³¹P nuclear magnetic resonance studies of bioenergetics and glycolysis in anaerobic Escherichia coli cells

³¹P nuclear magnetic resonance spectra of glycolyzing, anaerobic E. coli cells and their perchloric acid extracts were obtained at 145.7 MHz. Time-dependent intracellular concentrations of nucleoside di- and triphosphates, Pi, and sugar phosphates were measured during glycolysis with 2-min resolution, while intracellular and extracellular pH values were monitored simultaneously. Upon glucose addition, anaerobic E. coli cells rapidly produce acids and develop a transmembrane pH gradient (ΔpH). Glycolysis rates were calculated from the changes in the external pH. It was found that glycolysis rates are strongly dependent on internal pH, sharply decreasing when the pH drops below ~7.2. The ATPase inhibitor, dicyclohexylcarbodiimide (DCCD), prevented NTP hydrolysis and inhibited ΔpH formation. The uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), drastically reduced both the ΔpH and the NTP level. When the cells were previously treated with DCCD, FCCP collapsed the ΔpH while the NTP levels remained high. It is concluded that ATP produced by glycolysis is hydrolyzed by the membrane ATPase to generate a ΔpH and that FCCP stimulates ATP hydrolysis by ATPase and collapses the proton gradient.