Non-cooperative effects of glucose and 2-deoxyglucose on their metabolism in Saccharomyces cerevisiae studied by 1H-NMR and 13C-NMR spectroscopy.
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 degrees C in a standard pyrophosphate medium containing about 4.5 x 10(7) cells/ml. 1H-, 13C-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 dGlc6P/dGlc ratio were realised using [1-13C]dGlc and 13C-NMR spectroscopy. The rates of Glc consumption in the absence and in the presence of 5 mM dGlc and 20 mM dGlc were 29 +/- 0.03 mM/min (n = 3), 16 +/- 0.02 mM/min (n = 3), and 0.08 +/- 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 alpha and beta 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 dGlc6P/dGlc 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Herve
M
M
Département de Biologie Cellulaire et Moléculaire, CEN-Saclay, Gif-sur-Yvette, France.
Wietzerbin
J
J
Tran-Dinh
S
S
D002240:Carbohydrate Sequence; D002247:Carbon Isotopes; D003847:Deoxyglucose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014199:Trehalose / Q000378:metabolism
D002247:Carbon Isotopes; D011522:Protons; D003847:Deoxyglucose; D014199:Trehalose; D005947:Glucose