Accessibility of hepatocyte protein thiols to monobromobimane

Weis, Marianne; Cotgreave, I; Moore, Gregory A.; Norbeck, Kajsa; Moldéus, Peter

doi:10.1016/0167-4889(93)90171-k

Cited by 10 publications

(4 citation statements)

References 19 publications

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“…GSH (0-25 µM) treated with 250 µM monobromobimane and suspended in the same buffer was used as standard. Because this method has been shown with hepatocytes not to derivatize all protein thiols, analyses were also performed with 5 mM (final concentration) monobromobimane in the presence of 0.1 % Triton X-100 to maximize the exposure of thiol groups [19]. The total number of thiols detected under these conditions was three times that with the lower monobromobimane concentration, but in a control experiment it was found that the proportion of thiols lost was the same at both monobromobimane concentrations.…”

Section: Protein Thiolsmentioning

confidence: 99%

Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid

Carr

Winterbourn

1997

Biochemical Journal

103

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Neutrophils, when stimulated, generate reactive oxygen species including myeloperoxidase-derived HOCl. There is an associated decrease in reduced glutathione (GSH) concentration. We have shown that neutrophil GSH levels decrease on exposure to reagent HOCl, whereas the equivalent concentration of H2O2 had no effect. GSH loss occurred without cell lysis, was not reversible, and was accompanied by the loss of an equivalent proportion of the total protein thiols. No glutathione disulphide was formed. Studies with 35S-labelled cells indicated that much of the GSH lost was accounted for by mixed disulphides with protein and a product that co-migrated on HPLC with a novel compound formed in the reaction of HOCl and pure GSH. The properties of this compound are consistent with an intramolecular sulphonamide. Neutrophils stimulated with PMA lost 30-40% of their GSH and a similar proportion of protein thiols. Little glutathione disulphide was formed and the products were the same as seen with HOCl-treated cells. From the results and studies with inhibitors and scavengers, we conclude that HOCl was responsible for the GSH loss. Propargylglycine and buthionine sulphoximine, inhibitors of glutathione synthesis, enhanced GSH loss, but their effects were due to the production of long-lived chloramines that oxidized GSH with greater efficiency than HOCl, rather than to the inhibition of GSH synthesis. The lack of thiol selectivity by HOCl and irreversibility of oxidation means that GSH will provide limited antioxidant protection for thiol enzymes in stimulated neutrophils.

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Section: Protein Thiolsmentioning

confidence: 99%

Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid

Carr

Winterbourn

1997

Biochemical Journal

103

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“…Total free thiols were measured by a reaction with monobromobimane (mBBr) leading to the formation of a stable fluorescent adduct [25,26]. RKO cells were seeded in a 6-well plate at a density of 3.5 × 10 5 cells/well.…”

Section: Methodsmentioning

confidence: 99%

Cytotoxicity of 1,4-diamino-2-butanone, a putrescine analogue, to RKO cells: mechanism and redox imbalance

Soares

Boiani

Marnett

et al. 2013

Free Radical Research

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α-Aminocarbonyl metabolites (e.g., 5-aminolevulinic acid and aminoacetone) and the wide spectrum microbicide 1,4-diamino-2-butanone (DAB) have been shown to exhibit pro-oxidant properties. In vitro, these compounds undergo phosphate-catalyzed enolization at physiological pH and subsequent superoxide radical-propagated aerobic oxidation, yielding a reactive α-oxoaldehyde and H2O2. DAB cytotoxicity to pathogenic microorganisms has been attributed to the inhibition of polyamine biosynthesis. However, the role played in cell death by reactive DAB oxidation products is still poorly understood. This work aims to clarify the mechanism of DAB-promoted pro-oxidant action on mammalian cells. DAB (0.05-10 mM) treatment of RKO cells derived from human colon carcinoma led to a decrease in cell viability (IC50 ca. 0.3 mM DAB, 24 h incubation). Pre-addition of either catalase (5 μM) or aminoguanidine (20 mM) was observed to partially inhibit the toxic effects of DAB to the cells, while N-acetyl-L-cysteine (NAC, 5 mM) or reduced glutathione (GSH, 5 mM) provided almost complete protection against DAB. Changes in redox balance and stress response pathways were indicated by the increased expression of HO-1, NQO1 and xCT. Moreover, the observation of caspase 3 and PARP cleavage products is consistent with DAB-triggered apoptosis in RKO cells, which was corroborated by the partial protection afforded by the pan-caspase inhibitor z-VAD-FMK. Finally, DAB treatment disrupted the cell cycle in response to increased p53 and activation of ATM. Altogether, these data support the hypothesis that DAB exerts cytotoxicity via a mechanism involving not only polyamine biosynthesis but also by DAB oxidation products.

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“…The disadvantage, as compared with isoelectric focussing, is the rather low separation efficiency . 20 potential target proteins were identified by the specific labelling of the cysteines with monobromobimane (mBBr) (Kobrehel et al, 1992;Weis et al, 1993).…”

Section: Gel-based (2d Gel Electrophoresis) Labelling Methodsmentioning

confidence: 99%

Concepts and Approaches Towards Understanding the Cellular Redox Proteome

Ströher¹,

Dietz²

2006

Plant Biology

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The physiological activity of a significant subset of cell proteins is modified by the redox state of regulatory thiols. The cellular redox homeostasis depends on the balance between oxidation of thiols through oxygen and reactive oxygen species and reduction by thiol-disulfide transfer reactions. Novel and improved methodology has been designed during recent years to address the level of thiol/disulfide regulation on a genome-wide scale. The approaches are either based on gel electrophoresis or on chromatographic techniques coupled to high end mass spectrometry. The review addresses diagonal 2D-SDS-PAGE, targeted identification of specific redox-interactions, affinity chromatography with thioredoxins and glutaredoxins, gel-based and non-gel based labelling techniques with fluorophores (such as Cy3, Cy5, ICy), radioisotopes, or with isotope-coded affinity tags (ICAT), differential gel electrophoresis (DIGE) and combined fractional diagonal chromatography (COFRADIC). The extended methodological repertoire promises fast and new insight into the intricate regulation network of the redox proteome of animals, bacteria, and plants.

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Accessibility of hepatocyte protein thiols to monobromobimane

Cited by 10 publications

References 19 publications

Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid

Oxidation of neutrophil glutathione and protein thiols by myeloperoxidase-derived hypochlorous acid

Cytotoxicity of 1,4-diamino-2-butanone, a putrescine analogue, to RKO cells: mechanism and redox imbalance

Concepts and Approaches Towards Understanding the Cellular Redox Proteome

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