Effects of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate on phosphofructokinase from chicken erythrocytes

1. Phosphofructokinase (EC 2.7.1.11) from chicken erythrocytes is activated by fructose 2,6-bisphosphate, glucose 1,6-bisphosphate and AMP, and it is inhibited by 2,3-bisphosphoglycerate and inositol hexaphosphate. 2. The stimulatory effects produced by the two bisphosphorylated hexoses are additive and the effects produced by fructose 2,6-bisphosphate and by AMP are synergistic. 3. The activatory effect produced by fructose 2,6-bisphosphate is counteracted by fructose 1,6-bisphosphate. 4. The inhibition produced by both 2,3-bisphosphoglycerate and inositol hexaphosphate is released by fructose 2,6-bisphosphate. 5. It is concluded that, like phosphofructokinase from mammalian tissues, the enzyme from chicken erythrocytes can be modulated by the relative concentrations of those metabolites.     

Effects of fructose 1,6-bisphosphate on the activation of yeast phosphofructokinase by fructose 2,6-bisphosphate and AMP

Fructose 1,6-bisphosphate decreases the activation of yeast 6-phosphofructokinase (ATP:fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) by fructose 2,6-bisphosphate, especially at cellular substrate concentrations. AMP activation of the enzyme is not influenced by fructose 1,6-bisphosphate. Inorganic phosphate increases the activation by fructose 2,6-bisphosphate and augments the deactivation of the fructose 2,6-bisphosphate activated enzyme by fructose 1,6-bisphosphate. Because various states of yeast glucose metabolism differ in the levels of the two fructose bisphosphates, the observed interactions might be of regulatory significance.     

The effect of experimental hypothyroidism on phosphofructokinase activity and fructose 2,6-bisphosphate concentrations in rat heart.

Experimental hypothyroidism was induced in rats by the administration of NaClO4. Hearts from normal and hypothyroid rats were homogenized, and the extracts were assayed for phosphofructokinase-1 and phosphofructokinase-2 activity and fructose 2,6-bisphosphate concentrations. Hypothyroidism was associated with a drastic loss of phosphofructokinase-1 activity. A hyperbolic relationship between plasma thyroxine concentrations and phosphofructokinase-1 activity was found. As treatment with NaClO4 progressed, the decrease in blood thyroxine was faster than the decrease in enzyme activity. After prolonged hypothyroidism (a decrease in thyroxine of more than 10-fold), a 4-fold decrease in phosphofructokinase-1 activity was observed. In this metabolic condition 2-fold decreases in phosphofructokinase-2 activity and in fructose 2,6-bisphosphate were observed. A similar decrease in phosphofructokinase-1 activity in a partially purified preparation was found. The addition of L-thyroxine in the diet had little effect on phosphofructokinase-1 activity. However, exposure of minced pieces of hearts of hypothyroid rats to tri-iodothyronine for 5 h resulted in a clear increase in phosphofructokinase-1 activity, which was partially prevented by the simultaneous addition of cycloheximide. These results could account for the decrease in carbohydrate metabolism in heart from hypothyroid rats.     

Influence of fructose 2,6-bisphosphate on the aggregation properties of rat liver phosphofructokinase

The influence that fructose 2,6-bisphosphate (Fru-2,6-BP) has on the aggregation properties of rat liver phosphofructokinase has been studied by observing the fluorescence polarization of the enzyme covalently bound to the fluorescent probe pyrenebutyric acid. Fru-2,6-BP dramatically slows the dissociation of the high molecular weight aggregate forms of the enzyme when the enzyme is diluted to 3.2 micrograms/ml (4 X 10(-8) M subunits). Furthermore, Fru-2,6-BP is a strong promoter of reassociation to tetramer and larger forms if the enzyme has been previously allowed to dissociate to the dimer in its absence. Unlike many other positive effectors of liver phosphofructokinase, Fru-2,6-BP is also able to overcome the tendency of MgATP to promote tetramer formation and instead stabilize a very high degree of high molecular weight aggregate formation even in the presence of MgATP. The apparent affinity of liver phosphofructokinase for Fru-2,6-BP was measured by its ability to promote reassociation and compared to that for Fru-1,6-BP. The apparent dissociation constant for Fru-2,6-BP under these conditions is 36 microM, about 40-fold lower than the value of 1.4 mM measured for Fru-1,6-BP. Both ligands demonstrate synergism with the substrate Fru-6-P, which can lower the dissociation constant for Fru-2,6-BP 9-fold to 4 microM and that for Fru-1,6-BP 5-fold to 0.28 mM. These data are interpreted to suggest that influencing the aggregation state of rat liver phosphofructokinase may be one way in which Fru-2,6-BP achieves its effects on the enzyme in vivo.     

Influence of fructose 2,6-bisphosphate on the phosphofructokinase/fructose 1,6-bisphosphatase cycle

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 role of fructose 2,6-bisphosphate in the long-term control of phosphofructokinase in rat liver

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.     

Effects of 5-hydroxytryptamine, cyclic AMP, AMP, and fructose 2,6-bisphosphate on phosphofructokinase activity in Hymenolepis diminuta

1. 5-HT (10(-4) M) had no effect on the activity of phosphofructokinase in Hymenolepis diminuta. Concentrations of ATP above 33 microM inhibited PFK activity; AMP and cyclic AMP relieved this inhibition. 2. Local levels of cyclic AMP may be indirectly modulated by NaF, guanylyl imidophosphate, or 5-HT in the presence of GTP, which stimulates adenylyl cyclase activity x2 in H. diminuta homogenates. 3. Fructose 2,6-bisphosphate (F2BP), a physiological regulator of PFK activity in rat liver, also relieved ATP-induced inhibition of PFK. F2BP was present in supernatants from the worms at about 20 mumol/g wet wt. 4. 5-HT may cause an increase in the rate of glycolysis in H. diminuta by elevating either cyclic AMP and/or AMP levels; these nucleotides can in turn increase PFK activity.     

Reciprocal regulation of fructose 1,6-bisphosphatase and phosphofructokinase by fructose 2,6-bisphosphate in swine kidney

The effect of fructose 2,6-P₂, AMP and substrates on the coordinate inhibition of FBPase and activation of PFK in swine kidney has been examined. Fructose 2,6-P₂ inhibits the activity of FBPase and stimulates the activity of PFK in the presence of inhibitory concentrations of ATP. Under similar conditions 2.2 μM fructose 2,6-P₂ was required for 50% inhibition of FBPase and 0.04 μM fructose 2,6-P₂ restored 50% of the activity of PFK. Fructose 2,6-P₂ also enhanced the allosteric activation of PFK by AMP and it increased the extent of inhibition of FBPase by AMP. Fructose 2,6-P₂, AMP and fructose 6-P act cooperatively to stimulate the activity of PFK whereas the same latter two effectors and fructose 1,6-P₂ inhibit the activity of FBPase. Taken collectively, these results suggest that an increase in the intracellular level of fructose 2,6-P₂ during gluconeogenesis could effectively overcome the inhibition of PFK by ATP and simulataneously inactivate FBPase. When the level of fructose 2,6-P₂ is low, a glycolytic state would be restored, since under these conditions PFK would be inhibited by ATP and FBPase would be active.     

Hormonal control of fructose 2,6-bisphosphate concentration and of phosphofructokinase 2 in the rat liver during development

In fetal rat liver the concentration of fructose 2,6-bisphosphate is decreased by administration of glucagon. The glucagon effect, i.e., the phosphorylation state of phosphofructokinase 2, dominates over the substrate supply. Insulin was found to increase fructose 2,6-bisphosphate only when exogenous glucose is supplied simultaneously. The total activity of phosphofructokinase 2 exhibits remarkable developmental changes. It is high at term, moderate in the fetal as well as in the mature organ, and low during suckling. The level of the enzyme during development is controlled by pancreatic and adrenal hormones.     

Activation of muscle phosphofructokinase by fructose 2,6-bisphosphate and fructose 1,6-bisphosphate is differently affected by other regulatory metabolites

Fructose-2,6-P2 and fructose-1,6-P2 are strong activators of muscle phosphofructokinase. They have been shown to be competitive in binding studies, and it is generally thought that they affect the physical and catalytic properties of the enzyme in the same manner. However, there are indications in published data that the effects of the two fructose bisphosphates on phosphofructokinase are not identical. To examine this possibility, the kinetics of activation of rat skeletal muscle phosphofructokinase by the two fructose bisphosphates were compared in the presence of other regulatory metabolites. Citrate greatly increased the K0.5 of the enzyme for fructose-2,6-P2, with little effect on the maximum activation. In contrast, citrate greatly decreased the maximum activation by fructose-1,6-P2, with only a small effect on the K0.5. Changes in the concentrations of the inhibitor ATP or the activator AMP similarly altered the K0.5 for fructose-2,6-P2, but altered the maximum activation by fructose-1,6-P2. Finally, when fructose-1,6-P2 was added in the presence of a given concentration of fructose-2,6-P2, phosphofructokinase activity was decreased if the activation by fructose-2,6-P2 alone was greater than the maximum activation by fructose-1,6-P2 alone. These results are consistent with competition of the two fructose bisphosphates for the same binding site, but indicate that the conformational changes produced by their binding are different.     

Stimulation of yeast phosphofructokinase activity by Fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate is a powerful activator of yeast phosphofructokinase when assayed at pH levels of ≥7.0. Half maximal stimulation of enzyme activity occurs at 10^?7 M levels of Fru 2,6-P₂ concentration. This stimulating effect by Fru 2,6-P₂ can be synergistic to that exerted by AMP in counteracting the inhibition of phosphofructokinase activity by ATP. The affinity (S_0.5) of the yeast enzyme to fructose 6-phosphate changes from 1.5 mM in the absence of Fru 2,6-P₂ to 40 μM in its presence.     

Effects of vanadate on 6-phosphofructo 2-kinase activity and fructose 2,6-bisphosphate levels in cultured rat hepatocytes

The presence of vanadate in primary cultures of rat hepatocytes produced a significant increase in the concentration of fructose 2,6-bisphosphate and in the activity of 6-phosphofructo 2-kinase. Compared with insulin, vanadate had a more potent action on the metabolite increase, but a similar effect on the 6-phosphofructo 2-kinase activity. Both the insulin- and the vanadate-dependent enhancements of 6-phosphofructo 2-kinase were inhibited by cycloheximide which specifically blocks protein synthesis on the translational level, suggesting that the increase of the enzyme activity was due to induction rather than to a change in the catalytic activity.     

Effects of fructose 2,6-bisphosphate on phosphoglucomutase from plants

Fructose 2,6-bisphosphate affects phosphoglucomutase from plant and animal sources in a similar way. As previously found with rabbit muscle phosphoglucomutase, fructose 2,6-bisphosphate cannot substitute for glucose 1,6-bisphosphate as a cofactor in the reaction catalyzed by phosphoglucomutase from potato tubers, pea seeds, and string-beans. In the presence of glucose 1,6-bisphosphate, fructose 2,6-bisphosphate inhibits phosphoglucomutase from potato tubers. Activation of phosphoglucomutase from plant sources by fructose 2,6-bisphosphate reported by others was probably due to contamination of the commercial preparation of fructose 2,6-bisphosphate by glucose 1,6-bisphosphate.     

Specific activation by fructose 2,6-bisphosphate and inhibition by P-enolpyruvate of ascites tumor phosphofructokinase

Electrophoresis on 10 % acrylamide-SDS gels showed that incubation of rat liver plasma membranes with certain detergents induces a proteolysis. The results obtained using different detergents, temperatures and pH levels, as well as the action of some protease inhibitors are in favor of an enzymatically-induced proteolysis. Since the proteolytic activity remains unchanged, even after the peripheral proteins have been released, it is proposed that this activity may reflect one of the integral proteins functions.     

Effect of thiamin on the content of fructose 2,6-bisphosphate in Euglena

• 1.1. Thiamin deprived Euglena cells contained twice as high a concentration of Fru-2,6-P₂ in sufficient cells and the rate of the uptake of glucose from the medium was twice as great as that found in sufficient cells, indicating that Fru-2,6-P₂ is responsible for the glycolysis.
• 2.2. Moreover, incubation of thiamin deprived cells with increasing thiamin concentrations caused a decrease of Fru-2,6-P₂. The activity of Fru-6-P, 2-kinase was affected by thiamin and the activity of PFK-2 in thiamin deprived cells was 2.3 times greater than that observed in sufficient cells.
• 3.3. Incubating thiamin-deficient cells in a thiamin-containing medium, the activity of Fru-6-P, 2-kinase decreased rapidly and became the same activity as that found in thiamin sufficient cells.
• 4.4. In contrast, the two thiamin dependent 2-OGDC and PNOR activities showed a reverse relationship, being higher in thiamin-sufficient cells and lower in thiamin deficient cells.
• 5.5. We concluded that the carbohydrate metabolism of Euglena is regulated by Fru-2,6-P₂ through an intracellular concentration of thiamin based on the present evidence.

     

Effect of buffer solutions on activation of Shamouti orange pyrophosphate-dependent phosphofructokinase by fructose 2,6-bisphosphate

Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6-bisphosphate (Fru-2,6-P2) in the glycolytic reaction. The effect of activation by Fru-2,6-P2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: Ka = 2.77 +/- 0.3 nM in Hepes-NaOH and 7.75 +/- 1.49 nM in Tris-HCl. The presence of chloride ions (39 mM) in the Tris-HCl buffer inhibits PFP. Indeed, when using a Hepes-NaOH buffer and then adding 39 mM NaCl, Ka = 8.12 +/- 0.52 nM. The Ki for chloride ions is approximately 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru-2,6-P2 did not modify the velocity and the Vmax of the enzyme; however, its presence increased the affinity of the enzyme for Fru-1,6-P2 from 200 +/- 15.6 microM in absence of Fru-2,6-P2 to 89 +/- 10.3 microM in its presence (10 microM). In the presence of chloride (39 mM), the affinity for the substrate decreased with K(m) = 150 +/- 14 microM. The calculated Ki for chloride ions equals 56.9 mM. In both the glycolytic and the gluconeogenic reactions, Vmax is not affected; therefore, the inhibition mode of chloride is competitive.     

Inhibition of fructose bisphosphatase and stimulation of phosphofructokinase by a stable isosteric phosphonate analog of fructose 2,6-bisphosphate

We describe the synthesis of a mixture of D-manno- and D-gluco-2,5-anhydro-1-deoxy-1-phosphonohexitol 6-phosphate via a Horner-Emmons reaction of 2,3,5-tri-O-benzyl-beta-D-arabinofuranose followed by phosphorylation of the equivalent 6-position and subsequent deprotection. This mixture inhibits fructose-1,6-bisphosphatase; the concentration required for half-maximal effect in the presence of 25 microM AMP is approximately 6 microM. The mixture of analogs also stimulates 6-phosphofructo-1-kinase from rabbit liver; the concentration required to reach one-half Vmax was found to be ca. 25 microM at 0.25 mM fructose 6-phosphate and 50 microM AMP. These analogs have replaced the labile anomeric phosphate of fructose 2,6-bisphosphate with a stable methylenephosphonate, and could be of great interest due to their appropriate physiological effects and their chemical stability.