Fructose 1,6-bisphosphate. Kinetics of hydrolysis catalyzed by rabbit liver neutral fructose-1,6-bisphosphatase with Mn2+.
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Kinetic properties of pyruvate kinase of Neurospora crassa at physiological pH.
The kinetic properties of pyruvate kinase (EC 2.7.1.40) extracted from the mycelia of Neurospora crassa were examined at physiological pH to determine the role of the enzyme in the regulation of glycolysis. The velocity curve with the substrates, phosphoenolpyruvate and adenosine diphosphate, are hyperbolic. The effect of magnesium, potassium, or calcium on the enzyme is influenced by the pH but not to the extent that would change their role as cofactor or inhibitor. Adenosine triphosphate and citrate remain strong inhibitors even with changes in pH. Fructose-1,6-diphosphate and glucose-6-phosphate are the dual positive effectors at physiological pH. Valine is the only amino acid that inhibits the enzyme at a concentration range of valine found in the mycelial juice. Thus, the properties of the enzyme at physiological pH are significantly different from those observed at neutral pH of the usual assay conditions, but its role as a key regulator of glycolysis is unchanged.
Adenosine Triphosphate↗
Recovery of post-arrest cardiac performance: effect of fructose-1,6-diphosphate in the isolated rabbit heart.
The effect of fructose-1,6-diphosphate (FDP) on the recovery of post-arrest cardiac performance was assessed in isolated rabbit heart preparations subjected to cold ischemic cardioplegic arrest (CICA). Twenty-eight hearts were perfused with Krebs-Henseleit (KHBS) solution followed by 40 minutes of CICA. After CICA normothermic reperfusion with KHBS was restarted. The experimental preparations were divided in four groups of seven hearts each: one group served as control and the other three were perfused with KHBS supplemented with FDP (500 mg/l) given before CICA, after CICA or at both study points. Left ventricular end-diastolic pressure, left ventricular systolic pressure, dP/dt and perfusion pressure were measured at different pre- and post-arrest phases. Time analysis of recovery was also performed. The results demonstrated that FDP supplied prior to and after CICA: prevents the ventricular wall rigidity induced by the ischemic arrest, improves cardiac contractile force both in the basal condition and after cardioplegia, reduces the perfusion pressure, reduces the time to recovery of cardiac contractility. Our findings in the isolated rabbit heart are consistent with published data suggesting FDP may limit the impairment of cardiac dynamics induced by ischemia and improve the recovery after cardiac arrest.
Animals↗
[Fructose-1,6-diphosphate in the treatment of chronic heart failure].
Fructose-1,6-diphosphate (FDP) was given to 30 patients with chronic heart failure (CHF) caused by various kinds of heart diseases with the purpose to evaluate the effects of FDP on CHF patients. Definite hemodynamic and clinical improvement has been found in this group. CO increased by 1.61 +/- 0.31 L/Min (35%) (P less than 0.01) PCWP decreased by 5.5 +/- 1.08 mmHg (31%); mean PAP decreased by 5.8 +/- 2.07 mmHg (P less than 0.05). EF increased by 6.9 +/- 1.5 (15.1%) as shown by echocardiography and the peak effect of the drug appeared at 2 hours after administration. The results showed that FDP is effective in the treatment of heart failure, especially in patients with dysfunction of other organs.
Adult↗
The characteristics of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferases from Sansevieria trifasciata leaves and Phaseolus coccineus stems.
Three different molecular forms of pyrophosphate-dependent phosphofructokinase have been isolated: one from Sansevieria trifasciata leaves and two from Phaseolus coccineus stems. The form isolated from S. trifasciata has the molecular weight of about 115,000. The apparent molecular weights for the two forms from mung bean were approximately 220,000 and 450,000. All three forms have the same pH optima, an absolute requirement for Mg2+ ions both in the forward and reverse reaction, but differ in their sensitivity toward fructose 2,6-bisphosphate. Kinetic properties of the partially purified enzymes have been investigated in the presence and absence of fructose 2,6-bisphosphate. Pyrophosphate-dependent phosphofructokinase from S. trifasciata exhibited hyperbolic kinetics with all substrates tested. The saturation curves of the enzyme (form A) from mung bean for pyrophosphate, fructose 6-phosphate and fructose 1,6-bisphosphate were sigmoidal in the absence of fructose 2,6-bisphosphate. In the presence of fructose 2,6-bisphosphate these kinetics became hyperbolic.
Fabaceae↗
Changes in rat hepatic fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase activity during three days of consumption of a high protein diet or starvation.
Changes in plasma glucose, hepatic cyclic AMP, glycogen and fructose 2,6-bisphosphate (F-2,6-P2), and liver 6-phosphofructo-2-kinase (6-PF-2kinase), fructose 2,6-bisphosphatase (F-2,6-P2ase) and phosphoenolpyruvate carboxykinase (PEPCK) activities were examined in rats fed a low protein, high carbohydrate (HC) diet during 3 d of either starvation or feeding a high protein, carbohydrate-free (HP) diet. Under both HP feeding or starvation, liver cyclic AMP increased after 1 d and remained constant thereafter. Whereas plasma glucose was low during starvation, it was unaffected by HP feeding. In both experimental groups, liver glycogen fell after 1 d; thereafter it remained low on starvation, but increased progressively on HP diet reaching 70% of the HC-fed rats value on day 3. Under both experimental conditions, F-2,6-P2 fell 85% after day 1 and was unchanged thereafter. One day after the start of starvation or consumption of the HP diet, 6-PF-2kinase decreased, F-2,6-P2ase increased and 6-PF-2kinase/F-2,6-P2ase ratio decreased, but changes were significantly more important with the HP diet than with starvation. PEPCK activity increased in both experimental conditions, but the increase was greater on the HP diet than on starvation. These findings suggest that during the first 3 d the adaptative response of hepatic gluconeogenesis is higher with a HP diet than upon starvation.
Animals↗
Insulin-like effects of vanadate on glucokinase activity and fructose 2,6-bisphosphate levels in the liver of diabetic rats.
Streptozotocin diabetic rats showed more than a 4-fold increase in blood glucose levels, whereas hepatic glycogen, fructose 2,6-bisphosphate concentration, and 6-phosphofructo-2-kinase activity were decreased. The "total" 6-phosphofructo-2-kinase and the "active" (nonphosphorylated) form of the enzyme were decreased to a different extent, resulting in a fall of the "active"/"total" activity ratio. Vanadate administration for a 2-week period restored the altered values in the diabetic rats without modifying significantly in the control animals any of the parameters studied. Glucokinase activity was essentially lacking in the diabetic animals, and vanadate treatment restored the activity to about 65% of its control value, a good correlation between the recovery of the enzyme and the blood glucose level being observed. These results show an insulin-like effect of vanadate in the whole animal and suggest that insulin and vanadate possess similar actions on hepatic intracellular events.
Animals↗
Interaction of fructose 2,6-bisphosphate and AMP with fructose-1,6-bisphosphatase as studied by nuclear magnetic resonance spectroscopy.
The interaction of AMP and fructose 2,6-bisphosphate with rabbit liver fructose-1,6-bisphosphatase has been investigated by proton nuclear magnetic resonance spectroscopy (1H NMR). The temperature dependence of the line widths of the proton resonances of AMP as a function of fructose-1,6-bisphosphatase concentration indicates that the nucleotide C2 proton is in fast exchange on the NMR time scale while the C8 proton is exchange limit. The exchange rate constant, koff, has been calculated for the adenine C8 proton and is 1900 s-1. Binding of fructose 6-phosphate and inorganic phosphate, or the regulatory inhibitor, fructose 2,6-bisphosphate, results in a decrease in the dissociation rate constant for AMP from fructose-1,6-bisphosphatase, as indicated by the sharpened AMP signals. A temperature dependence experiment indicates that the AMP protons are in slow exchange when AMP dissociates from the ternary complex. The rate constant for dissociation of AMP from the enzyme.AMP.fructose 2,6-bisphosphate complex is 70 s-1, 27-fold lower than that of AMP from the binary complex. These results are sufficient to explain the enhanced binding of AMP in the presence of fructose 2,6-bisphosphate and, therefore, the synergistic inhibition of fructose-1,6-bisphosphatase observed with these two regulatory ligands. Binding of fructose 2,6-bisphosphate to the enzyme results in broadening of the ligand proton signals. The effect of AMP on the binding of fructose 2,6-bisphosphate to the enzyme has also been investigated. An additional line width broadening of all the fructose 2,6-bisphosphate protons has been observed in the presence of AMP. The assignment of these signals to the sugar was accomplished by two-dimensional proton-proton correlated spectra (two-dimensional COSY) NMR. From these data, it is concluded that AMP can also affect fructose 2,6-bisphosphate binding to fructose-1,6-bisphosphatase.
Adenosine Monophosphate↗
Relationship between thiol group modification and the binding site for fructose 2,6-bisphosphate on rabbit liver fructose-1,6-bisphosphatase.
A thiol group present in rabbit liver fructose-1,6-bisphosphatase is capable of reacting rapidly with N-ethylmaleimide (NEM) with a stoichiometry of one per monomer. Either fructose 1,6-bisphosphate or fructose 2,6-bisphosphate at 500 microM protected against the loss of fructose 2,6-bisphosphate inhibition potential when fructose-1,6-bisphosphatase was treated with NEM in the presence of AMP for up to 20 min. Fructose 2,6-bisphosphate proved more effective than fructose 1,6-bisphosphate when fructose-1,6-bisphosphatase was treated with NEM for 90-120 min. The NEM-modified enzyme exhibited a significant loss of catalytic activity. Fructose 2,6-bisphosphate was more effective than the substrate in protecting against the thiol group modification when the ligands are present with the enzyme and NEM. 100 microM fructose 2,6-bisphosphate, a level that should almost saturate the inhibitory binding site of the enzyme under our experimental conditions, affords only partial protection against the loss of activity of the enzyme caused by the NEM modification. In addition, the inhibition pattern for fructose 2,6-bisphosphate of the NEM-derivatized enzyme was found to be linear competitive, identical to the type of inhibition observed with the native enzyme. The KD for the modified enzyme was significantly greater than that of untreated fructose-1,6-bisphosphatase. Examination of space-filling models of the two bisphosphates suggest that they are very similar in conformation. On the basis of these observations, we suggest that fructose 1,6-bisphosphate and fructose 2,6-bisphosphate occupy overlapping sites within the active site domain of fructose-1,6-bisphosphatase. Fructose 2,6-bisphosphate affords better shielding against thiol-NEM modification than fructose 1,6-bisphosphate; however, the difference between the two ligands is quantitative rather than qualitative.
Adenosine Monophosphate↗
The inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate is enhanced by EDTA and diminished by zinc(II).
The sensitivity of the Mg(II)-dependent activity of rabbit liver fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) to inhibition by fructose 2,6-bisphosphate (Fru-2,6-P2) was enhanced by EDTA and diminished to negligible levels by 0.5-2 microM Zn(II) added as another FBPase inhibitor. Fru-2,6-P2 was more efficient in the presence of the synergistic effector AMP: still, the Fru-2,6-P2 concentration inhibiting 50% changed from 3 microM (with EDTA) to higher than 50 microM (with Zn(II]. On the other hand, the Zn(II)-dependent FBPase activity was inhibited by Fru-2,6-P2 to a much lesser extent than the Mg(II)-dependent activity.
Adenosine Monophosphate↗
Age-dependent changes in rat hepatic fructose 2, 6-bisphosphate, 6-phosphofructo-2-kinase/fructose 2, 6-bisphosphatase and pyruvate kinase activity in response to a high protein diet or starvation.
Plasma insulin (I), glucagon (G) and glucose, hepatic glycogen, fructose 2, 6-bisphosphate (F2, 6-P2), fructose 1, 6-bisphosphate, phosphoenolpyruvate, and some liver key enzymes involved in glycolysis (6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (6-PF-2kinase/F-2,6-P2ase), activity ratio (velocity at suboptimal substrate concentration/maximum velocity) of pyruvate kinase (PK-L] and in gluconeogenesis (phosphoenolpyruvate carboxykinase activity) have been compared in young (2 months) and old (16 months) rats upon starvation or transition to a high protein (HP) diet. In the 10 and 24 hours after the dietary switch, plasma glucose decreased less and hepatic glycogen was less depleted in the old rats. The ratios of plasma I/G and of hepatic 6-PF-2kinase/F-2,6-P2ase were higher in the old rats and their decrease delayed at both time points, as was the concentration of hepatic F-2,6-P2 and the activity ratio of PK-L (before and after removal of endogenous noncovalent factors). The consistency of these differences indicate that the mechanisms for control of glycolysis/gluconeogenesis are similar in young and old rats, but it appears that in old rats starved or fed HP diet, the switch from glycolysis to gluconeogenesis is delayed. This suggests that as a result of the slowness of the hormonal changes the process of phosphorylation/dephosphorylation, which is so important in the short-term regulation of the glycolysis/gluconeogenesis pathway, may be impaired with age.
Aging↗
Regulation of protein synthesis in rabbit reticulocyte lysate. Glucose 6-phosphate is required to maintain the activity of eukaryotic initiation factor (eIF)-2B by a mechanism that is independent of the phosphorylation of eIF-2 alpha.
Previous studies from other laboratories, using rabbit reticulocyte lysate filtered through Sephadex G-25 or G-50, have demonstrated that glucose 6-phosphate is required to maintain active rates of translation, but its mechanism of action is currently unsettled. We have tested whether glucose 6-phosphate is required to prevent activation of the hemin-controlled translational repressor and the phosphorylation of the smallest or alpha subunit of eukaryotic initiation factor 2 (eIF-2). We have found that antibody to the hemin-controlled translational repressor can completely restore protein synthesis in reticulocyte lysate, filtered through Sephadex G-25, that is incubated in the absence of hemin and presence of glucose 6-phosphate, but cannot restore protein synthesis in such lysate incubated in the presence of hemin and absence of glucose 6-phosphate. We have also found, using a modification of the method of Matts and London [1984) J. Biol. Chem. 259, 6708-6711) to measure the ability of gel-filtered lysate to dissociate and exchange GDP from eIF-2.GDP, that this endogenous eIF-2B activity is reduced to the same low level in the presence of hemin and absence of glucose 6-phosphate as it is in the absence of hemin and presence of glucose 6-phosphate. Although there is a low level of phosphorylation of eIF-2 alpha in gel-filtered lysate given hemin but no glucose 6-phosphate, it cannot account for the loss of eIF-2B activity, since this phosphorylation is removed by antibody to the hemin-controlled translational repressor or isocitrate, which do not restore protein synthesis or eIF-2B activity, and not by fructose 1,6-diphosphate, which does partially restore protein synthesis and eIF-2B activity. These findings suggest that sugar phosphates may exert a direct effect on eIF-2B and may be required for its proper function. Additional support for this conclusion is our finding that protein synthesis and eIF-2B activity in partially hemin-deficient lysate can be restored by high levels of glucose 6-phosphate or fructose 1,6-diphosphate without a reduction in the level of phosphorylated eIF-2 alpha, suggesting that such levels of sugar phosphate may permit restoration of normal function with a limiting amount of eIF-2B.
Animals↗
ACTH stimulates fructose 2,6-bisphosphate synthesis and glycolysis in Y-1 adrenal tumor cells.
The effect of ACTH on glycolysis has been studied in Y-1 tumor adrenal cells. ACTH caused a sustained increase in the liberation of lactate as well as a stimulation of both basal and glucose-induced fructose 2,6-bisphosphate content. ACTH produces changes also in the activities of phosphofructokinase-1 and phosphofructokinase-2. The addition of Ca2+ or dibutyryl cyclic AMP did not modify neither lactate production nor fructose 2,6-bisphosphate levels. The results suggest that fructose 2,6-bisphosphate regulates ACTH-induced glycolysis at the phosphofructokinase-1 step, although the biochemical mechanism of phosphofructokinase-2 activation remains elusive.
Adrenal Gland Neoplasms↗
Vanadate raises fructose 2,6-bisphosphate concentrations and activates glycolysis in rat hepatocytes.
In rat hepatocytes, vanadate increases fructose 2,6-bisphosphate (Fru-2,6-P2) in a time- and dose-dependent manner, and counteracts the decrease in this metabolite caused by glucagon, forskolin or exogenous cyclic AMP. Vanadate does not directly modify the activity of 6-phosphofructo-2-kinase, even though it can counteract the inactivation of this enzyme caused by glucagon. Furthermore, vanadate raises the yield of 3H2O from [3-3H]glucose, indicating that it increases the flux through 6-phosphofructo-1-kinase. Moreover, vanadate in hepatocytes incubated in the presence of glucose increases the production of both lactate and CO2. Therefore vanadate has insulin-like effects on the glycolytic pathway in rat hepatocytes. These results clearly contrast with our previous observation that vanadate exerts glycogenolytic non-insulin-like effects on glycogen synthase and phosphorylase.
Animals↗
Control of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle by sn-glycerol 3-phosphate.
The kinetics of PFK-2 and FBPase-2 from rat liver were investigated with respect to the substrates and the effector sn-glycerol 3-phosphate. PFK-2 exhibits a hyperbolic response with respect to its substrates Fru 6-P and ATP. The inhibition of the activity of PFK-2 by sn-glycerol 3-phosphate could be described by assuming competition with Fru 6-P at the catalytic site. sn-Glycerol 3-phosphate activates the FBPase-2 and is capable of reversing partially the inhibition of the enzyme by Fru 6-P. The dynamics of the PFK-2/FBPase-2 cycle has been investigated in an enzyme system composed of PFK-2/FBPase-2, creatine kinase and creatine phosphate. sn-Glycerol 3-phosphate was found to decrease the quasi-stationary concentration of Fru 2,6-P2. The control of the PFK-2/FBPase-2 cycle by sn-glycerol 3-phosphate turned out most efficient at high concentrations of both sn-glycerol 3-phosphate and Fru 6-P. In addition, sn-glycerol 3-phosphate was found to increase the concentration control coefficient of Fru 2,6-P2 with respect to Fru 6-P.
Adenosine Triphosphate↗
Effects of bile duct ligation and dietary change on hepatic carbohydrate metabolism in rats.
Effects of bile duct ligation and dietary change on hepatic carbohydrate metabolism in rats were studied by analyzing the levels of fructose 2,6-bisphosphate (fructose-2,6-P2) and other metabolites. There was no significant difference in the levels of fructose-2,6-P2 and glycolytic intermediates between rats with and without bile duct ligation when rats were in a fed state. However, the levels of fructose-2,6-P2, hexose monophosphates, and triose phosphates increased and the level of plasma glucose decreased in rats with bile duct ligation in a starved state. The crossover analysis of gluconeogenic intermediates in rats with bile duct ligation showed accumulations of hexose monophosphates together with fructose-2,6-P2 in spite of the starved state of rats, indicating an impaired conversion of glucose 6-phosphate to glucose. Despite the activation of the fructose 6-phosphate-fructose 1,6-bisphosphate futile cycle as indicated by these findings, the energy charge levels were not reduced. The block at the level of glucose production may explain the relative predominance of insulin effect on glycolysis.
Adenine Nucleotides↗
Hemolytic anemia and red blood cell metabolic disorder attributable to low phosphorus intake in cows.
Hypophosphatemia was induced in 2 cows by reducing phosphorus content in their feed after parturition. Serum inorganic phosphorus (Pi) values decreased to 1 mg/dl within 10 days after parturition; and RBC adenosine 5'-triphosphate (ATP) and reduced glutathione values decreased to 50 and 70% of baseline values, respectively. Methemoglobin concentration was moderately higher than normal. These changes preceded the onset of hemolysis, and anemia progressed with decreases in PCV, hemoglobin concentration, and RBC counts. Serum Pi resumed its normal value when anemia was most severe. This RBC disorder was confirmed to be characteristic of hemolytic anemia in cows resulting from hypophosphatemia. The RBC glycolytic intermediates, total triose phosphate (combined glyceraldehyde-3-phosphate and dihydroxyacetone phosphate content) and fructose-1,6-diphosphate, greatly increased in vivo and in vitro with decreases in serum or plasma Pi and RBC ATP. From our results, we concluded that inadequate Pi in the plasma impairs the function and viability of RBC by hindering the production of ATP via disturbance of reactions at the glyceraldehyde-3-phosphate dehydrogenase step.
Adenosine Triphosphate↗
Interaction of ADP and fructose-2,6-bisphosphate with phosphofructokinase-1 from yeast.
ADP was found to activate or, depending on the experimental conditions, to inhibit yeast phosphofructokinase-1. In the absence of AMP and fructose-2,6-bisphosphate ADP increases the apparent affinity of the enzyme to fructose-6-phosphate. At low ATP concentrations the maximum activity with respect to fructose-6-phosphate decreases in the presence of ADP, while at high ATP a significant increase of the maximum activity by ADP is observed. In the presence of fructose-2,6-bisphosphate and AMP only the inhibiting effect of ADP persists. The data may be interpreted in terms of a hyperbolic inhibition mechanism.
Adenosine Diphosphate↗