Probing cellular protein targets of H2O2 with fluorescein-conjugated iodoacetamide and antibodies to fluorescein.
Recent studies suggest that H2O2, at subtoxic concentrations generated in response to the activation of a variety of cell surface receptors, functions as an intracellular messenger. However, the intracellular targets of H2O2 action have not been identified. A procedure to detect proteins with reactive cysteine residues susceptible to oxidation by intracellularly generated H2O2 is now described. This approach is based on the labeling of proteinaceous cysteine with 5-iodoacetamidofluorescein at pH 5.5 and immunoblot analysis of the labeled proteins with antibodies specific to fluorescein. With this procedure, many proteins in human A431 cells were shown to contain reactive cysteines and to be readily oxidized by H2O2 generated in response to cellular stimulation with epidermal growth factor. One of these H2O2-sensitive proteins was identified as protein tyrosine phosphatase 1B.
Wu
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Y
Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Kwon
K S
KS
Rhee
S G
SG
D000906:Antibodies; D015153:Blotting, Western; D002395:Catecholamines / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005452:Fluoresceins; D006801:Humans; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D048288:Imidazolines; D008970:Molecular Weight; D016877:Oxidants / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010206:Papain / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011233:Precipitin Tests; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002395:Catecholamines; D005452:Fluoresceins; D048288:Imidazolines; D016877:Oxidants; D011506:Proteins; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; C015296:5-iodoacetamidofluorescein; D006861:Hydrogen Peroxide; C011775:(3,4-dihydroxyphenylamino)-2-imidazoline; D017027:Protein Tyrosine Phosphatases; D010206:Papain; D003545:Cysteine