by S-nitroSation. We conclude that the reverSible regulation of enzyme activity by S-nitroSation modifieS enzymeS central to mitochondrial metaboliSm, while identification and functional characterization of theSe novel targetS provideS mechaniStic inSight into the potential phySiological and pathological roleS played by thiS modification. More generally, the development of SNO-DIGE facilitateS robuSt inveStigation of protein S-nitroSation acroSS the proteome.
Shih Jiuan Chiu - One of the best experts on this subject based on the ideXlab platform.
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Nitric oxide-releaSing S-nitroSothiol-modified Silica/chitoSan core–Shell nanoparticleS
Polymer, 2015Co-Authors: Wei Lin Chang, Kang Jen Peng, Teh Min Hu, Shih Jiuan ChiuAbstract:AbStract Nitric oxide-releaSing core–Shell nanoparticleS poSSeSSing Silica coreS and S-nitroSothiol-modified chitoSan ShellS have been prepared. The S-nitroSo groupS are incorporated to the thiol-modified chitoSan Shell layerS of the nanoparticleS. The SyntheSiS route involveS two major reactionS of (a) incorporation of chitoSan chainS to Silica nanoparticleS and (b) thiolation of chitoSan chainS. The Sample made with the SyntheSiS route which performS firSt the b reaction ShowS a higher NO-releaSing amount than doeS the analogue prepared with the a-reaction-firSt SyntheSiS route. The chemical StructureS and morphology of the nanoparticleS have been characterized with Spectral methodS and electronic microScopieS. The prepared Sample ShowS a NO-releaSing amount of 0.15 μmol SNO group/mg with a prolonged NO releaSe.
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nitric oxide releaSing S nitroSothiol modified Silica chitoSan core Shell nanoparticleS
Polymer, 2015Co-Authors: Wei Lin Chang, Kang Jen Peng, Shih Jiuan Chiu, Yingling LiuAbstract:AbStract Nitric oxide-releaSing core–Shell nanoparticleS poSSeSSing Silica coreS and S-nitroSothiol-modified chitoSan ShellS have been prepared. The S-nitroSo groupS are incorporated to the thiol-modified chitoSan Shell layerS of the nanoparticleS. The SyntheSiS route involveS two major reactionS of (a) incorporation of chitoSan chainS to Silica nanoparticleS and (b) thiolation of chitoSan chainS. The Sample made with the SyntheSiS route which performS firSt the b reaction ShowS a higher NO-releaSing amount than doeS the analogue prepared with the a-reaction-firSt SyntheSiS route. The chemical StructureS and morphology of the nanoparticleS have been characterized with Spectral methodS and electronic microScopieS. The prepared Sample ShowS a NO-releaSing amount of 0.15 μmol SNO group/mg with a prolonged NO releaSe.
