Phosphorus-31 Nuclear Magnetic Resonance Studies of the Effect of Oxygen upon Glycolysis in Yeast

31P NMR spectra were obtained at 145.7 MHz on suspensions of Saccharomyces cerevisiae cells grown on glucose, raffinose, acetate, and ethanol carbon sources. The NMR spectra were measured either under the growth conditions (i.e., using cells suspended in the growth media in the presence of the growth carbon source and 02) or after feeding glucose under anaerobic and aerobic conditions. The NMR spectra allowed determination of intracellular pH (pHin), as well as the intracellular concentrations of P (Pin), ATP, and other phosphorylated metabolites. In measurements performed during the steady state of glycolysis after addition of glucose, changes in Pin levels or pHin were not observed upon oxygenation with glucose-repressed cells; however, in response to oxygen, derepressed cells showed a severalfold reduction in Pin concentration and an increase of pH'” by ~0.2-0.4 pH unit. According to in vitro data [Banuelos, M., Gancedo, C., & Gancedo, J. M. (1977) J. Biol. Chem. 252, 6394-6398], these particular changes of intracellular conditions should decrease the Kma, of phosphofructokinase by a factor of ~ 3 and could thereby contribute appreciably to the regulation of this enzyme in the presence of oxygen. The pH”1 values of Saccharomyces cerevisiae cells supplied with 02 and the growth carbon source were found to be between 7.3 and 7.5; measurements made at extracellular pH values between 3.5 and 7.2 showed that under these conditions the pHin values of these cells vary only by 0.1 pH unit. The 31P NMR spectra were also measured during derepression of glucose-repressed cells; it was observed that initially intracellular pH values were ~6.7 and increased to ~7.2 slowly over a period of ~60 min. The time course of a concentrated suspension of yeast cells showed a sudden increase in pHin approximately 4 min after glucose addition and time-dependent changes in P, fructose 1,6-bisphosphate, and ATP concentrations.