Bosworth 2009 Proc Natl Acad Sci U S A: Difference between revisions
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{{Publication | {{Publication | ||
|title=Bosworth CA, Toledo JC Jr, Zmijewski JW, Li Q, Lancaster JR (2009) Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide. Proc. Natl. Acad. Sci. USA 106: 4671-4676. | |title=Bosworth CA, Toledo JC Jr, Zmijewski JW, Li Q, Lancaster JR (2009) Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide. Proc. Natl. Acad. Sci. USA 106: 4671-4676. | ||
|authors=Bosworth CA, Toledo JC Jr, Zmijewski JW, Li Q, Lancaster JR ย | |authors=Bosworth CA, Toledo JC Jr, Zmijewski JW, Li Q, Lancaster JR | ||
|year=2009 | |year=2009 | ||
|journal=Proc. Natl. Acad. Sci. | |journal=Proc. Natl. Acad. Sci. | ||
|abstract=Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (โขNO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of โขNO with | |abstract=Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (โขNO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of โขNO with O<sub>2</sub>, reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of โขNO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (<8 ฮผM) O<sub>2</sub> concentrations and exhibits zero-order dependence on โขNO concentration. Indeed, RSNO formation is not inhibited even at O<sub>2</sub> levels <1 ฮผM. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by >50%. One possible metal-dependent, O<sub>2</sub>-independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of โขNO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by โ50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free โขNO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells. | ||
|keywords= Iron,ย Nitrosation, Reactive nitrogen species,ย Reactive oxygen species, Chelatable iron | |keywords=Iron,ย Nitrosation, Reactive nitrogen species,ย Reactive oxygen species, Chelatable iron | ||
|info=[http://www.ncbi.nlm.nih.gov/pubmed/19261856 PMID: 19261856] | |info=[http://www.ncbi.nlm.nih.gov/pubmed/19261856 PMID: 19261856] | ||
}} | }} | ||
{{Labeling | {{Labeling | ||
|instruments=Oxygraph-2k | |instruments=Oxygraph-2k | ||
|topics=Respiration; OXPHOS; ETS Capacity | |articletype=Protocol; Manual | ||
|discipline=Mitochondrial Physiology, Biomedicine, Environmental Physiology; Toxicology | |||
|organism=Yeast; Fungi | |||
|preparations=Intact Cell; Cultured; Primary | |||
|injuries=RONS; Oxidative Stress | |||
|kinetics=ADP; Pi, Oxygen, Reduced Substrate; Cytochrome c | |||
|topics=Respiration; OXPHOS; ETS Capacity, Mitochondrial Biogenesis; Mitochondrial Density, Amino Acid | |||
|enzymes=Marker Enzyme | |||
}} | }} |
Revision as of 09:39, 17 September 2010
Bosworth CA, Toledo JC Jr, Zmijewski JW, Li Q, Lancaster JR (2009) Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide. Proc. Natl. Acad. Sci. USA 106: 4671-4676. |
Bosworth CA, Toledo JC Jr, Zmijewski JW, Li Q, Lancaster JR (2009) Proc. Natl. Acad. Sci.
Abstract: Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (โขNO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of โขNO with O2, reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of โขNO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (<8 ฮผM) O2 concentrations and exhibits zero-order dependence on โขNO concentration. Indeed, RSNO formation is not inhibited even at O2 levels <1 ฮผM. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by >50%. One possible metal-dependent, O2-independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of โขNO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by โ50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free โขNO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells. โข Keywords: Iron, Nitrosation, Reactive nitrogen species, Reactive oxygen species, Chelatable iron
Labels:
Stress:RONS; Oxidative Stress"RONS; Oxidative Stress" is not in the list (Cell death, Cryopreservation, Ischemia-reperfusion, Permeability transition, Oxidative stress;RONS, Temperature, Hypoxia, Mitochondrial disease) of allowed values for the "Stress" property. Organism: Yeast; Fungi"Yeast; Fungi" is not in the list (Human, Pig, Mouse, Rat, Guinea pig, Bovines, Horse, Dog, Rabbit, Cat, ...) of allowed values for the "Mammal and model" property.
Preparation: Intact Cell; Cultured; Primary"Intact Cell; Cultured; Primary" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. Enzyme: Marker Enzyme"Marker Enzyme" is not in the list (Adenine nucleotide translocase, Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, Inner mt-membrane transporter, Marker enzyme, Supercomplex, TCA cycle and matrix dehydrogenases, ...) of allowed values for the "Enzyme" property. Regulation: Respiration; OXPHOS; ETS Capacity"Respiration; OXPHOS; ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Mitochondrial Biogenesis; Mitochondrial Density"Mitochondrial Biogenesis; Mitochondrial Density" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property., Amino Acid"Amino Acid" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.
HRR: Oxygraph-2k