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31P-NMR saturation transfer studies of aerobic Escherichia coli cells

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Abstract

31P-NMR measurements of saturation transfer have been used to measure the flux between Pi and ATP in Escherichia coli cells respiring on an endogenous carbon source. Measurements were made in the wild type and in cells genetically modified to give a 5-fold higher concentration of the F1F0-ATP synthase. The flux in the two cell types was not significantly different. This, together with studies using inhibitors specific for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase and the ATP synthase, suggests that the observed flux arises predominantly from glycolytic rather than ATP synthase activity. Although this conclusion is in disagreement with previous experiments on E. coli, it is in agreement with recent experiments on yeast.

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      Resonance saturation also underlies recent Chemical Exchange Saturation Transfer (CEST) studies for advanced protein conformational studies, notably on “invisible” minor species [848,849]. In the case of living cells, 31P-NMR saturation transfer was applied in the 1980’s to quantify rates of inorganic phosphate or ATP consumption in E. coli [850], in human erythrocytes [851], in breast cancer cells [852] and also the fluxes towards glucose-6-phosphate or fructose-1,6-bis-phosphate in aerobic and anaerobic conditions in S. cerevisiae [746,853-855]. In parallel, a number of groups used 31P-resonance saturation transfer to measure ATP and phosphocreatine consumption and replenishment, hence measuring creatine or ATPase kinase activity in rabbit [856] and rat hearts [857-862], in frog [863] and cat muscles [864] and in rat brain [865].

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    Present address; National Institute for Environmental Studies, Yatabe, Tsukuba, Ibaraki 305, Japan.

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