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Similarity of activation of yeast phosphofructokinase by AMP and fructose-2,6-bisphosphate.

Phosphofructokinase from yeast is effectively activated by AMP and fructose-2,6-bisphosphate by increasing the affinity of the enzyme to fructose-6-phosphate and the maximum activity toward this substrate. The enzyme is activated by AMP and fructose-2, 6-bisphosphate both at high and at low concentrations of ATP. The half maximum stimulation concentrations of AMP and fructose-2, 6-bisphosphate are about 200 microM and 2 microM, respectively. At saturating concentrations of AMP and fructose-2, 6-bisphosphate similar maximum activities were observed in the dependence of enzyme activity on the concentrations of fructose-6-phosphate. The fructose-6-phosphate affinity is more enhanced by fructose-2, 6-bisphosphate than by AMP.

Adenosine Monophosphate↗

Dissipative structures for an allosteric model. Application to glycolytic oscillations.

An allosteric model of an open monosubstrate enzyme reaction is analyzed for the case where the enzyme, containing two protomers, is activated by the product. It is shown that this system can lead to instabilities beyond which a new state organized in time or in space (dissipative structure) can be reached. The conditions for both types of instabilities are presented and the occurrence of a temporal structure, consisting of a limit cycle behavior, is determined numerically as a function of the important parameters involved in the system. Sustained oscillations in the product and substrate concentrations are shown to occur for acceptable values of the allosteric and kinetic constants; moreover, they seem to be favored by substrate activation. The model is applied to phosphofructokinase, which is the enzyme chiefly responsible for glycolytic oscillations and which presents the same pattern of regulation as the allosteric enzyme appearing in the model. A qualitative and quantitative agreement is obtained with the experimental observations concerning glycolytic self-oscillations.

Adenosine Monophosphate↗

Cyclofructins with six to ten beta-(1-->2)-linked fructofuranose units: geometries, electrostatic profiles, lipophilicity patterns, and potential for inclusion complexation.

Cyclofructins composed of six (1, "CF6") to ten (5, "CF10") beta-(1-->2)-linked fructofuranose units were subjected to conformational analysis using Monte Carlo simulations based on the PIMM91 force-field. Breaking the molecular symmetry partially by alternating inclination of the spiro-type anellated fructofuranoses relative to the crown ether ring core, i.e. the 3-OH groups pointing either towards or away from the molecular center, substantially lowers the strain energy of the cyclofructins. The global energy-minimum geometries of CF6, CF8, and CF10 exhibit Cn/2 rotational symmetry, whilst the odd-membered macrocycles in CF7 and CF9 adopt C1 symmetry. Identical conformations of the solid-state geometry of CF6 (1) and its computer-generated form manifest the reliability of the computational analysis. The molecular surfaces calculated for the energy-minimum structures establish a disk-type shape for CF6 (1), CF7 (2), and CF8 (3), whereas further ring enlargement to CF9 (4) and CF10 (5) leads to torus-shaped molecules with through-going cavities. Color-coded projection of the molecular lipophilicity patterns (MLPs) and the electrostatic potential profiles (MEPs) onto these surfaces cogently displays the crown ether-like properties, favoring the complexation of metal cations via strong electrostatic interactions through the 3-OH groups located on the hydrophilic molecular side. The central cavities of CF9 and CF10 are characterized not only by significantly enhanced hydrophobicity, but also by highly negative electrostatic potentials around the narrow aperture of the tori made up by the 3-OH/4-OH groups, and positive potentials on the opposite rim. Accordingly, CF9 and CF10 are capable to form inclusion complexes, the cavity of the latter being approximately as large as the one of alpha-cyclodextrin. Calculation of the inclusion complex geometries of CF9 with beta-alanine and of CF10 with p-aminobenzoic acid revealed the guest to be deeply incorporated into the respective cavities, masking the guest's hydrophobic parts. Analysis of the electrostatic interactions at the interface of the zwitter-ionic guests with the oppositely polarized hosts predicts a high degree of regiospecificity for complex formation.

Carbohydrate Conformation↗

Preconditioning preserves mitochondrial function and glycolytic flux during an early period of reperfusion in perfused rat hearts.

OBJECTIVE: The purpose of the present study was to examine the effects of preconditioning on glycolysis and oxidative phosphorylation during reperfusion in perfused rat hearts. METHODS: Preconditioning was induced by 5 min of ischemia and 5 min of reperfusion before 40 min of sustained ischemia and subsequent 30 min of reperfusion. Tissue energy metabolite levels, mitochondrial oxygen consumption capacity and adenine nucleotide translocator content of the perfused hearts were assessed at 40 min of ischemia, 5 and 30 min of reperfusion. RESULTS: Preconditioning improved the postischemic recovery of rate x pressure product (92.5 +/- 8.7 vs. 24.9 +/- 1.2% for non-preconditioned group) and high-energy phosphate content (ATP and CrP; 39.5 +/- 2.0 and 96.2 +/- 4.9% of initial vs. 24.1 +/- 0.9 and 56.1 +/- 4.3% of initial for the non-preconditioned group). The mitochondrial oxygen consumption capacity and the adenine nucleotide translocator content of the non-preconditioned heart were decreased by sustained ischemia and remained decreased throughout reperfusion. Preconditioning prevented these decreases. The tissue lactate level of the non-preconditioned heart was high throughout reperfusion (16.5-fold vs. basal), whereas in the preconditioned heart it returned to the basal level within a few minutes of reperfusion. Furthermore, the ratios of [fructose 1,6-bisphosphate]/([glucose 6-phosphate] + [fructose 6-phosphate]) at 5-min reperfusion were higher (2.2-fold) than those of the non-preconditioned heart. CONCLUSIONS: The results suggest that preconditioning preserves the capacity for normal mitochondrial function and the facilitation of glycolysis during reperfusion, which may play an important role in the improvement of postischemic contractile function and high-energy phosphate content.

Adenosine Triphosphate↗

Glucose delivery is a major determinant of glucose utilisation in the ischemic myocardium with a residual coronary flow.

BACKGROUND: Experimental data from isolated rat hearts suggest that glycolysis in severe myocardial ischemia is inhibited by accumulation of glycolytic metabolites. In contrast, positron emission tomography (PET) in patients with myocardial ischemia records a 'mismatch' between the decreased coronary flow in viable ischemic tissue and an increased fluorodeoxyglucose (18FDG) signal. To resolve this contradiction, we investigated glucose uptake at very low coronary flows in the isolated rat heart model. METHODS: Rates of glucose uptake were measured in the isolated Langendorff-perfused Wistar rat heart, at control (12-15 ml/g wet wt/min) and low coronary flows (0.1, 0.2 and 0.5 ml/g wet wt/min) and at a range of glucose concentrations (2.75, 5.5, 11 and 22 mM). RESULTS: The steady-state rate of glucose uptake versus glucose concentration could be described by a double rectangular hyperbola at each coronary flow. Glucose uptake fell to levels significantly below control at low coronary flows. However, the extraction of glucose (glucose uptake as % of glucose delivered) rose sharply, from 1% at control coronary flows, to 25-30% at low coronary flows. Crossover analysis of glycolytic intermediates in freeze-clamped tissue indicated little inhibition at any specific site, although phosphofructokinase activity was restricted when glycolytic substrate availability was high. Insulin and preconditioning both increased glucose uptake with 11 mM glucose, possibly by increasing membrane transporter density and thus increasing glucose delivery to the cytosol. CONCLUSIONS: Despite the reduction in absolute glucose uptake at low coronary flow-rates, the extraction of glucose was greatly increased, possibly following GLUT4 translocation. Delivery of glucose to the glycolytic pathway appears to be a major controlling site of glycolysis in low-flow ischemia. Downstream regulation is then distributed along the pathway with no one site exerting greater inhibition than reduced glucose delivery.

Animals↗

Glucose flux and the redox state of pyridine dinucleotides in the rat lens.

The concentration of alpha-glycerophosphate (GP) appeared to increase and readily reached a new steady state in lenses incubated with KCN or under hyperglucosic condition. This increase can be explained by the change in NADH/NAD ratio under each condition. The relative ratio of pyridine dinucleotides (NADH/NAD) was calculated from equilibrium equations of two NAD-linked enzymes, lactate dehydrogenase (LDH) and alpha-glycerophosphate dehydrogenase (GPDH). The change in NADH/NAD ratio based on biochemical assays correlates well with that estimated from GPDH reaction. This indicates that measurements of pyridine dinucleotides (e.g. in vivo redox fluorometry) can be used to demonstrate lens metabolic status.

Animals↗

A deeper investigation on carbohydrate cycling in Sinorhizobium meliloti.

Recycling of triose-phosphate and pentose-phosphate was previously reported on glucose in Sinorhizobium meliloti, a polysaccharide-synthesizing bacterium, but the metabolic basis of such processes remained unclear. In this work, we have used (13)C-labelling strategies to demonstrate that carbohydrate cycling in this organism is independent of the gluconate bypass, the alternative pathway for glucose assimilation involving its periplasmic oxidation into gluconate. Furthermore, carbohydrate cycling in S. meliloti is also observed on fructose, making the situation in this bacterium significantly different from that depicted for alginate-synthesizing species.

Carbohydrate Metabolism↗

Bifunctional phosphomannose isomerase/GDP-D-mannose pyrophosphorylase is the point of control for GDP-D-mannose biosynthesis in Helicobacter pylori.

In this report a recombinant bifunctional phosphomannose isomerase/GDP-D-mannose pyrophosphorylase from Helicobacter pylori has been studied. The enzyme catalyzes the first and third steps of GDP-D-mannose biosynthesis from D-fructose-6-phosphate. The first step, isomerization from D-fructose-6-phosphate to D-mannose-6-phosphate, is found to be rate-limiting in GDP-D-mannose biosynthesis due to feedback inhibition. The inhibition is of non-competitive (mixed) type. As the enzyme is found only in bacteria probably participating in capsular polysaccharide biosynthesis, it could be a specific therapeutic target against bacterial infection.

Bacterial Proteins↗

Chloroplast class I and class II aldolases are bifunctional for fructose-1,6-biphosphate and sedoheptulose-1,7-biphosphate cleavage in the Calvin cycle.

Class I and class II aldolases are products of two evolutionary non-related gene families. The cytosol and chloroplast enzymes of higher plants are of the class I type, the latter being bifunctional for fructose-1,6- and sedoheptulose-1,7-P2 in the Calvin cycle. Recently, class II aldolases were detected for the cytosol and chloroplasts of the lower alga Cyanophora paradoxa. The respective chloroplast enzyme has been shown here to be also bifunctional for fructose-1,6- and sedoheptulose-1,7-P2. Kinetics, also including fructose-1-P, were determined for all these enzymes. Apparently, aldolases are multifunctional enzymes, irrespective of their class I or class II type.

Cell Compartmentation↗

Tetramer-dimer conversion of phosphofructokinase from Thermus thermophilus induced by its allosteric effectors.

Phosphofructokinase (PFKase) was purified from an extreme thermophile. Thermus thermophilus. Allosteric natures of T. thermophilus PFKase is similar to those of Bacillus stearothermophilus PFKase, that is, hyperbolic plots of the activity versus concentration of fructose 6-phosphate (F6P) were changed into a sigmoidal shape by the addition of phosphoenolpyruvate (PEP), while further addition of ADP caused it to revert to a hyperbolic shape. The native T. thermophilus PFKase has an Mr of 148,000 consisting of four 36,500 subunits. However, it exists as a two-subunit form of Mr 74,000 in the presence of PEP. The two-subunit form was catalytically inactive. The four-subunit enzyme was regenerated by addition of either F6P or Mg.ADP, or by removal of PEP from the solution. This reversible dissociation was observed within a wide range of pH (6.5 to 8.4) and temperature (4 degrees C to 65 degrees C). Thus, unlike PFKase from other sources, the allosteric kinetics of T. thermophilus PFKase can be explained well, at least qualitatively, by the dynamic equilibrium between the active four-subunit form and inactive two-subunit form that is modulated by PEP, F6P and Mg.ADP. Parallel suppression of the PEP-induced conversion in molecular form and kinetics by high concentrations of sulfate and phosphate supports the above explanation. Also, the observation that the degree of PEP inhibition was dependent on the protein concentration of the PFKase in the assay solution is consistent with the presence of this equilibrium.

Adenosine Diphosphate↗

The appearance of a critical input concentration of fructose 6-phosphate in the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase cycle.

Stationary states of the fructose 6-phosphate/fructose 2,6-bisphosphate cycle were investigated in relation to the input concentration of fructose 6-phosphate. Below a critical input concentration of fructose 6-phosphate very low levels of fructose 2,6-bisphosphate were obtained. Above this point the fructose 2,6-bisphosphate changes in direct proportion to the input of fructose 6-phosphate. Phosphorylation of the enzyme causes an increase of the critical input concentration of fructose 6-phosphate. The control coefficients for fructose 2,6-bisphosphate have their maximum at the critical input concentration of fructose 6-phosphate.

Fructosephosphates↗

Glutamine: fructose-6-phosphate amidotransferase activity and gene expression are regulated in a tissue-specific fashion in pregnant rats.

We examined whether regulation of glutamine: fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme of the hexosamine pathway, is tissue specific and if so whether such regulation occurs at the level of gene expression. We compared GFA activity and expression and levels of UDP-hexosamines and UDP-hexoses between insulin-sensitive (liver and muscle) tissues and a glucose-sensitive (placenta) tissue from 19 day pregnant streptozotocin diabetic and non-diabetic rats. In pregnant non-diabetic rats GFA activities averaged (1521+/-75 pmol/mg protein x min) in the placenta, 895+/-74 in the liver and 81+/-11 in muscle (p<0.001 between each tissue). In the diabetic rats, GFA activities were approximately 50% decreased both in the liver (340+/-42 pmol/mg protein x min, p<0.05 vs control rats) and in skeletal muscle (46+/-3, p<0.05) compared to control rats. In the placenta, GFA activities were identical between diabetic (1519+/-112 pmol/mg protein x min) and non-diabetic (1521+/-75) animals. In the liver, the reduction in GFA activity could be attributed to a significant decrease in GFA mRNA concentrations, while GFA mRNA concentrations were similar in the placenta between diabetic and non-diabetic animals. UDP-N-acetylglucosamine (UDP-GlcNAc), the end product of the hexosamine pathway, was significantly reduced in the liver and in skeletal muscle but similar in the placenta between diabetic and non-diabetic rats. In summary, GFA activity and expression and the concentration of UDP-GlcNAc are decreased in the liver but unaltered in the placenta, although GFA activity is almost 2-fold higher in this tissue than in the liver. These data provide the first evidence for tissue specific regulation of GFA and for its regulation at the level of gene expression.

Animals↗

Effects of thyroid status on glucose cycling by isolated rat hepatocytes.

The effects of alterations in thyroid status on glucose metabolism have been investigated in rat hepatocytes. Addition of 10 or 40 mmol/L glucose induced increases in respiration rate that were significantly larger in cells from hyperthyroid rats than from hypothyroid animals. The responses of hepatocytes from euthyroid rats were intermediate. In cells from hyperthyroid rats, most of the increase occurred upon addition of 10 mmol/L glucose, with only a further small stimulation resulting when glucose concentration was increased to 40 mmol/L. For a given glucose concentration, glycolytic rates, determined by measuring release of tritium from [6-3H]glucose, were comparable in all thyroid states. Studies with 10 mmol/L [2-3H]glucose showed that cycling between glucose-6-phosphate and glucose was almost twofold higher in euthyroid and hyperthyroid states as compared with the hypothyroid state, although the magnitude of the increase in cycling rate was only approximately 0.2 mumol glucose.min-1.g-1. When 40 mmol/L [2-3H]glucose was added, over 44% of the glucose that was phosphorylated to glucose-6-phosphate was cycled back to glucose, but this cycling was independent of thyroid status. Cycling between fructose-1,6-bisphosphate and fructose-6-phosphate was negligible in all thyroid states. Rates of glycogen synthesis were comparable in hypothyroid and hyperthyroid states and slightly less than in the euthyroid state. Glycolytically formed pyruvate was cycled back to glucose in hepatocytes from hypothyroid, euthyroid, and hyperthyroid rats. During a 60-minute incubation period, cycling to glucose in the presence of 10 mmol/L or 40 mmol/L glucose was up to twofold higher in cells from euthyroid and hyperthyroid rats than in hepatocytes from hypothyroid animals. The measured increases in cycling rates induced by thyroid hormone were small and in theory could have been satisfied by a much smaller increase in respiration rate than was observed.

Animals↗