The Effect of pH on Glycolysis and Phosphofructokinase Activity in Cultured Cells and Synaptosomes

: The effect of [H+] on the rate of glycolysis was investigated in glioma C6 and fibroblast BHK‐21 cells and in synaptosomes from rat brain. The rates of lactate production at an extracellular pH (pHe) of 6.2, 7.4, and 7.8 were correlated with intracellular [ATP], [ADP], and [Pi] ([ATP]i, [ADP]i, and [Pi]i, respectively) and, when relevant, creatine phosphate (PCr) as well as with the levels of several glycolytic intermediates. In C6 cells cytosolic [H+] was measured simultaneously together with [Ca2+], [K+], [Na+], and membrane potentials. In all three systems studied, an increase in [H+]e suppressed whereas a fall enhanced the rate of lactate generation. Changes in pHe produced no simple correlation between the amount of lactate formed and alterations either in the absolute [ATP], [ADP], [Pi], and [PCr] or their ratios but did correlate with the levels of glycolytic intermediates. Higher [fructose‐1,6‐bisphosphate] and [glyceraldehyde‐3‐phosphate] and lower [glucose‐6‐phosphate] and [fructose‐6‐phosphate] accompanied faster glycolytic activity. Addition of rotenone markedly enhanced glycolysis at all pHe values studied. The increases were larger at higher [H+] so that the rate of lactate generation was only slightly lower at pH 6.2 than at 7.4 or 7.8. With rotenone present, [ATP] (and where relevant [PCr]) fell and [ADP] and [Pi] rose under all pHe conditions. Simultaneously [glucose‐6‐phosphate] and [fructose‐6‐phosphate] decreased whereas [fructose‐1,6‐bisphosphate] and [glyceraldehyde‐3‐phosphate] increased; the levels of the last two were similar at pH 6.2 and 7.4. Alterations in concentrations of cations other than H+ were small and unlikely to contribute to the regulation of glycolysis. It is concluded that (a) under intracellular conditions, in the presence of a high [ATP] and a low [ADP] and [AMP], a fall in [H+] powerfully inhibits phosphofructokinase; lactate production correlates with the levels of glycolytic intermediates. (b) A small decline in cellular energy state is sufficient to release phosphofructokinase inhibition by protons. (c) Once activated by the decrease in energy level, the steady‐state glycolytic rate is dependent on other factors, such as glycolytic intermediates and enzymes.