Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.