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PubMed1985

Nuclear magnetic resonance studies of fructose 2,6-bisphosphate and adenosine 5'-monophosphate interaction with bovine liver fructose-1,6-biphosphatase.

1H and 31P nuclear magnetic resonance was used to investigate the interaction of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) with bovine liver fructose-1,6-bisphosphatase. Mn2+ bound to fructose-1,6-bisphosphatase was used as a paramagnetic probe to map the active and AMP allosteric sites of fructose-1,6-bisphosphatase. Distances between enzyme-bound Mn2+ and the phosphorus atoms at C-6 of fructose-6-P and alpha-methyl-D-fructofuranoside 1,6-bisphosphate were identical, and the enzyme-Mn to phosphorus distance determined for the C-6 phosphorus atom of Fru-2,6-P2 was very similar to these values. Likewise, the enzyme-Mn to phosphorus distances for Pi, the C-1 phosphorus atom of alpha-methyl-D-fructofuranoside 1,6-bisphosphate, and the C-2 phosphorus atom of Fru-2,6-P2 agreed within 0.5 A. The distance between enzyme-bound Mn2+ and the phosphorus atom of AMP was significantly shorter than the distances obtained for any of the aforementioned ligands, but the presence of Fru-2,6-P2 caused the enzyme-Mn to phosphorus distance for AMP to lengthen markedly. NMR line broadening of AMP protons was studied at various temperatures. The dissociation rate constant was found to be greater than 20 s-1. It was concluded that Fru-2,6-P2 strongly affects the interaction of AMP with fructose-1,6-bisphosphatase and that the sugar most likely acts at the active site of the enzyme.

Adenosine Monophosphate↗
PubMed1986

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

The inhibition of pig liver fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate has been investigated over a wide range of substrate concentration by measuring the release of labelled inorganic phosphate from [1-32P]fructose 1,6-bisphosphate. The activity of the enzyme can be inhibited completely by fructose 2,6-bisphosphate. The inhibiting effect is most pronounced at low substrate concentrations. The results have been analyzed in terms of a mathematical model assuming a competitive interaction of fructose 1,6-bisphosphate and fructose 2,6-bisphosphate at the catalytic site as well as a synergistic cooperation of the two hexose bisphosphates at an inhibiting allosteric site of the enzyme.

Animals↗
PubMed1983

Fructose 2,6-bisphosphate activates the cAMP-dependent phosphorylation of yeast fructose-1,6-bisphosphatase in vitro.

Fructose-1,6-bisphosphatase purified from Saccharomyces cerevisiae is phosphorylated in vitro by a cAMP-dependent protein kinase. The phosphorylation reaction incorporates 1 mol of phosphate/mol of enzyme and is greatly stimulated by fructose 2,6-bisphosphate. Fructose 2,6-bisphosphate acts upon fructose-1,6-bisphosphatase, not on the protein kinase. The phosphorylation of fructose 1,6-bisphosphatase lowers its activity by about 50%. The characteristics of the phosphorylation reaction in vitro show that this modification is responsible for the inactivation of fructose-1,6-bisphosphatase observed in vivo.

Cyclic AMP↗
PubMed1995

Production of fructose and fructose-3-phosphate in maturing rat lenses.

PURPOSE: A large increase in glycation of crystallins between 1 and 8 months has been demonstrated in lenses obtained from aging rats. The objective of this study was to investigate if an age-associated increase in the levels of any of the phosphorylated and nonphosphorylated sugars in the aging rat lenses could be correlated with this increase. METHODS: Lenses were obtained from Sprague-Dawley rats ranging in age from 2 to 20 months. Trichloroacetic extracts of these tissues were analyzed by using 31P-NMR for sugar phosphates and high-pressure liquid chromatography equipped with an electrochemical detector for sugars and polyols. RESULTS: Although no elevation in the lenticular glucose levels was observed, an age-associated increase in the concentrations of polyol pathway-associated metabolites--sorbitol, fructose, sorbitol-3-phosphate, and fructose-3-phosphate--was detected. In contrast, no significant changes were observed in glycolytic or pentose shunt metabolites. CONCLUSION: Aging lenses accumulate increased concentrations of fructose and fructose-3-phosphate. Because fructose-3-phosphate is a potent glycating agent and a potential in vivo source of 3-deoxyglucosone, its accumulation in the lens, along with fructose, may be a contributing factor in the age-associated increase of nonenzymatic glycation in rat lenses.

Aging↗
PubMed1994

Shared active sites of fructose-1,6-bisphosphatase. Arginine 243 mediates substrate binding and fructose 2,6-bisphosphate inhibition.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, Arg-243 was replaced by alanine using techniques of site-specific mutagenesis and the cloned pig kidney enzyme expressed in Escherichia coli. Compared with wild-type enzyme, kinetic parameters of the altered enzyme characterizing catalytic efficiency, magnesium binding, and inhibition by AMP differed but by less than an order of magnitude; affinity for substrate fructose 1,6-bisphosphate was 10-fold poorer, and affinity for inhibitor fructose 2,6-bisphosphate was 1000-fold poorer. Molecular dynamics simulations were undertaken to determine possible alterations in active sites of the enzyme due to replacement of Arg-243 by Ala and suggested that in the mutant enzyme loss of one cationic group leads to reorganization of the active site especially involving lysine residues 269 and 274. The differences in properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase and confirm on a functional basis the shared active site in this important metabolic enzyme.

Adenosine Monophosphate↗
PubMed2001

Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes.

The role of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) in developing leaves was studied using wild-type tobacco (Nicotiana tabacum L.) and transformants with decreased expression of PFP. (i) The leaf base, which is the youngest and most actively growing area of the leaf, had 2.5-fold higher PFP activity than the leaf tip. T3 transformants, with a 56-95% decrease in PFP activity in the leaf base and an 87-97% decrease in PFP activity in the leaf tip, were obtained by selfing and re-selfing individuals from two independent transformant lines. (ii) Other enzyme activities also showed a gradient from the leaf base to the leaf tip. There was a decrease in PFK and an increase in fructose-6-phosphate,2-kinase and plastidic fructose-1, 6-bisphosphatase, whereas cytosolic fructose-1,6-bisphosphatase activity was constant. None of these gradients was altered in the transformants. (iii) Fructose-2,6-bisphosphate (Fru2,6bisP) levels were similar at the base and tip of wild-type leaves in the dark. Illumination lead to a decrease in Fru2,6bisP at the leaf tip and an increase in Fru2,6bisP at the leaf base. Compared to wild-type plants, transformants with decreased expression of PFP had up to 2-fold higher Fru2,6bisP at the leaf tip in the dark, similar levels at the leaf tip in the light, 15-fold higher levels at the leaf base in the dark, and up to 4-fold higher levels at the leaf base in the light. (iv) To investigate metabolic fluxes, leaf discs were supplied with 14CO2 in the light or [14C]glucose in the light or the dark. Discs from the leaf tip had higher rates of photosynthesis than discs from the leaf base, whereas the rate of glucose uptake and metabolism was similar in both tissues. Significantly less label was incorporated into neutral sugars, and more into anionic compounds, cell wall and protein, and amino acids in discs from the leaf base. Metabolism of 14CO2 and [14C]glucose in transformants with low PFP was similar to that in wild-type plants, except that synthesis of neutral sugars from 14CO2 was slightly reduced in discs from the base of the leaf. (v) These results reveal that the role of PFP in the growing cells in the base of the leaf differs from that in mature leaf tissue. The increase in Fru2,6bisP in the light and the high activity of PFP relative to cytosolic fructose-1,6-bisphosphatase in the base of the leaf implicate PFP in the synthesis of sucrose in the light, as well as in glycolysis. The large increase in Fru2,6bisP at the base of the leaf of transformants implies that PFP plays a more important role in metabolism at the leaf base than in mature leaf tissue. Nevertheless, there were no major changes in carbon fluxes, or leaf or plant growth in transformants with below 10% of the wild-type PFP activity at the leaf base, implying that large changes in expression can be compensated by changes in Fru2,6-bisP, even in growing tissues.

Biological Transport↗
PubMed2002

Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells.

The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media. 31P-NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6-bisphosphate content of each line was unaffected by 2-deoxyglucose and increased during growth on phosphate-limited media. Short-term treatment of callus with 2-deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long-term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U-14C]glucose in response to incubation with 2-deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate-depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6-bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate-deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the carbohydrate content changed in response to growth on phosphate-depleted media, it is concluded that an increase in flux through pyrophosphate:fructose-6-phosphate 1-phosphotransferase is not a major component of the metabolic response of heterotrophic tobacco cells to phosphate deficiency.

Journal Article↗
PubMed1984

The rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle.

Substrate cycling of fructose 6-phosphate through reactions catalysed by 6-phosphofructokinase and fructose-1,6-bisphosphatase was measured in skeletal muscles of the rat in vitro. The rate of this cycle was calculated from the steady-state values of the 3H/14C ratio in hexose monophosphates and fructose 1,6-bisphosphate after the metabolism of either [5-3H,6-14C]glucose or [3-3H,2-14C] glucose. Two techniques for the separation of hexose phosphates were studied; t.l.c. chromatography on poly(ethyleneimine)-cellulose sheets or ion-exchange chromatography coupled with enzymic conversion. These two methods gave almost identical results, suggesting that either technique could be used for determination of rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling. It was found that more than 50% of the 3H was retained in the fructose 1,6-bisphosphate; it is therefore probable that previous measurement of cycling rates, which have assumed complete loss of 3H, have underestimated the rate of this cycle. The effects of insulin, adrenaline and adrenergic agonists and antagonists on rates of fructose 6-phosphate/fructose 1,6-bisphosphate cycling were investigated. In the presence of insulin, adrenaline (1 microM) increased the cycling rate by about 10-fold in epitrochlearis muscle in vitro; the maximum rate under these conditions was about 2.5 mumol/h per g of tissue. The concentration of adrenaline that increased the cycling rate by 50% was about 50 nM. This effect of adrenaline appears to be mediated by the beta-adrenergic receptor, since the rate was increased by beta-adrenergic agonists and blocked by beta-adrenergic antagonists. From the knowledge of the precise rate of this cycle, the possible physiological importance of cycling is discussed.

Animals↗
PubMed1981

Mannose 6-, fructose 1-, and fructose 6-phosphates inhibit human natural cell-mediated cytotoxicity.

In vitro human natural cell-mediated cytotoxicity (NCMC) to K-562, Molt-4, and F-265 cells is inhibited in a dose-dependent manner by mannose 6-phosphate, fructose 1-phosphate and fructose 6-phosphate. This inhibition is not observed with mannose, glucose, fucose, glucose 6-phosphate, mannose 1-phosphate, galactose 1-phosphate, or galactose 6-phosphate. Preincubation of the effector cells, obtained from fresh whole blood, with mannose-6-phosphate, fructose-1-phosphate, or fructose-6-phosphate did not inhibit cytotoxicity, which indicated that these hexose phosphates are not nonspecifically toxic towards the effector lymphocytes. Mannose-6-phosphate and the stereochemically similar fructose-1-phosphate are more potent inhibitors than fructose-6-phosphate in terms of concentration required and time of onset of effect. Inhibition of cytotoxicity by mannose-6-phosphate varied with target cell type: F-265 is protected at much lower concentrations of mannose-6-phosphate (less than 1 mM) than is either Molt-4 or K-562. The inhibition of NCMC is also observed with the inhibitors of lysosomal function, NH4Cl, and chloroquine. The presence of a functional mannose-6-phosphate receptor on target cells was demonstrated: (i) Gelonin, a seed protein that inactivates the eukaryotic ribosome but is nontoxic to intact cells, was covalently linked to monophosphopentamannose, and this conjugate ws toxic to both K-562 and F-265 target cells, the latter being by far the more sensitive; and (ii) chloroquine, NH4Cl, and mannose-6-phosphate all inhibited the toxicity of gelonin-monophosphopentamannose. These results suggest either that a cytolytic lymphokine contains a hexose phosphate residue and may be taken up by target cells through the lysosomal/mannose 6-phosphate pathway or that such a residue is involved in target cell-effector cell recognition.

Cytotoxicity, Immunologic↗
PubMed1984

Effects of diets on concentrations of 6-phosphogluconate and fructose 2,6-bisphosphate in rat livers and an assay of fructose 2,6-bisphosphate with an improved method.

We determined the effects of diets that have different lipogenic potentials on hepatic concentrations of 6-phosphogluconate and fructose 2,6-bisphosphate, both of which activate hepatic phosphofructokinase. Diets high in carbohydrate increased concentrations of both effectors compared to a high protein (gluconeogenic) diet. The concentration of 6-phosphogluconate was associated with the lipogenic nature of the diet, and the range of its concentration matched that over which phosphofructokinase responds to 6-phosphogluconate in vitro. In contrast, the concentration of fructose of 2,6-bisphosphate was not associated with the lipogenic potential of the diets. Fructose 2,6-bisphosphate was either absent from liver or its concentration was 10- to 30-fold higher than the concentration that gives the maximal activation of phosphofructokinase in vitro. The results indicate that fructose 2,6-bisphosphate and 6-phosphogluconate have different roles in the regulation of phosphofructokinase. Fructose 2,6-bisphosphate may be involved in switching hepatic carbohydrate metabolism between gluconeogenesis and glycolysis, whereas changes in the concentration of 6-phosphogluconate may coordinate the disposition of glucose 6-phosphate between the oxidative branch of the hexosemonophosphate pathway and glycolysis. In the course of our studies, we improved an enzymatic assay for fructose 2,6-bisphosphate.

Animals↗
PubMed1986

The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Energetics of the phosphoryl group transfer from phosphoenolpyruvate to fructose.

Energy coupling to fructose transport in Rhodopseudomonas sphaeroides is achieved by phosphorylation of the membrane-spanning fructose-specific carrier protein, EFruII. The phosphoryl group of phosphoenolpyruvate is transferred to EFruII via the cytoplasmic component SF (soluble factor). The standard free enthalpy of hydrolysis of the two phosphorylated proteins has been estimated from isotope exchange measurements in chemical equilibrium. The delta G degrees for SF-P is -60.5 kJ/mol. The standard free enthalpy for hydrolysis of EII-P is -37.9 kJ/mol, but -45.2 kJ/mol when SF is still complexed to it, as in the overall reaction. Therefore the standard free enthalpy of hydrolysis of SF X EII-P is 70% of the standard free enthalpy of hydrolysis of P-enolpyruvate. The measurements reveal two regulation sites in the system. First, the phosphorylation of SF is inhibited by pyruvate when the concentration ratio of pyruvate/P-enolpyruvate becomes too high. Second, a low concentration of internal fructose prevents the phosphorylation of the carrier by the internal fructose-1-P pool when the concentration of the latter becomes too high or the phosphorylation rate by P-enolpyruvate too slow. Furthermore comparison of the isotope exchange and the overall phosphotransferase reaction kinetics leads to the conclusion that binding of fructose to the carrier is a slow step relative to the phosphoryl group transfer from EFruII to fructose.

Catalysis↗
PubMed1983

Assessment of a futile cycle involving reconversion of fructose 6-phosphate to fructose 1,6-bisphosphate during gluconeogenic growth of Escherichia coli.

In gluconeogenesis, fructose 6-phosphate is formed from fructose 1,6-bisphosphate, and if fructose 1,6-bisphosphate were reformed by the phosphofructokinase reaction there would be a "gluconeogenic futile cycle." We assessed the extent of this cycling in Escherichia coli growing on glycerol 3-phosphate, using a medium containing 32Pi. Fructose 1,6-bisphosphate coming from glycerol 3-phosphate should be unlabeled, but any coming from fructose 6-phosphate should contain label from the gamma-position of ATP. The amount of labeling of the 1-position of fructose 1,6-bisphosphate was only 2 to 10% of that of the gamma-position of ATP in a series of isogenic strains differing in phosphofructokinases (Pfk-1, Pfk-2, or Pfk-2). In control experiments with glucose 6-phosphate instead of glycerol 3-phosphate, the two positions were equally labeled. Thus, although the presence of Pfk-2 causes gluconeogenic impairment (Daldal et al., Eur. J. Biochem., 126:373-379, 1982), gluconeogenic futile cycling cannot be the reason.

Adenosine Triphosphate↗
PubMed1978

[Behavior of various blood constituents (glucose, fructose, insulin, lactate, pyruvate, free fatty acids, inorganic phosphate) and the half-life of monosaccharides in plasma after i.v infusion of glucose, fructose, galactose and invert sugar solutions in ruminants. 3. Studies in sheep].

Monosaccharide half-life as well as the effects of intravenous infusion of 0.5 g/kg body weight glucose, fructose, and invert sugar solutions on insulin levels in blood plasma as well as on pyruvate and lactate concentrations in the blood were measured in two adult sheep weighing 72 kg and 65 kg. The half-life values for glucose were 16 minutes and 19 minutes and that for fructose 20 minutes. Glucose half-life, following infusion of invert sugar, was 35 minutes or 25 minutes, while that of fructose was 16 minutes in either case. Infusion of fructose solution was followed by a somewhat sizeable and prolonged rise of the glucose levels in blood plasma. Infusion of glucose solution resulted only in short-time increase of fructose concentrations in blood plasma. The highest insulin rise in blood plasma was found to have taken place in response to infusion of fructose solution. Pyruvate and lactate concentrations underwent merely minor changes. Reference is made to the potential benefit of using invertose solution in the treatment of ketosis (cf. second communication).

Animals↗
PubMed2000

Overexpression and purification of fructose-1-phosphate kinase from Escherichia coli: application to the assay of fructose 1-phosphate.

Fructose 1-phosphate is a metabolite that plays a regulatory role in liver and is best measured using an assay based on its conversion to fructose 1,6-bisphosphate by a bacterial fructose-1-phosphate kinase (Fru1PK). The open reading frame encoding Escherichia coli Fru1PK has been introduced in an expression plasmid (pET3a) based on the T7 promoter-driven system, which was used to overexpress the enzyme. The conditions for the production of soluble Fru1PK were optimized. The purification procedure used involved ammonium sulfate precipitation and chromatography on DEAE-Sepharose and was aimed mostly at stabilizing the enzyme and at freeing Fru1PK from bacterial contaminants that could interfere in the fructose 1-phosphate assay. From a 1-liter culture, more than 50 mg protein is obtained. This preparation can be used in an enzymatic assay that measures specifically fructose 1-phosphate in tissue extracts.

Animals↗
PubMed1987

Comparative use of glucose and fructose in cultured fibroblasts from patients with hereditary fructose intolerance.

The utilization of fructose and glucose by fibroblast cultures obtained from patients with hereditary fructose intolerance (HFI) was studied in comparison with fibroblast controls. The cell growth, the time course of D-glucose or D-fructose uptake and the consumption of fructose were similar for both HFI and control cells. Some results showed significant differences between these two cell types: HFI cells consumed less glucose, produced less lactate and contained less glycogen than control cells. Furthermore, significantly less [U-14C]D-glucose and [U-14C]D-fructose was incorporated into lipids in HFI cells than in control cells. The mechanisms responsible for these differences observed between the two cell types are not known.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1969

Renal fructose-metabolizing enzymes: significance in hereditary fructose intolerance.

In patients with hereditary fructose intolerance, which is characterized by deficient aldolase activity toward fructose-1-phosphate, fructose induces a renal tubular dysfunction that implicates only the proximal convoluted tubule. Because normal metabolism of fructose by way of fructose-1-phosphate requires fructokinase, aldolase "B," and triokinase, the exclusively cortical location of these enzymes indicates that the medulla is not involved in the metabolic abnormality presumably causal of the renal dysfunction.

Animals↗
PubMed1984

Detection of heterozygotes for fructose 1,6-diphosphatase deficiency by measuring fructose 1,6-diphosphatase activity in their cultured peripheral lymphocytes.

The fructose 1,6-diphosphatase activities in peripheral lymphocytes from the parents of a patient with fructose 1,6-diphosphatase deficiency were lower than the mean value of normal controls, but the value of the mother overlapped lower values for normal controls. The fructose 1,6-diphosphatase activities in lymphocytes of normal adults and the parents increased progressively during in vitro culture, but no enzyme activity could be detected in the lymphocytes of the patient even after culture. None of the values for the parents overlapped those of normal controls on either day 5 or 10 of culture. Thus, it seems probable that heterozygotes for fructose 1,6-diphosphatase deficiency can be distinguished from normal individuals by measuring the fructose 1,6-diphosphatase activity in their cultured lymphocytes.

Adult↗
PubMed1982

The affinity of rabbit muscle fructose-bisphosphatase for fructose bisphosphate.

This paper reports that microM concentrations of fructose bisphosphate are titrated by rabbit muscle fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) when the enzyme concentration is varied in the range which secures measurable initial velocities of reaction: a result that can only be explained by supposing that the enzyme has a greater affinity for fructose bisphosphate than suggested by Fernando, J., Enser, M., Pontremoli, S. and Horecker, B.L. (1968) Arch. Biochem. Biophys. 126, 599-606. The results also suggest that the keto form of the substrate may be the preferred configuration and that the enzyme is inhibited by magnesium-bound fructose bisphosphate.

Animals↗