Journal
BIOCHEMICAL JOURNAL
Volume 370, Issue -, Pages 935-943Publisher
PORTLAND PRESS
DOI: 10.1042/BJ20021614
Keywords
apoptosis; diamide; GSH; NADPH; thiol
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Funding
- Telethon [324/BI] Funding Source: Medline
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Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells (G6pdDelta), obtained by transient Cre recombinase expression in a G6pd-loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 1.1.1.42). These G6pdDelta cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pdDelta cells show a high ratio of NADPH to NADP(+) and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pdDelta but not for wild-type ES cells, NADPH and GSH in G6pdDelta cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP(+)] ratio, does not occur in G6pdDelta cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.
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