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

Abstract

31P NMR 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 tri-phosphates, Pi and sugar phosphates were measured during glycolysis with 2 min resolution, while intracellular and extra-cellular pH values were monitored simultaneously. Upon glucose addition, anaerobic E. coli cells rapidly produce acids and develop a transmembrane pH gradient (.DELTA.pH). Glycolysis rates were calculated from the changes in the external pH. Glycolysis rates are strongly dependent on internal pH, sharply decreasing when the pH drops below .apprx. 7.2. The ATPase inhibitor, dicyclohexylcarbodiimide (DCCD), prevented NTP [nucleoside triphosphate] hydrolysis and inhibited .DELTA.pH formation. The uncoupler, carbonyl cyanide p-triflouromethoxyphenyl hydrazone (FCCP), drastically reduced both the .DELTA.pH and the NTP level. When the cells were previously treated with DCCD, FCCP collapsed the .DELTA.pH while the NTP levels remained high. Apparently, ATP produced by glycolysis is hydrolyzed by the membrane ATPase to generate a .DELTA.pH and that FCCP stimulates ATP hydrolysis by ATPase and collapses the proton gradient.