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Fructose-1,6-diphosphate (FDP) counteracts the inhibition of contractile strength of rabbit cardiac muscle induced by KCl "in vitro" and the valinomycin induced depolarization. FDP also protects in a dose-dependent fashion, up to 100 mg/kg, different strains of mice from the lethal effect of KCl that is due to a complete atrio-ventricular block and related to a high value of plasma potassium level. A protective effect of FDP is observed when increasing doses of (+/-) propranolol are injected before maximum non lethal dose of KCl.
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The effects of 5 sulfonylureas on fructose-2,6-bisphosphate (F-2,6-P2) formation using isolated perfused rat liver were examined. All sulfonylureas examined stimulated dose-dependent formation of the activator in a limited range of the concentration. A maximum effect on F-2,6-P2 formation was observed at the concentration of 10(-3) M of tolbutamide or chlorpropamide, at 10(-4) M of gliclazide or acetohexamide and at 10(-6) M of glibenclamide. These concentrations of sulfonylurea correspond with those in blood when therapeutical doses of the drug are administered orally. Sulfonamide and biguanide did not show any stimulatory effect on F-2,6-P2 level. The results demonstrate that stimulation of liver F-2,6-P2 formation is a common characteristic of sulfonylureas and suggest strongly that one of the extrapancreatic actions of sulfonylurea is stimulation of F-2,6-P2 formation followed by enhancement of glycolysis and inhibition of gluconeogenesis in the liver.
The effects of tolbutamide and pancreatic hormones on liver fructose-2,6-bisphosphate (F-2,6-P2) formation were examined using isolated rat hepatocytes. Glucagon decreased the F-2,6-P2 level in a dose-dependent manner. Insulin (greater than 10(-9) M) increased the F-2,6-P2 level reduced by glucagon (less than 10(-9) M), but did not show a stimulatory effect on this activator formation in the absence of glucagon. On the other hand, tolbutamide increased the F-2,6-P2 level in hepatocytes regardless of the presence or absence of glucagon. Tolbutamide (2 mM) stimulation on liver F-2,6-P2 formation was enhanced by the concomitant addition of insulin (10(-8) M) in the presence of glucagon (3 X 10(-11) M). These observations suggest that the regulatory effect of tolbutamide on liver F-2,6-P2 level is independent of that of insulin.
Numerous interventions have been used to protect organ systems and cellular viability from the lethal injury accompanying hypoperfusion and ischemia. Some measures have been directed toward improving perfusion, while others have attempted to enhance the metabolic processes. Our work has focused for the most part on augmenting the anaerobic carbohydrate utilization in ischemic and hypoperfused tissues with fructose-1,6-diphosphate (FDP). Such an approach is based on the premise that exogenous FDP will restore the activity of glycolysis, which has been inhibited by acidosis, by intervening in the Embden-Meyerhoff pathway both as a metabolic regulator and as a high-energy substrate. We have tested this hypothesis in more than 1,000 animals subjected to shock or regional ischemia, and the results appear to confirm our presumption. In myocardial infarction, FDP improves hemodynamic parameters, attenuates ECG-proven ischemic injury and dysrhythmias, prevents ATP and creatine phosphate depletion from ischemic myocardium, reduces infarct size, and increases survival. In hemorrhagic, traumatic, and endotoxin shock, FDP improves hemodynamics, attenuates organ injury, and increases survival. Recently we demonstrated that FDP attenuates the reperfusion ischemic tissue injury by inhibiting the generation of oxygen free radicals by neutrophils. In normal volunteers, this agent increases carbohydrate utilization, while administration of a like amount of glucose produces no effect. In patients with myocardial infarction, FDP appears to have the same effect as that observed in the animal model. When this agent is administered to patients in traumatic, hemorrhagic, or septic shock, hemodynamic and pulmonary function are significantly improved. In patients with adult respiratory distress syndrome, similar beneficial effects have been observed with FDP administration.(ABSTRACT TRUNCATED AT 250 WORDS)
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On the basis of kinetic activation assays, the apparent affinity of muscle phosphofructokinase for fructose 2,6-bisphosphate was about 9-fold greater than that for fructose 1,6-bisphosphate, which in turn was about 10 times higher than that for glucose 1,6-bisphosphate. Equilibrium binding experiments showed that both fructose bisphosphates bind to phosphofructokinase with negative cooperativity; the affinity for fructose 2,6-bisphosphate was about 1 order of magnitude greater than the affinity for fructose 1,6-bisphosphate. Binding of fructose 2,6-bisphosphate to phosphofructokinase was antagonized by fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and vice versa. Both fructose bisphosphates promoted aggregation of the enzyme to higher polymers as indicated by sucrose density gradient centrifugation. Other indicators of phosphofructokinase conformation such as thiol reactivity and maximum activation of in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase gave identical results in the presence of fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, or glucose 1,6-bisphosphate, indicating a common conformation is produced by all three ligands. It is concluded that the sugar bisphosphates bind to a single site on the enzyme.
Minimum values for the content of covalent intermediates in the equilibria of muscle aldolase with its cleavable substrates have been determined by acid denaturation/precipitation. Ribulose 1,5-bisphosphate, a nonsubstrate that binds well to aldolase in the native state, does not form a covalent complex that is acid precipitable. The insoluble protein complexes with substrates fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, representing approximately 50% and approximately 60% of total bound substrate, are much more stable in acid and alkali than that with substrate 5-deoxyfructose 1,6-bisphosphate, suggesting that they have the form of protein-bound N-glycosides. Whether such complexes exist on the enzyme in the native state in addition to being formed subsequent to denaturation is unresolved. Both the acid-precipitable and nonprecipitable forms of fructose 1,6-bisphosphate are converted to triose phosphate products at the same rate, providing no kinetic evidence for a pool that is not on the main reaction path. Total fructose 1,6-bisphosphate liganded to enzyme returns to the free solution about 9 times for each net cleavage reaction. It is still not clear whether this is limited by the cleavage step or by release of glyceraldehyde phosphate.
We explored the stereospecificity of the fructose 2,6-bisphosphate site of rabbit muscle 6-phosphofructo-1-kinase by determination of the activation constants (Ka) of several structurally locked analogues of this potent metabolic regulator. Under the assay conditions used, the Ka of fructose 2,6-bisphosphate was 0.12 microM. The most effective synthetic analogues and their Ka's were 2,5-anhydro-D-mannitol 1,6-bisphosphate (2.9 microM), 1,4-butanediol bisphosphate (6.6 microM), hexitol 1,6-bisphosphate (40 microM), and 2,5-anhydro-D-glucitol 1,6-bisphosphate (47 microM). Ten other bisphosphate compounds were much less effective as activators of the enzyme. These findings indicate that, unlike its active site, this allosteric site of 6-phosphofructo-1-kinase does not require the furanose ring. Its basic requirement seems to be a compound with two phosphate groups approximately 9 A apart. Although the free hydroxy groups of the activator do not seem to be essential, their presence enhances appreciably the affinity of the ligand for this regulatory site.
Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was purified from the mung bean Phaseolus aureus. The enzyme is activated by fructose 2,6-bisphosphate at nanomolar concentrations. The enzyme exhibits Michaelis-Menten kinetics, and the reaction mechanism, deduced from initial velocity studies in the absence of inhibitors as well as product and dead-end inhibition studies, is rapid equilibrium random in the presence and absence of fructose 2,6-bisphosphate. In the direction of fructose 6-phosphate phosphorylation, saturating fructose 2,6-bisphosphate (1 microM) increases V congruent to 9-fold and increases V/KMgPPi and V/KF6P about 30-fold. In the reverse direction (phosphate phosphorylation), the same concentration of activator has little if any effect on V or the Km for inorganic phosphate (Pi) and Mg2+ but does increase V/KFBP about 42-fold. No changes were observed in any of the other rate constants. The binding affinity of fructose 2,6-bisphosphate to all enzyme forms is identical. The activator site of the mung bean PPi-PFK binds fructose 2,6-bisphosphate with a Kact of 30 nM with the 2,5-anhydro-D-glucitol 1,6-bisphosphate (the most effective analogue) 33-fold less tightly. Of the alkanediol bisphosphate series, 1,4-butanediol bisphosphate exhibited the tightest binding (Kact congruent to 3 microM). These and a series of other activating analogues are discussed in relation to the activator site.
The fluorescent nucleotide analogue formycin 5'-monophosphate (FMP) inhibits rabbit liver fructose-1,6-bisphosphatase (I50 = 17 microM, Hill coefficient = 1.2), as does the natural regulator AMP (I50 = 13 microM, Hill coefficient = 2.3), but exhibits little or no cooperativity of inhibition. Binding of FMP to fructose-1,6-bisphosphatase can be monitored by the increased fluorescence emission intensity (a 2.7-fold enhancement) or the increased fluorescence polarization of the probe. A single dissociation constant for FMP binding of 6.6 microM (4 sites per tetramer) was determined by monitoring fluorescence intensity. AMP displaces FMP from the enzyme as evidenced by a decrease in FMP fluorescence and polarization. The substrates, fructose 6-phosphate and fructose 1,6-bisphosphate, and inhibitors, methyl alpha-D-fructofuranoside 1,6-bisphosphate and fructose 2,6-bisphosphate, all increase the maximal fluorescence of enzyme-bound FMP but have little or no effect on FMP binding. Weak metal binding sites on rabbit liver fructose-1,6-bisphosphatase have been detected by the effect of Zn2+, Mn2+, and Mg2+ in displacing FMP from the enzyme. This is observed as a decrease in FMP fluorescence intensity and polarization in the presence of enzyme as a function of divalent cation concentration. The order of binding by divalent cations is Zn2+ = Mn2+ greater than Mg2+, and the Kd for Mn2+ displacement of FMP is 91 microM. Methyl alpha-D-fructofuranoside 1,6-bisphosphate, as well as fructose 6-phosphate and inorganic phosphate, enhances metal-mediated FMP displacement from rabbit liver fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)
The concentration of fructose 6-phosphate required to produce half-maximal velocity of rat liver phosphofructokinase at pH 9 (Ka) has been measured at 110 different combinations of MgATP and fructose 2,6-bisphosphate (Fru-2,6-BP) concentrations spanning the range 0.1-100 mM and 0.003-100 microM, respectively. The data have been evaluated by nonlinear regression to an equation resulting from a linked-function analysis of an enzyme capable of binding three ligands simultaneously at separate sites. In addition, the data have been fit to equations, derived from the linked-function expression, that would result if various combinations of antagonistic ligands were unable to bind to the enzyme simultaneously, even at high concentration, either because they compete for a single binding site or because they bind exclusively to different conformational forms of the enzyme. The complete linked-function equation is able to predict the Ka for rat liver phosphofructokinase as a function of any Fru-2,6-BP and/or MgATP concentration significantly better than any of the alternatives examined, particularly at high concentrations of one or both modifier ligands. The free energy couplings between all three possible pairs of ligands are of quite moderate magnitude, especially when the multiplicity of binding sites for each ligand that actually exists on the functional enzyme is considered. Therefore, we conclude that any explanation of the action of Fru-2,6-BP and MgATP by a model more elaborate than the simple linked-function case considered herein cannot be simplified by assuming that the properties of rat liver phosphofructokinase result from an equilibrium of limiting conformational states that exhibit exclusive binding properties.
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