Revised Manuscript Received May 18, 1987 abstract: 31P nuclear magnetic resonance(NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. Our results have revealed that the reactions catalyzed by glycer-aldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A. Intracellular phosphate (P¡) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state saturation and were used in combination with ST data to determine P¡ consumption rates. 13C NMR and 02 electrodemeasurements were also conducted to monitor changes in rates of glucose consumption and 02 consumption, respectively, under the various metabolic conditions examined. Our results suggest that GAPDH/PGK-catalyzed P¡-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobicand aerobic glucose-fed yeast. Kinetics through GAPDH/PGK were found to depend on metabolic conditions. The coupled systemappears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol. Additionally, mitochondrialATPase activity appears to be responsible for the transfer observed in iodo-acetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally.

31P NMR1 magnetization-transfer techniques have been used to measure steady-state kinetics of enzyme-catalyzed reactions in a variety of living systems [for reviews, see Alger and Shulman (1984) and Koretsky and Weiner (1984)]. In particular, saturation-transfer experiments performed by se-lectively saturating the-phosphate of ATP and monitoring the change in the intracellular phosphate intensity (P; in) have measured phosphate-ATP exchange reactions in vivo. In yeast (Alger et al., 1982; Campbell et al., 1985; Brindle & Krikler, 1985), the Langendorf perfused heart (Matthews et al., 1981; Kingsley-Hickman et al., 1986), the perfused and in situ rat kidney (Freeman et al., 1983), and the in situ rat brain (Shoubridge et al., 1982), rates of oxygen consumption were determined and combined with the ST-measured P¡ con-sumption rates to determine P/O ratios. The P/O ratio is