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PubMed1988

Involvement of the conserved histidine-188 residue in the L-lactate dehydrogenase from Thermus caldophilus GK24 in allosteric regulation by fructose 1,6-bisphosphate.

The conserved histidine-188 residue of the L-lactate dehydrogenase of Thermus caldophilus GK 24, which is allosterically activated by fructose 1,6-bisphosphate, has been exchanged to phenylalanine by site-specific mutagenesis. In the mutant enzyme the strong stimulatory effect of fructose 1,6-bisphosphate is abolished. The analysis of the pH dependence of the activity indicates that the positive charge of the conserved His-188 residue is important for the interaction of the enzyme with the allosteric effector.

Binding Sites↗
PubMed2001

Neuroprotection and intracellular Ca2+ modulation with fructose-1,6-bisphosphate during in vitro hypoxia-ischemia involves phospholipase C-dependent signaling.

The neuroprotectant fructose-1,6-bisphosphate (FBP) preserves cellular [ATP] and prevents catastrophic increases in [Ca2+]i during hypoxia. Because FBP does not enter neurons or glia, the mechanism of protection is not clear. In this study, we show that FBP's capacity to protect neurons and stabilize [Ca2+]i during hypoxia derives from signaling by a phospholipase-C-intracellular Ca2+-protein kinases pathway, rather than Ca2+ chelation or glutamate receptor inhibition. FBP reduced [Ca2+]i changes in hypoxic hippocampal neurons, regardless of [Ca2+]e, and preserved cellular integrity as measured by trypan blue or propidium iodide exclusion and [ATP]. FBP also prevented hypoxia-induced increases in [Ca2+]i when glucose was absent and when [Ca2+]e was increased to negate Ca2+ chelation by FBP. These protective effects were observed equally in postnatal day 2 (P2) and P16 neurons. Inhibiting glycolysis with iodoacetate eliminated the protective effects of FBP in P16 neurons. FBP did not alter Ca2+ influx stimulated by brief applications of NMDA or glutamate during normoxia or hypoxia, but did reduce the increase in [Ca2+]i produced by 10 min of glutamate exposure during hypoxia. Because FBP increases basal [Ca2+]i and stimulates membrane lipid hydrolysis, we tested whether FBP's protective action was dependent on phospholipase C signaling. The phospholipase C inhibitor U73122 prevented FBP-induced increases in [Ca2+]i and eliminated FBP's ability to stabilize [Ca2+]i and increase survival during anoxia. Similarly, FBP's protection was eliminated in the presence of the mitogen/extracellular signal protein kinase (MEK) inhibitor U0126. We conclude that FBP may produce neuroprotection via activation of neuroprotective signaling pathways that modulate Ca2+ homeostasis.

Adenosine Triphosphate↗
PubMed2002

Activation of the neuroprotective ERK signaling pathway by fructose-1,6-bisphosphate during hypoxia involves intracellular Ca2+ and phospholipase C.

The mechanism of the neuroprotective action of the glycolytic pathway intermediate fructose-1,6-bisphosphate (FBP) may involve activation of a phospholipase-C (PLC) dependent MAP kinase signaling pathway. In this study, we determined whether FBP's capacity to decrease delayed cell death in hippocampal slice cultures is dependent on PLC signaling or activation of the intracellular Ca(2+)-MEK/ERK neuroprotective signaling cascade. FBP (3.5 mM) reduced delayed death from oxygen/glucose deprivation in CA1, CA3 and dentate neurons in slice cultures. The phospholipase-C inhibitor U73122 and the MEK1/2 inhibitor U0126 prevented this protection. In hippocampal and cortical neurons, FBP increased phospho-ERK1/2 (p42/44) immunostaining during hypoxic, but not normoxic conditions. Increased phospho-ERK immunostaining was dependent on PLC and also on MEK 1/2, an upstream regulator of ERK. Further, we found that FBP enhancement of phospho-ERK immunostaining depended on [Ca(2+)](i): PLC inhibition and the IP(3) receptor blocker xestospongin C prevented FBP from increasing [Ca(2+)](i) and increasing phospho-ERK levels. However, while FBP-induced increases in [Ca(2+)](i) were blocked by xestospongin and a PLC inhibitor, [Ca(2+)](i) increases induced by the neuroprotective growth factor BDNF were not prevented. We conclude that during hypoxia FBP initiates a series of neuroprotective signals which include PLC activation, small increases in [Ca(2+)](i), and increased activity of the MEK/ERK signaling pathway.

Animals↗
PubMed2003

Fructose-1,6-bisphosphate preserves intracellular glutathione and protects cortical neurons against oxidative stress.

Fructose-1,6-bisphosphate (FBP), an endogenous intermediate of glycolysis, protects the brain against ischemia-reperfusion injury. The mechanisms of FBP protection after cerebral ischemia are not well understood. The current study was undertaken to determine whether FBP protects primary neurons against hypoxia and oxidative stress by preserving reduced glutathione (GSH). Cultures of pure cortical neurons were subjected to oxygen deprivation, a donor of nitric oxide and superoxide radicals (3-morpholinosydnonimine), an inhibitor of glutathione synthesis (L-buthionine-sulfoximine) or glutathione reductase (1,3-bis(2-chloroethyl)-1-nitrosourea) in the presence or absence of FBP (3.5 mM). Neuronal viability was determined using an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. FBP protected neurons against hypoxia-reoxygenation and oxidative stress under conditions of compromised GSH metabolism. The efficacy of FBP depended on duration of hypoxia and was associated with higher intracellular GSH concentration, an effect partly mediated via increased glutathione reductase activity.

Animals↗
PubMed1996

Fructose-1,6-bisphosphate after hypoxic ischemic injury is protective to the neonatal rat brain.

Fructose-1,6-bisphosphate (FBP) has been shown to attenuate central nervous system injury in adult animals. We evaluated whether FBP given after an ischemic-hypoxic insult is protective to the developing brain in a neonatal rat model of hypoxia-ischemia. Postnatal day 7 rat pups were subjected to focal ischemia followed by global hypoxia and then administered either FBP or saline intraperitoneally. A dose of 500 mg/kg or greater of FBP significantly reduced the amount of injury such that 55% of FBP- vs. 17% of saline-treated rats had no injury; 6% of FBP- and 47% of saline-treated rats had severe damage (P = 0.004). There was less infarcted brain in FBP-treated rats (12 +/- 11% vs. 37 +/- 32%; P = 0.005); and fewer FBP-treated rats had > 30% ipsilateral cortical injury (12% of FBP- vs. 50% of saline-treated rats; P = 0.002). FBP lowered serum calcium levels during the first 24 h after the insult without significant changes in ionized calcium or osmolarity. These results indicate that FBP treatment administered systemically after hypoxia-ischemia reduces CNS injury in neonatal rats.

Animals↗
PubMed1997

Effects of neuroprotective dose of fructose-1,6-bisphosphate on hypoxia-induced expression of c-fos and hsp70 mRNA in neonatal rat cerebrocortical slices.

In situ hybridization (ISH) measurements of c-fos and hsp70 expression were made in brain slice studies of hypoxia, with or without fructose-1,6-bisphosphate (FBP) pretreatment. Each experiment used eighty 350 microns thick cerebrocortical slices, obtained from twenty 7-day old rats. Thirty minute periods of hypoxia were followed by 8 h of hyperoxic perfusion. Slices were removed at eight predetermined times, and processed for ISH and immunohistochemistry. In three of six hypoxia experiments, slices were pretreated for 60 min with 2 mM FBP, a condition known to maintain ATP level in brain slices during hypoxia. In three other hypoxia experiments slices received no pretreatment. In two control experiments slices were perfused for 11.5 h without hypoxia. In control experiments, hsp70 mRNA was barely detectable in slices at all times, although moderate c-fos mRNA expression occurred at 1 h after decapitation. Hypoxia produced a modest but statistically significant increase in c-fos mRNA and hsp70 mRNA induction 4 h following reoxygenation. At all times after hypoxia, FBP pretreatment reduced expression of c-fos and hsp70 mRNA. The absence of hsp70 mRNA in control slices suggests that intracellular protein denaturation was minimal in this preparation. In slices made hypoxic, the decrease in c-fos and hsp70 mRNA caused by FBP pretreatment suggests ameliorated progression towards injury. Immunohistochemistry showed no HSP70 protein at any time following hypoxia, with or without FBP pretreatment, presumably due to delayed HSP70 protein synthesis, or to a block in translation, as observed in vivo in other studies.

Animals↗
PubMed1998

Reversal of ischemic-induced chronic memory dysfunction in aging rats with a free radical scavenger-glycolytic intermediate combination.

Rats were subjected to bilateral carotid artery occlusion (2-VO) or sham occlusion (No-VO) and tested 12 weeks for visuo-spatial memory (VSM) function. After 14 weeks, 2-VO rats (N = 4) showing severe visuo-spatial memory impairment were given dimethyl sulfoxide (DMSO)-fructose 1,6-diphosphate (FDP) i.p. for seven days and retested on the water maze. After DMSO-FDP, a 54% improvement in their VSM was seen which nearly reached control No-VO values. Untreated 2-VO (N = 4) and No-VO (N = 8) rats showed no significant changes in their VSM. DMSO-FDP treatment was discontinued and rats were retested on the water maze but improvement was lost and VSM function regressed to pretreatment levels. Immunohistochemical examination showed minimal neuronal damage in all 2-VO rats and slight loss of microtubule associated protein-2. Glial fibrillary acidic protein immunostaining increase was observed only in untreated 2-VO rats. The results indicate that a DMSO-FDP combination improves VSM secondary to chronic brain hypoperfusion.

Aging↗
PubMed1986

Structures of D-threo-2,5-hexodiulose 1-phosphate and D-threo-2,5-hexodiulose 1,6-bisphosphate (5-keto-D-fructose mono- and bis-phosphate) in solution by 13C-N.M.R. spectroscopy.

The mono- (2) and bis-phosphate (3) derivatives of D-threo-2,5-hexodiulose (1) (5-keto-D-fructose) were synthesized enzymically and purified by anion-exchange chromatography. The proportions, sizes of ring, and anomeric configurations were determined by F.t. 31P- and 13C-n.m.r. spectroscopy. Compound 2 was found to exist preponderantly (70-78%) in the beta-pyranose form with the remainder existing in the 2R,5R-furanose form. Compound 3 assumes two different furanose forms in solution, one (77-84%) being the 2R,5R-furanose form and the other the 2S,5R-furanose form.

Carbohydrate Conformation↗
PubMed2002

Chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone from D-fructose 1,6-diphosphate.

The selective chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDF) from D-fructose 1,6-diphosphate in the presence of reduced nicotinamide-adenine-dinucleotides (NAD(P)H) was investigated by means of HPLC-DAD and HPLC-UV-MS/MS. The temperature optimum for HDF formation was 30 degrees C, whereas the pH value (pH 3-10) and chemical nature of the buffer had no significant influence. A linear correlation of reaction time and D-fructose 1,6-diphosphate concentration with the obtained HDF yield was observed. Proteins appeared to have a stabilizing effect. The NAD(P)H were mandatory, even in the presence of protein, implying a non-enzymatic hydride-transfer to an unknown intermediate which finally leads to the selective formation of HDF. The hydride-transfer was confirmed by the application of selectively pro-4R or pro-4S deuterium labeled NADH resulting in each case in the formation of HDF exhibiting a deuterium labeling of approx 30% and employment of [4R,S-(2)H(2)]-NADH led to a deuterium labeling of approx 66%. The incubation of [1-(13)C]-D-fructose 1,6-diphosphate with [4R,S-(2)H(2)]-NADH revealed that the hydride is transferred to C-5 or C-6 of the D-fructose 1,6-diphosphate skeleton. Thus, a chemical HDF formation from D-fructose 1,6-diphosphate under physiological reaction conditions was shown and for the first time to our knowledge a non-enzymatic hydride-transfer from NADH to a carbohydrate structure was demonstrated.

Deuterium↗
PubMed1984

Increase of intraerythrocytic fructose-1,6-diphosphate after incubation of whole human blood with fructose-1,6-diphosphate.

The incubation of whole blood with fructose-1,6-diphosphate (FDP) entails a statistically significant increase of intraerythrocytic FDP together with a decrease of blood glucose. The increase is not significant when equimolar amounts of fructose plus twice molar phosphate are used. The effect of FDP is decreased in the presence of an excess of oxygen. FDP added to the whole blood is removed from plasma by the activity of plasma enzymes and by the presence of blood cells as well. No specific interaction of FDP with plasma proteins seems to occur and the effects of FDP addition last longer than is compatible with the presence of FDP in the plasma.

Adolescent↗
PubMed1986

Bisphosphorylated metabolites of glycerate, glucose, and fructose: functions, metabolism and molecular pathology.

2,3-Bisphosphoglycerate, glucose 1,6-P2 and fructose 2,6-P2 have been recognized as regulatory signals implicated in the control of metabolism, oxygen affinity of red cells and other cellular functions. The alterations of their metabolism constitute a novel area in molecular pathology. The concentration of 2,3-bisphosphoglycerate in erythrocytes changes in a number of pathological conditions. An inherited deficiency of the multifunctional enzyme involved in the synthesis and breakdown of 2,3-bisphosphoglycerate in erythrocytes has been reported. The levels of glucose 1,6-P2 are reduced in the liver and in the muscle of rats with experimentally induced diabetes. In muscle of genetically dystrophic mice a decrease in the levels of glucose 1,6-P2 has been found, probably resulting from enhancement of glucose 1,6-P2 phosphatase activity. Fructose 2,6-P2 levels are decreased in the liver of experimental diabetic mice and rats, and elevated in the liver of genetically obese animals.

Diphosphoglyceric Acids↗
PubMed2002

Clotrimazole decreases glycolysis and the viability of lung carcinoma and colon adenocarcinoma cells.

Glycolysis is known to be the primary energy source in most cancer cells. We investigated here the effect of clotrimazole (1-(alpha-2-chlorotrityl)imidazole), the antifungal azole derivative, which was recently recognized as calmodulin antagonist, on the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis, and on ATP content and cell viability in LL/2 Lewis lung carcinoma cells and CT-26 colon adenocarcinoma cells. We found that clotrimazole induced a significant, dose- and time-dependent reduction in the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate, ATP, and cell viability. These findings suggest that clotrimazole causes a reduction in glycolysis and ATP levels, which eventually leads to cell destruction after 3 h of treatment. Since cell proliferation was also reported to be inhibited by calmodulin antagonists, this substance is most promising agent in treatment of cancer by inhibiting both cell proliferation and the glycolytic supply of ATP required for cancer cell growth.

Adenocarcinoma↗
PubMed1999

Taxol (paclitaxel) induces a detachment of phosphofructokinase from cytoskeleton of melanoma cells and decreases the levels of glucose 1,6-bisphosphate, fructose 1,6-bisphosphate and ATP.

Glucose utilization through glycolysis, which is the primary energy source in cancer cells, is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Here we report of a novel mechanism of action of taxol (paclitaxel; Baccatin III N-benzyl-beta-phenylisoserine ester), the anti-microtubule agent with remarkable anticancer activity. We show that taxol affects both levels of regulation of glycolysis in melanoma cells; it decreases the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two allosteric stimulatory signal molecules of glycolysis, and also causes a detachment of phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11), the rate-limiting enzyme of glycolysis, from the cytoskeleton of B16 melanoma cells. These effects of taxol were dose-dependent, and preceded the decrease in ATP levels and cell viability. Thus, taxol not only inhibits the essential dynamic processes of microtubule network, but also reduces glycolysis, through the novel mechanisms described here.

Adenosine Triphosphate↗
PubMed1986

Purification of the bovine lens isozymes which reduce fructose diphosphate to sorbitol diphosphate.

Three isozymes of an enzyme which reduce fructose 1, 6-diphosphate (FDP) to sorbitol 1, 6-diphosphate (SDP) in the presence of NADH have been purified from bovine lens. The isozymes were fractionated by acid precipitation of the lens homogenate followed by DE-52 column chromatography. This step separated the FDP reducing activity into three major peaks, peak 1, peak 2, and peak 3. Each of these peaks were further purified by affinity chromatography using Reactive blue-2-agarose, Sephadex G-150 gel filtration, and DE-52 column chromatography. Polyacrylamide disc gel electrophoresis demonstrated the presence of one major isozyme and one minor isozyme in each of the three peaks. The Km values for FDP were 8.0, 5.7, and 4.7 mM for peaks 1, 2, and 3 respectively. The reaction product SDP was characterized by nuclear magnetic resonance spectroscopy. All the isozymes utilized pyruvate as substrate with the Km for peaks 1, 2, and 3 being 0.63, 0.20, and 0.09 mM respectively. These studies therefore indicate that FDP reducing activity and lactate dehydrogenase activity co-purify and may be expressed by the same enzyme protein.

Animals↗
PubMed1987

Evidence for gluconeogenesis in the amphibian retina.

Evidence for the existence of a gluconeogenic pathway was provided in the amphibian retina. It was found that [3H]glutamate was converted to [3H]glucose derived from [3H]glutamate was incorporated into glycogen. The rate for this incorporation was found to be essentially the same in both light- and dark-adapted retinas: 0.147 vs. 0.142 nmol (mg protein X 2 hr)-1, respectively. However, the rate of incorporation was found to decline progressively with time. The rate for the incorporation of label derived from glutamate into glycogen was found to be considerably less than that for [3H]glucose: 10.2 nmol (mg protein X 2 hr)-1. The activity of a key gluconeogenic enzyme, fructose-1,6-bisphosphatase, also was demonstrated in retinal supernatants, approximately 1 nmol (mg X min)-1, and the activity of this enzyme was found to be inhibited both by adenosine monophosphate and by fructose-2,6-bisphosphate.

Adaptation, Ocular↗
PubMed2000

Cells overexpressing fructose-2,6-bisphosphatase showed enhanced pentose phosphate pathway flux and resistance to oxidative stress.

Changes in the content of fructose-2,6-bisphosphate, a modulator of glycolytic flux, also affect other metabolic fluxes such as the non-oxidative pentose phosphate pathway. Since this is the main source of precursors for biosynthesis in proliferating cells, PFK-2/FBPase-2 has been proposed as a potential target for neoplastic treatments. Here we provide evidence that cells with a low content of fructose-2,6-bisphosphate have a lower energy status than controls, but they are also less sensitive to oxidative stress. This feature is related to the activation of the oxidative branch of the pentose phosphate pathway and the increased production of NADPH.

Animals↗
PubMed2002

The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis.

The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P(2)), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P(2)). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P(2) present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P(2) appeared to be entirely abrogated in the mutated enzymes confirming our predictions.

Allosteric Regulation↗
PubMed1997

Analysis of phosphofructokinase subunits and isozymes in ascites tumor cells and its original tissue, murine mammary gland.

Phosphofructokinase (PFK) subunits and isozymes have been examined in ascites tumor cells and murine mammary gland, the tissue from where this tumor originated. Ascites tumor was found to contain predominantly the C-type subunit, whereas the L-type subunit was more abundant in mammary gland. An altered M-type subunit of lower electrophoretic mobility was found in both cell types and no M4 homotetramers were detected in either of them. Characteristic regulatory properties of ascites tumor PFK, i.e. insensitivity to fructose-1,6-P2 activation and inhibition by P-enolpyruvate, were also observed in the mammary gland isozyme. The nature of these properties and the contribution of the distinct subunit types to fructose-1,6-P2 activation are discussed.

Animals↗