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At least 109 records · Page 6Linked to original sources
PubMed1991

Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase.

The classical, alpha/beta-subunit form (Q2) of green tomato pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), a cytosolic enzyme functional in carbohydrate metabolism, was rapidly inactivated on incubation with the oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Analysis of the DTNB-treated sample by a fluorescence procedure revealed that inactivation was accompanied by oxidation of sulfhydryl groups, primarily on the alpha-subunit. Phosphate metabolites--fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, Pi, and PPi--protected against DTNB inactivation to varying degrees. The Km values for fructose 6-phosphate and PPi were not changed by DTNB treatment, but the capability for activation by fructose 2,6-bisphosphate was severely diminished. The oxidative inactivation of PFP was reversed by dithiothreitol, but not by monothiols (reduced glutathione or beta-mercaptoethanol). Reactivation was accompanied by restoration of the ability to undergo activation by fructose 2,6-bisphosphate. The findings suggest that sulfhydryl groups are essential for the activation of PFP by fructose 2,6-bisphosphate and raise the possibility that a reversible change in their redox status can take place under certain conditions. Evidence that this is the case was obtained with a preparation from wheat flour which, in the absence of an added oxidant, required reduction by a dithiol for activation by fructose 2,6-bisphosphate (dithiothreitol and reduced thioredoxin h).

Dithionitrobenzoic Acid↗
PubMed1985

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.

Adenosine Monophosphate↗
PubMed2003

Fluorescence study of ligand binding to potato tuber pyrophosphate-dependent phosphofructokinase: evidence for competitive binding between fructose-1,6-bisphosphate and fructose-2,6-bisphosphate.

The intrinsic fluorescence of potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was used as an indicator of conformational changes due to ligand binding. Binding of the substrates and the allosteric activator fructose-2,6-bisphosphate was quantitatively compared to their respective kinetic effects on enzymatic activity. PFP exhibited a relatively high affinity for its isolated substrates, relative to the enzyme's respective K(m) (substrate) values. There are two distinct types of fructose-1,6-bisphosphate interaction with PFP, corresponding to catalytic and activatory binding. Activatory fructose-1,6-bisphosphate binding shares several characteristics with fructose-2,6-bisphosphate binding, indicating that both ligands compete for the same allosteric activator site. Activation by fructose-1,6-bisphosphate or fructose-2,6-bisphosphate was exerted primarily on the forward (glycolytic) reaction by greatly increasing the enzyme's affinity for fructose-6-phosphate. Binding of substrates and effectors to PFP and PFP kinetic properties were markedly influenced by assay pH. Results indicate an increased glycolytic role for PFP during cytosolic acidification that accompanies anoxia stress.

Binding, Competitive↗
PubMed1973

Effect of administration of the fructose on the glycogenolytic action of glucagon. An investigation of the pathogeny of hereditary fructose intolerance.

1. The mechanism by which the administration of fructose to patients with hereditary fructose intolerance makes them unresponsive to the hyperglycaemic action of glucagon was studied. In four patients, a 10-fold increase in the urinary excretion of cyclic AMP was induced by glucagon, but this effect was drastically decreased by the previous administration of fructose (250mg/kg). Further, the intravenous injection of 6-N,2'-O-dibutyryl cyclic AMP did not cause an increase in the blood glucose during fructose-induced hypoglycaemia. 2. The administration of a large dose of fructose (5g/kg) to mice decreased markedly both the concentration of ATP and the increase in the concentration of cyclic AMP caused by glucagon in the liver. Other ATP-depleting agents had a similar effect and a linear correlation could be drawn between the concentration of ATP and the change in cyclic AMP concentration; a half-maximal effect was obtained for a concentration of ATP close to the K(m) value of adenylate cyclase. 3. The administration of fructose to mice caused the inactivation of phosphorylase in the liver, but this effect was easily reversed by glucagon. 4. At a concentration of 10mm-fructose 1-phosphate and 1.5mm-P(i), purified liver phosphorylase a was inhibited by 70%. This inhibition appears to be a likely explanation for the unresponsiveness to glucagon of patients with hereditary fructose intolerance.

Journal Article↗
PubMed1988

Purification and characterization of myocardial fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase.

Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit Mr of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,2-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by cAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The Km of the kinase for fructose-6-P and ATP was 70 microM and 260 microM, respectively for both the 58,000 and the 54,000 enzymes. Km for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 microM, respectively. The enzyme was phosphorylated by fructose-2,6-P2 but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.

Animals↗
PubMed1980

Adaptative changes of activity of enzymes involved in fructose metabolism in the liver and jejunal mucosa of rats following fructose feeding.

The adaptative response of a diet containing 60% fructose on the activity of those enzymes which are involved in the metabolism of fructose was measured in the liver and in the jejunal mucosa of rats over a period of 12 days. Control animals received isocaloric amounts of glucose or starch. Under fructose feeding there was a marked increase in the activity of fructose-1-phosphate aldolase (3-fold), ketohexokinase (2--3-fold), and triokinase (3-fold) in the jejunal mucosa. In the liver, however, a significant increase in enzyme activity could only be seen for triokinase (2--3-fold), whereas the activity of the other enzymes measured were only slightly or not at all altered. The activity of the three enzymes mentioned above were elevated to a maximum within 3 days after feeding the fructose diet. In the following time of observation no major further changes occurred. The results show that fructose feeding in comparison to a glucose or starch containing diet leads to a marked adaptative increase in the activity of those enzymes, which are involved in the breakdown of fructose, only in the jejunal mucosa.

Alcohol Oxidoreductases↗
PubMed1983

A radioisotopic method for fructose-1-phosphate aldolase assay that facilitates diagnosis of hereditary fructose intolerance.

A sensitive new method in which D-[U-14C]fructose-1-phosphate is used for fructose-bisphosphate aldolase (EC 2.1.2.13) assay is described. The radioactive fructose-1-phosphate compound was prepared from [U-14C]fructose by use of partly purified fructokinase (EC 2.7.1.4). With this method we measured normal values for aldolase in human liver (2.4-10.0 nmol/min per mg of protein), kidney (3.6-3.8), and intestine (4.2-10.0) as well as Km values for fructose-1-phosphate (approximately 1.0-2.2 mmol/L). In patients with hereditary fructose intolerance the aldolase activity in liver and intestine was less than 10% of normal values. The Lineweaver-Burk plots for data from patients with hereditary fructose intolerance were hyperbolic, indicating a structural alteration in the enzyme.

Adult↗
PubMed1996

Effects of fructose ingestion on sorbitol and fructose 3-phosphate contents of erythrocytes from healthy men.

To investigate the effect of fructose ingestion on sorbitol and fructose 3-phosphate (F3P) in erythrocytes, we administered 50 g fructose with and without treatment with an aldose reductase inhibitor, epalrestat, to seven healthy, normal-glucose-tolerant, male volunteers aged 20-43 years. The same subjects were given 50 g glucose on another day. The sorbitol and F3P contents in their erythrocytes increased significantly, reaching peak levels at 60 min and 180 min, respectively, following fructose in gestion. On the other hand, glucose ingestion did not cause any statistically significant change in sorbitol content in their erythrocytes, although it significantly elevated their F3P content. Treatment with epalrestat had no significant effect on incremental changes in erythrocyte sorbitol and F3P content following fructose ingestion. This suggests that oral fructose may be converted directly to sorbitol and F3P in erythrocytes instead of being converted via glucose. Thus, the dietary intake of fructose may affect the concentrations of sorbitol and F3P in erythrocytes in normal men.

Adult↗
PubMed1983

Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases.

Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.

Adenosine Monophosphate↗
PubMed1984

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate.

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.

Adenosine Monophosphate↗
PubMed1988

Allosteric inhibition of Dictyostelium discoideum fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate.

It has been found that the inhibition of Dictyostelium discoideum fructose-1,6-bisphosphatase by fructose 2,6-P2 greatly diminished when the pH was raised to the range 8.5-9.5, which resulted in a marked decrease of the affinity for the inhibitor with no change in the Km for the substrate. This provides evidence for the involvement of an allosteric site for fructose 2,6-P2. Moreover, the fact that excess substrate inhibition also decreased at the pH values for minimal fructose 2,6-P2 inhibition, and was essentially abolished in the presence of fructose 2,6-P2, strongly suggests that this inhibition takes place by binding of fructose 1,6-P2 as a weak analogue of the physiological effector fructose 2,6-P2.

Allosteric Regulation↗
PubMedDate not supplied

Responses of insulin to oral glucose and fructose loads in marginally copper-deficient rats fed starch or fructose.

The purpose of this study was to assess the effects of dietary fructose either alone or in combination with marginal copper deficiency in weanling male rats exposed to their respective diets for only 2 wk. This short duration of exposure to inadequate copper intake prevents progressive morbidity brought about by increasing periods of exposure to dietary copper deprivation. Weanling male rats were fed a copper-deficient (0.6 microgram Cu/g) or a copper-adequate (6.0 micrograms Cu/g) diet containing 62% fructose or 62% starch for 2 wk. Either an oral glucose or an oral fructose tolerance test was conducted after an overnight fast. Insulin levels were elevated by either oral glucose or oral fructose at fasting and at 30 min postload in rats fed fructose compared with those fed starch. Despite high levels of plasma, insulin blood glucose was not reduced. Marginal copper deficiency had no effect on either plasma insulin or blood glucose. Data identify fructose as the sole agent responsible for inducing adverse changes in glucose metabolism. Two weeks of fructose consumption was sufficient to produce these changes.

Animals↗
PubMed1983

On the mechanism of inhibition of neutral liver fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate.

The inhibitory effect of fructose 2,6-biphosphate on fructose 1,6-bisphosphatase was reinvestigated in order to solve the apparent contradiction between competition with the substrate and the synergism with AMP, a strictly noncompetitive inhibitor. The effect of fructose 2,6-bisphosphate was compared to that of other ligands of the enzyme, which, like the substrate and methyl (alpha + beta)fructofuranoside 1,6-bisphosphate bind to the active site or which, like AMP, bind to an allosteric site. An increase in temperature or pH, or the presence of sulfosalicylate, lithium or higher concentrations of magnesium as well as partial proteolysis by subtilisin increased [I]0.5 for fructose 2,6-bisphosphate and AMP without affecting Km. With the exception of the pH change, all these conditions were also without effect on the affinity of the enzyme for the competitive inhibitor, methyl (alpha + beta)fructofuranoside 1,6-bisphosphate. These observations can be explained by assuming that fructose 2,6-bisphosphate has no affinity for the active site of fructose 1,6-bisphosphatase but binds to an allosteric site which is different from the AMP site. Fructose 2,6-bisphosphate is therefore classified as an allosteric competitive inhibitor and a model is proposed which explains its synergism with AMP as well as the various cooperative effects.

Adenosine Monophosphate↗
PubMed1991

Kinetics of the conformational transition of the spinach chloroplast fructose-1,6-bisphosphatase induced by fructose 2,6-bisphosphate.

The activation of oxidized chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate and magnesium previously described at pH 7.5 [Soulié et al. (1988) Eur. J. Biochem. 176, 111-117] has now been studied at pH 8, the pH which prevails under light conditions in the chloroplast stroma. The process obeys a hysteretic mechanism but the rate of activation is considerably increased with half-times down to 50 s and the apparent dissociation constant of fructose 2,6-bisphosphate from the enzyme is lowered from 1 mM at pH 7.5 to 3.3 microM at pH 8. The process is strictly metal-dependent with a half-saturation concentration of 2.54 mM for magnesium. The conformational transition postulated in our hysteretic model has been investigated through both the spectrophometric and chemical modification approaches. The activation of the enzyme by fructose 2,6-bisphosphate in the presence of magnesium results in a slow modification of the ultraviolet absorption spectrum of the enzyme with an overall increase of 3% at 290 nm. The same treatment leads to the protection of two free sulfhydryls and an increased reactivity of one sulfhydryl group/enzyme monomer to modification by 5,5'-dithiobis(2-nitrobenzoic acid). The titration of the exposed cysteinyl residue prevents the relaxation of enzyme species induced by fructose 2,6-bisphosphate to the native form. The activation of chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate is discussed both with respect to the understanding of the overall regulation properties of the enzyme and to a possible physiological significance of this process.

Binding Sites↗
PubMed1985

Activation of fructose-1,6-bisphosphatases by monovalent cations and its relationship with a fructose-2,6-bisphosphate allosteric site.

The effects of potassium ions on pig kidney fructose-1,6-bisphosphatase activity have been studied. At low (non-inhibitory) concentrations of fructose-1,6-bisphosphate K+ shows an inhibitory effect and the apparent Km for fructose-1,6-bisphosphate increases as the concentration of monovalent cation increases. The inhibition by high substrate concentrations is decreased by addition of the potassium ions. Modification of a highly reactive cysteine residue with cyanate or N-ethylmaleimide results in the loss of activation of the enzyme by K+. Significant protection to the loss of potassium activation and substrate inhibition is afforded by the presence of low concentrations of fructose-2,6-bisphosphate or inhibitory levels of fructose-1,6-bisphosphate. Non-inhibitory concentrations of the substrate give partial protection against the loss of monovalent cation activation. The inhibitor AMP markedly increases the reactivity of the cysteine residue. The carbamoylated enzyme is not inhibited by excess of Mg2+ as compared to native enzyme. The results suggest that K+ decreases the affinity of the enzyme for fructose-1,6-bisphosphate at both the catalytic site and an allosteric site for fructose-2,6-bisphosphate. Furthermore, they lead to the proposal that monovalent cations activation could be due to the removal of both Mg2+ and substrate inhibitions.

Animals↗
PubMed1984

Inhibition of fructose-1,6-bisphosphatase from pig liver by fructose-6-phosphate.

The inhibition of the neutral form of fructose-1,6-bisphosphatase from pig liver by fructose-6-phosphate was investigated in the substrate concentration range of 0.05-500 microM by determination of the rate of formation of labelled inorganic phosphate from [1-32P]fructose-1,6-bisphosphate. The inhibition of the enzyme by fructose-6-phosphate is biphasic, the extent of inhibition decreases with increasing substrate concentrations. Even at high concentrations of fructose-6-phosphate the enzyme is not inhibited completely. The results were interpreted in terms of the model of MONOD, WYMAN and CHANGEUX [10] by assuming a weak competition of fructose-6-phosphate and fructose-1,6-bisphosphate at the catalytic site and a cooperation of the two ligands at the same allosteric site.

Animals↗
PubMed1981

Inhibition of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate.

Rat liver fructose-1,6-bisphosphatase, which was assayed by measuring the release of 32P from fructose 1,6-[1-32P]bisphosphate at pH 7.5, exhibited hyperbolic kinetics with regard to its substrate. beta-D-Fructose 2,6-bisphosphate, an activator of hepatic phosphofructokinase, was found to be a potent inhibitor of the enzyme. The inhibition was competitive in nature and the Ki was estimated to be 0.5 microM. The Hill coefficient for the reaction was 1.0 in the presence and absence of fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate also enhanced inhibition of the enzyme by the allosteric inhibitor AMP. The possible role of fructose 2,6-bisphosphate in the regulation of substrate cycling at the fructose-1,6-bisphosphatase step is discussed.

Adenosine Monophosphate↗
PubMed1983

Oscillations in the phosphofructokinase--fructose 1,6-bisphosphatase cycle. II. Influence of fructose 1,6-bisphosphatase on the character of oscillatory states.

The dynamic behaviour of an open futile cycle composed of phosphofructokinase and fructose 1,6-bisphosphatase has been investigated in a homogeneous reconstituted enzyme system in which the two enzymes cooperate with pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerase. By the kinetic cooperation of phosphofructokinase and fructose 1,6-bisphosphatase various types of dynamic patterns can be generated. In the absence of fructose 1,6-bisphosphatase oscillations do not occur. As experimentally demonstrated, in a definite region of maximum activities of fructose 1,6-bisphosphatase sustained oscillations originate while at higher concentrations of the enzyme damped oscillations appear. At increasing concentrations of fructose 1,6-bisphosphatase damping is strengthened. Finally, at very high concentrations of fructose 1,6-bisphosphatase the metabolites relax monotonously to the stationary states. The observed oscillatory phenomena are mainly caused by the antagonistic effects of AMP on the kinetics of phosphofructokinase and fructose 1,6-bisphosphatase.

Adenylate Kinase↗