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Imaging Dynamic Redox Changes in Mammalian Cells with Green Fluorescent Protein Indicators
Ist Teil von
The Journal of biological chemistry, 2004-05, Vol.279 (21), p.22284-22293
Ort / Verlag
United States: American Society for Biochemistry and Molecular Biology
Erscheinungsjahr
2004
Link zum Volltext
Quelle
Alma/SFX Local Collection
Beschreibungen/Notizen
Changes in the redox equilibrium of cells influence a host of cell functions. Alterations in the redox equilibrium are precipitated
by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive
green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator. Ratios of fluorescence
from excitation at 400 and 490 nm indicate the extent of oxidation and thus the redox potential while canceling out the amount
of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific
proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 (GFP with mutations
C48S, S147C, and Q204C) and roGFP2 (the same plus S65T) with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes
during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H 2 O 2 -stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid.