The Fluorescein-derived Dye Aminophenyl Fluorescein Is a Suitable Tool to Detect Hypobromous Acid (HOBr)-producing Activity in Eosinophils

Flemmig, Jörg; Zschaler, Josefin; Remmler, Johannes; Arnhold, Jürgen

doi:10.1074/jbc.m112.364299

Cited by 40 publications

(34 citation statements)

References 70 publications

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“…23,24 Here the HOCl-derived oxidation of APF was followed at 25 °C by measuring the fluorescence intensity at 522 nm (excitation at 488 nm). The fluorescence spectrophotometer (Hitachi F-7000) was equipped with a thermostatic cell holder for quartz cuvettes with a path length of 10 mm.…”

Section: Methodsmentioning

confidence: 99%

Transiently Produced Hypochlorite Is Responsible for the Irreversible Inhibition of Chlorite Dismutase

et al. 2014

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Chlorite dismutases (Clds) are heme b-containing prokaryotic oxidoreductases that catalyze the reduction of chlorite to chloride with the concomitant release of molecular oxygen. Over time, they are irreversibly inactivated. To elucidate the mechanism of inactivation and investigate the role of the postulated intermediate hypochlorite, the pentameric chlorite dismutase of “Candidatus Nitrospira defluvii” (NdCld) and two variants (having the conserved distal arginine 173 exchanged with alanine and lysine) were recombinantly produced in Escherichia coli. Exchange of the distal arginine boosts the extent of irreversible inactivation. In the presence of the hypochlorite traps methionine, monochlorodimedone, and 2-[6-(4-aminophenoxy)-3-oxo-3H-xanthen-9-yl]benzoic acid, the extent of chlorite degradation and release of molecular oxygen is significantly increased, whereas heme bleaching and oxidative modifications of the protein are suppressed. Among other modifications, hypochlorite-mediated formation of chlorinated tyrosines is demonstrated by mass spectrometry. The data obtained were analyzed with respect to the proposed reaction mechanism for chlorite degradation and its dependence on pH. We discuss the role of distal Arg173 by keeping hypochlorite in the reaction sphere for O–O bond formation.

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Section: Methodsmentioning

confidence: 99%

Transiently Produced Hypochlorite Is Responsible for the Irreversible Inhibition of Chlorite Dismutase

et al. 2014

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“…Cells were harvested using cold 1 mL PBS containing 5 mM EDTA, and peroxynitrite was measured by flow cytometry26. For measurement of NO using diaminofluorescein-FM diacetate (DAF-FM DA), cells were incubated with 10 μM DAF FM-DA and 10 μM BAPTA-AM in 450 μL KRB Buffer (89 mM NaCl, 4.7 mM KCl, 25 mM NaHCO 3 , 1.2 mM MgSO 4 -7H 2 O, 1.2 mM KH 2 PO 4 , 2.5 mM CaCl 2 -2H 2 O, 11.1 mM D-Glucose, 20 mM Hepes-Na, pH7.4) for 1 hr, then incubated with CCCP (10 μM) for 1 hr, and finally cells were harvested using cold 1 mL PBS containing 5 mM EDTA and NO was measured by flow cytometry27.…”

Section: Methodsmentioning

confidence: 99%

S-nitrosylation regulates mitochondrial quality control via activation of parkin

Ozawa

Komatsubara

Nishimura

et al. 2013

Sci Rep

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Parkin, a ubiquitin E3 ligase of the ring between ring fingers family, has been implicated in mitochondrial quality control. A series of recent reports have suggested that the recruitment of parkin is regulated by phosphorylation. However, the molecular mechanism that activates parkin to induce mitochondrial degradation is not well understood. Here, and in contrast to previous reports that S-nitrosylation of parkin is exclusively inhibitory, we identify a previously unrecognized site of S-nitrosylation in parkin (Cys323) that induces mitochondrial degradation. We demonstrate that endogenous S-nitrosylation of parkin is in fact responsible for activation of its E3 ligase activity to induce aggregation and degradation. We further demonstrate that mitochondrial uncoupling agents result in denitrosylation of parkin, and that prevention of denitrosylation restores mitochondrial degradation. Our data indicates that NO both positive effects on mitochondrial quality control, and suggest that targeted S-nitrosylation could provide a novel therapeutic strategy against Parkinson's disease.

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“…Active MPO is unique in generating HOCl in the presence of H 2 O 2 . APF detects HOCl in living cells, as HOCl oxidizes the nonfluorescent substrate into fluorescent APF (22). First, we verified the APF detection system using human neutrophils (n ϭ 5).…”

Section: Mpo Acquired By Ecs Is Enzymatically Active As Shownmentioning

confidence: 99%

“…Assessment of MPO Activity Using Aminophenyl Fluorescein, Flow Cytometry, and Fluorescence Microscopy-The flow cytometry assay was performed as described recently (22). ECs or neutrophils were loaded for 30 min with 200 l of aminophenyl fluorescein (APF) (final concentration 5 M).…”

Section: Methodsmentioning

confidence: 99%

β2 Integrin-mediated Cell-Cell Contact Transfers Active Myeloperoxidase from Neutrophils to Endothelial Cells

Jerke

Rolle

Purfürst

et al. 2013

Journal of Biological Chemistry

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Background: How endothelial cells (ECs) could acquire exogenous neutrophil myeloperoxidase (MPO) is unknown. Results: ECs acquired enzymatically active MPO directly from neutrophils, via ␤2 integrin-mediated cell-cell contact independent of extracellular MPO release. Conclusion: Neutrophils directly transfer MPO to ECs by cell-cell contact. Significance: Direct delivery of MPO to ECs may contribute to the vascular pathology and dysfunction seen in atherosclerosis and vasculitides.

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The Fluorescein-derived Dye Aminophenyl Fluorescein Is a Suitable Tool to Detect Hypobromous Acid (HOBr)-producing Activity in Eosinophils

Cited by 40 publications

References 70 publications

Transiently Produced Hypochlorite Is Responsible for the Irreversible Inhibition of Chlorite Dismutase

Transiently Produced Hypochlorite Is Responsible for the Irreversible Inhibition of Chlorite Dismutase

S-nitrosylation regulates mitochondrial quality control via activation of parkin

β2 Integrin-mediated Cell-Cell Contact Transfers Active Myeloperoxidase from Neutrophils to Endothelial Cells

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