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Phosphorylation of purified yeast fructose-1,6-bisphosphatase was studied using purified preparations from yeast of two different cyclic AMP-independent protein kinases and a cyclic AMP-dependent protein kinase. Incorporation of 32P into fructose-1,6-bisphosphatase could be demonstrated only with the cyclic AMP-dependent protein kinase. Phosphorylation of fructose-1,6-bisphosphatase was stimulated by 3 microM fructose-2,6-bisphosphate and inhibited by 1 mM 5'-AMP.
Treatment of rat liver-type pyruvate kinase with rabbit liver cathepsin B at pH 7.0 caused loss of activity in the standard assay with 0.6 mM of phosphoenolpyruvate. The modified enzyme exhibited about 10% of the original activity when assayed with 2.0 mM of the substrate. No detectable change in the subunit molecular weight of the enzyme occurred during inactivation. On addition of 4 microM fructose 1,6-bisphosphate the activity of the treated enzyme was restored to that of the original enzyme. Limited proteolysis of the enzyme by cathepsin B appears to enhance the requirement for the positive effector, fructose 1,6-bisphosphate.
The phosphofructokinase stabilizing factor, believed to be a peptide of molecular weight 3,800 (Dunaway G.A. and Segal H.L., 1976, J. Biol. Chem. 251, 2323-2329), shares many chemical and biological properties with fructose 2,6-bisphosphate. It co-migrated with it upon gel filtration in the molecular weight range 300-400 or 3,000-4,000 depending upon the ionic strength of the solution. Fructose 2,6-bisphosphate is the most potent phosphofructokinase stabilizing agent present in the liver of a fed rat. Its disappearance during fasting and diabetes could account for the faster rate of degradation of phosphofructokinase reported to occur under these conditions. The effect of starvation to decrease by 60% the phosphofructokinase content of the liver is, however, for its greatest part, related to a non-specific decrease in liver mass.
In order to investigate a possible regulatory role of fructose 2,6-bisphosphate in early developmental stages, where profound changes in the carbohydrate metabolism are known to occur, this effector was estimated in fetal and postnatal rat liver. Polyphasic changes of the hepatic fructose 2,6-bisphosphate levels were found, which could be correlated to alterations in the glucose metabolism. A minimum in the hepatic fructose 2,6-bisphosphate level at the -3rd day coincides with the initiation of glycogen synthesis and its increase two hours after birth concurs with glycogen mobilization.
The level of fructose 2,6-bisphosphate is markedly decreased in the rat V.renal gland in diabetes, falling to 23% of the control value. There is parallel decrease in the flux of 14C-labelled glucose through the glycolytic route and tricarboxylic acid cycle. Only minimal changes in hexokinase (EC 2.7.1.1.), a 22% decrease in Type I hexokinase of the soluble fraction, were observed, highlighting the probable significant involvement of fructose 2,6-bisphosphate in the regulation of glycolysis in the adrenal. In contrast, there was evidence for a marked rise in the flux of glucose through the pentose phosphate pathway, which may be linked to enhanced corticoid synthesis in the diabetic state.
Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 microM, 50% inhibition was observed at 4.8 microM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (Ki = 16 microM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate - fructose-1,6-bisphosphatase active site interaction.
The flux of glucose through the pentose phosphate pathway, important in relation to the provision of ribose 5-phosphate for nucleotide and RNA synthesis, was decreased by 70% in the diabetic rat heart in parallel with a similar decreased flux through the glycolytic route. A common factor linking the decreased flux through these alternative routes is the known fall in cardiac hexokinase; in these experiments there is a 50% decrease in Type II hexokinase (EC 2.7.1.1.) in both soluble and particulate fractions. The level of fructose 2,6-bisphosphate, a regulator of phosphofructokinase activity, is decreased by 20% in the alloxan diabetic rat heart, this may be a significant additional factor in the marked decrease in the flux of glucose through the glycolytic route in the myocardium in diabetes.
Partially purified trehalose phosphorylase (EC 2.4.1.64) from Euglena gracilis SM-ZK was inhibited by fructose 2,6-bisphosphate in both synthetic and degradative directions. Ki value for trehalose phosphorolysis was 1.2 microM and that for trehalose synthesis was 0.5 microM. Functions of fructose 2,6-bisphosphate in Euglena, particularly in the regulative mechanism of the two reserve carbohydrates, paramylon and trehalose, are discussed.
Fructose 2,6-bisphosphate levels were assayed in freeze-clamped livers of anesthesized rats. All doses of pentobarbital that were effective in anesthesizing the rats caused a significant decrease in fructose 2,6-bisphosphate levels. Injection of pentobarbital also resulted in decreased 6-phosphofructo 2-kinase activity and increased fructose 2,6-bisphosphatase activity measured in dialyzed (NH4)2SO4-treated liver extracts but with no change in pyruvate kinase activity. It was concluded that the anesthesia-induced decrease in fructose 2,6-bisphosphate levels results at least in part from increased phosphorylation of 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase.
Hepatic phosphofructokinase, isolated in a medium containing 100 mM (NH4)2SO4, can be activated by ATP. This metabolite-induced activation was investigated in view of the suggestion that it is related to phosphorylation of phosphofructokinase. The results obtained do not support this interpretation. Inhibitors of protein phosphatases (NaF) and kinases (the Mg++-chelator, ethylene diamine tetraacetic acid) did not affect the recovery of phosphofructokinase. In contrast, media of high ionic strength reduced the phosphofructokinase activity and rendered the enzyme sensitive to ATP-induced activation. Activation was also induced by other known effectors of phosphofructokinase (nucleoside triphosphates, fructose bisphosphates) and was not dependent on Mg++-ions. It is suggested that activation represents ligand-induced reversal of the inactivation of phosphofructokinase which occurs at high ionic strength. The differential sensitivity of phosphofructokinase from fed or starved animals to inactivation and reactivation is discussed.
Hyperglycemic mice with streptozotocin diabetes were divided into two groups according to the presence or absence of ketosis. No difference in blood glucose level between two groups was observed in this experiment. However, hepatic fructose-2,6-P2 level and fructose-6-P,2-kinase activity were decreased only in ketotic diabetic mice. Similar decreases in those indices were observed in 48-h starved normal mice. In ketotic diabetes, insulinization for 24 h was required to normalize fructose-2,6-P2 level and fructose-6-P,2-kinase activity, while glucose administration normalized altered fructose-2,6-P2 metabolism in starvation only in 30 min. Hepatic cyclic AMP was increased neither in ketotic nor in non-ketotic diabetic mice. These results indicate that the decrease in hepatic fructose-2,6-P2 level in diabetes is apparently related to the occurrence of ketosis, but not to hyperglycemia. The mechanisms of the decrease in fructose-6-P,2-kinase activity in ketotic diabetes and starvation are discussed.
In a reconstituted enzyme system multiple stationary states and oscillatory motions of the substrate cycle catalyzed by phosphofructokinase and fructose 1,6-bisphosphatase are significantly influenced by fructose 2,6-bisphosphate. Depending on the initial conditions, fructose 2,6-bisphosphate was found either to generate or to extinguish oscillatory motions between glycolytic and gluconeogenic states. In general, stable glycolytic modes are favored because of the efficient activation of phosphofructokinase by this effector. The complex effect of fructose 2,6-bisphosphate on the rate of substrate cycling correlates with its synergistic cooperation with AMP in the activation of phosphofructokinase and inhibition of fructose 1,6-bisphosphatase.
The cooperation of phosphofructokinase-2 and fructose-2,6-bisphosphatase is investigated. Experimentally derived rate laws of the kinase and bisphosphatase activities introduced into the respective differential equations permitted to describe the time evolution of fructose-2,6-bisphosphate to quasi-stationary levels. The two enzyme activities were found to exert strong temperature dependence. The quasi-stationary levels of fructose-2,6-bisphosphate, however, are independent on temperature.
The alpha- and beta-anomers of arabinose 1,5-bisphosphate and ribose 1,5-bisphosphate were tested as effectors of rat liver 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase. Both anomers of arabinose 1,5-bisphosphate activated the kinase and inhibited the bisphosphatase. The alpha-anomer was the more effective kinase activator while the beta-anomer was the more potent inhibitor of the bisphosphatase. Inhibition of the bisphosphatase by both anomers was competitive, and both potentiated allosteric inhibition by AMP. beta-Arabinose 1,5-bisphosphate was also more effective in decreasing fructose 2,6-bisphosphate binding to the enzyme. Neither anomer of ribose 1,5-bisphosphate affected 6-phosphofructo-1-kinase or fructose-1,6-bisphosphatase, indicating that the configuration of the C-2 (C-3 in Fru 2,6-P2) hydroxyl group is important for biological activity. These results are also consistent with arabinose 1,5-bisphosphate binding to the active site and thereby enhancing the interaction of AMP with the allosteric site.
Our report presents data on the phosphorylation of muscle phosphofructokinase by Ca2+-activated, phospholipid-dependent protein kinase. We have found a stoichiometrical phosphorylation (about 1.5 mol per mol subunit), and a low apparent Km (about 0.7 microM). These data speak in favor of a physiological role for the reaction, as does the fact that phosphofructokinase from a new species (rat) was successfully phosphorylated. On the other hand we present the hitherto unpublished circumstance that the phosphorylation is inhibited by conditions that stabilise the activity of phosphofructokinase. This fact makes us question the true significance of this reaction.
The levels of fructose 2,6-P2 and 6-phosphofructo 2-kinase have been found to be decreased in the liver of both ketotic and non-ketotic diabetic rats, a good correlation between fall of hepatic fructose 2,6-P2, ketonemia and glycemia being observed. The "total" 6-phosphofructo 2-kinase activity and the "active" (non-phosphorylated) from of the enzyme were decreased to a different extent, resulting in a fall of the "active"/"total" activity ratio. Hepatic levels of glucose 1,6-P2 were lowered only in ketotic diabetes. Insulin treatment normalized all the values studied. Insulin administration to control rats decreased the hepatic levels of fructose 2,6-P2 and did not affect glucose 1,6-P2 levels. It also decreased the "active" form of 6-phosphofructo 2-kinase, without significantly altering the "total" activity.