Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate

Abstract

Optimal conditions for the reversible inactivation of crystalline rabbit muscle phosphofructokinase [EC 2.7.1.11] by homogeneous rabbit liver fructose-1,6-bisphosphatase [EC 3.1.3.11] were studied. At higher enzyme levels (to 530 .mu.g/ml of phosphofructokinase) the 2 proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HCl, 2 mM potassium phosphate and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23 and 0.degree. C. Inactivation did not require ATP or Mg2+ in the incubation mixture, but 2.7 mM ATP or greater was required in the assay to keep phosphofructokinase in an inactive form. Inorganic phosphate, (NH4)2SO4 and AMP, when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase, had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320,000 g of enzyme. At lower phosphofructokinase concentrations (0.2-2 .mu.g/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. Inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of ATP was decreased. Inactivation was seen at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose-1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Direct interaction between phosphofructokinase and fructose-1,6-bisphosphatase was not seen. Under approximately physiological conditions, the presence of fructose-1,6-bisphosphatase may cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.