Non‐cooperative effects of glucose and 2‐deoxyglucose on their metabolism in Saccharomyces cerevisiae studied by ¹H‐NMR and ¹³C‐NMR spectroscopy

Abstract

The reciprocal effects of 2-deoxy-D-glucose (dGlc) and glucose (Glc) on the aerobic metabolism of Glc and dGlc in Glc-grown repressed Saccharomyces cerevisiae were studied at 30°C in a standard pyrophosphate medium containing about 4.5 X lo7 cells/ml. 'H-, 'T-NMR and biochemical techniques were used for quantitative evaluation of Glc and dGlc metabolites. The detection of intracellular dGlc and the determination of the intracellular dGlc6PldGlc ratio were realised using [l-'3C]dGlc and 'T-NMR spectroscopy. The rates of Glc consumption in the absence and in the presence of 5 mM dGlc and 20 mM dGlc were 29 -t-0.03 mM/min (n = 3), 16 2 0.02 mM/min (n = 3), and 0.08 -t-0.01 mM/min (n = 3), respectively. This means that the Glc consumption is reduced about 41% and 70% in the presence of 5 mM and 20 mM dGlc, respectively. When dGlc is the unique carbon source, only a and p anomers of dGlc6P were formed. Their quantities are equivalent and reach the maximum values within 1 h of incubation and then decrease gradually. By contrast, Glc favours the consumption of dGlc and the synthesis of dGlc6P, dideoxy-trehalose (dGlc-dGlc), deoxy-trehalose (dGlc-Glc). In the presence of Glc, dGlc6P reaches a plateau after 1 h or 2 h of incubation while the quantities of trehalose (Glc-Glc), dGlc-dGlc, dGlc-Glc, which are small at 1 h, rapidly increase with time. The reasons why dGlc and Glc exert opposite effects on their metabolism are discussed.The production of Glc-Glc decreases with increasing the external dGlc concentration or the dGlc/ Glc ratio. The effect of dGlc on the biosynthesis of Glc-Glc can be explained by the competition of dGlc and Glc with respect to hexokinase. Although Glc favours the synthesis of dGlc6P, the maximum concentration of dGlc6P shows little dependence on the external dGlc concentration as long as glucose is not exhausted. The internal dGlc6PldGlc ratio at equilibrium, about 4.7 ? 0.7, is also found to be independent of the dGlc Concentration in the suspension. Only a small fraction of dGlc6P disappears to give rise to the formation of dGlc-dGlc and dGlc-Glc. At equilibrium the inverse reaction from dGlc6P to dGlc may be important to compensate for the fast reaction of dGlc phosphorylation by hexokinase. At least nine series of experiments were conducted and showed that, in pyrophosphate media and for incubation times less than 4 h, dGlc-dGlc was not observed in the absence of Glc. However, as for dGlc6P, the production of dGlc-dGlc does not depend very much on the external concentration of dGlc and Glc. The production of dGlc-Glc is proportional to the Glc concentration but insensitive to the dGlc concentration. Apart from dGlc6P, dGlc-Glc is the most abundant metabolite of dGlc and Glc. The synthesis of this compound results in an interaction between the precursors arising from two different carbon sources. Thus the effect of a drug strongly depends on the physiological state of cells. The presence of an energy source such as Glc was shown to be able to stimulate the drug consumption and the formation ...

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

“…We expected that the inhibition of glycolytic oscillations by 2-DG would, similar to what is observed after inhibition with iodoacetate ( Fig. 5), be accompanied by a dramatic decrease in the intracellular ATP because of its inhibition of the phosphorylation of glucose to glucose 6-phosphate (38). Instead we observed an almost stationary but moderately decreased ATP concentration, indicating that glycolysis is still active.…”

“…Iodoacetate is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (37). 2-DG is phosphorylated in a reaction catalyzed by hexokinase (38). It is generally assumed that the 2-DG-6-phosphate formed cannot be metabolized further, and hence ATP production by the lower part of glycolysis is inhibited, resulting in a decrease in glucose consumption and accumulation of intracellular glucose (22,38).…”

“…2-DG is phosphorylated in a reaction catalyzed by hexokinase (38). It is generally assumed that the 2-DG-6-phosphate formed cannot be metabolized further, and hence ATP production by the lower part of glycolysis is inhibited, resulting in a decrease in glucose consumption and accumulation of intracellular glucose (22,38). Addition of 20 mM iodoacetate to a suspension of yeast cells with an oscillating glycolysis resulted in a fast decline in intracellular ATP and also in a decrease in the NADH fluorescence corresponding to a slow oxidation of NADH as shown in Fig.…”

Detection of modifications in the glucose metabolism induced by genetic mutations in Saccharomyces cerevisiae by 13C‐ and 1H‐NMR spectroscopy

“…NMR has already been successfully used to investigate the effect of perturbing conditions on cellular metabolism such as drugs on yeast [1,4–7], different physiological conditions in cardiac cells [8–13], in renal cells [14], in cerebral tissues [15], in liver [16], HIV infection in epithelial cells [17] and cellular stress [18,19]. In these studies, a substrate was metabolized and the modifications induced by the various perturbations in cellular metabolism were clearly detected.…”

On the mechanism of oscillations in neutrophils

“…To support that glycolysis is not involved in the oscillations we added 2-deoxyglucose (2-DOG), which is phosphorylated by hexokinase. Phosphorylated 2-DOG cannot be metabolized further, and the cell is therefore depleted of ATP [30,31]. When we incubated cells with 2-DOG for 10 min before stimulation we did not observe any change in the oscillations in H 2 O 2 production, which indicates that glycolysis is not involved in the oscillations.…”