In vivo phosphorus-31 NMR saturation transfer studies of ATPase kinetics in Saccharomyces cerevisiae

Abstract

Phosphorus-31 saturation transfer NMR techniques have been employed to measure the unidirectional Pi consumption rate by respiration competent suspensions of the yeast Saccharomyces cerevisiae while the levels of ATP, ADP, and Pi are constant. These experiments are performed by saturating the ATP gamma phosphate resonance and observing the changes in the Pi resonance intensity while the yeast are respiring on endogenous substrates. The unidirectional Pi consumption rate is 3.5 +/- mumol s-1 (g of wet cells)-1. The rate is reduced 10-fold upon addition of oligomycin (80 micrograms/ML), suggesting that at least 90% of the Pi consumption activity is due to the mitochondrial F1-F0 ATPase. We have not been able to conclusively assign the remaining 10%. When the yeast are glycolyzing anaerobically, the unidirectional Pi consumption rate was 1.0 +/- 0.2 mumol s-1 (g of wet cells)-1. At most, 80% of this is due to Pi consumption by the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase leaving a residual activity of at least 0.2 mumol s-1 (g of wet cells)-1. Thus the activity in the oligomycin-inhibited cells under respiratory conditions and the nonglycolytic activity in anaerobic cells are equal to within the experimental errors. Furthermore the unidirectional rate of Pi consumption during anaerobic glycolysis is insensitive to oligomycin. These data suggest that the mitochondrial adenosinetriphosphatase is not turning over during anaerobic glycolysis. Possible explanations for this inhibition are discussed.