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PubMed1990

The role of fructose 2,6-bisphosphate in glycolytic oscillations in extracts and cells of Saccharomyces cerevisiae.

Fructose 2,6-bisphosphate is physiologically one of the most potent activators of yeast 6-phosphofructo-1-kinase. The glycolytic oscillation observed in cell-free cytoplasmic extracts of the yeast Saccharomyces cerevisiae responds to the addition of fructose 2,6-bisphosphate in micromolar concentrations by showing a pronounced decrease of both the amplitude and the period. The oscillations can be suppressed completely by 10 microM and above of this activator but recovers almost fully (95%) to the unperturbed state after 3 h. Fructose 2,6-bisphosphate shifts the phases of the oscillations by a maximal +/- 60 degrees. Oscillations in concentration of endogenous fructose 2,6-bisphosphate in the extract were also observed. Fructose 2,6-bisphosphate alters the dynamic properties of 6-phosphofructo-1-kinase which are vital for its role as the 'oscillophore'. However, the minute amount (approximately 0.3 microM) of endogenous fructose 2,6-bisphosphate and the phase relationship of its oscillations compared with other metabolites indicate that this activator is not an essential component of the oscillatory mechanism. Further support for this conclusion is the observation of sustained oscillations in both the extracts and a population of intact cells of a mutant strain (YFA) of S. cerevisiae with no detectable fructose 2,6-bisphosphate (less than 5 nM).

Adenosine Monophosphate↗
PubMed1991

Irreversible transitions in the 6-phosphofructokinase/fructose 1,6-bisphosphatase cycle.

The dynamics of the fructose 6-phosphate fructose-1,6-bisphosphate cycle operating in an open and homogeneous system reconstituted from purified enzymes was extensively studied. In addition to 6-phosphofructokinase and fructose-1,6-bisphosphatase, pyruvate kinase, adenylate kinae and glucose-6-phosphate isomerase were involved. In that multi-enzyme system, the main source of non-linearity is the reciprocal effect of AMP on the activities of 6-phosphofructokinase and fructose-1,6-bisphosphatase. Depending upon the experimental parameter values, stable attractors, various types of multiple states and sustained oscillations were shown to occur. In the present report we show that irreversible transitions are also likely to occur for realistic operating conditions. Two parameters of the system, that is the adenylate energy charge of the influx and the fructose-1,6-bisphosphatase maximal activity, are potential candidates to provoke such irreversible transitions from one steady state to the other: (a) when varying the maximal activity of fructose-1,6-bisphosphatase, the system can jump irreversibly from a low to a high stable steady state, and (b) when the adenylate energy charge of the influx is the changing parameter, irreversible transitions occur from a high stable steady state to a stable oscillatory state (limit cycle motion). This behavior can be predicted by constructing the loci of limit points and Hopf bifurcation points.

Fructose-Bisphosphatase↗
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↗
PubMed1992

Unusual amino acid substitution in the anion-binding site of Lactobacillus plantarum non-allosteric L-lactate dehydrogenase.

In Lactobacillus plantarum non-allosteric L-lactate dehydrogenase (L-LDH), the highly conserved His188 residue, which is involved in the binding of an allosteric effector, fructose 1,6-bisphosphate [Fru(1,6)P2], in allosteric L-LDH is uniquely substituted by an Asp. The mutant L. plantarum L-LDH, in which Asp188 is replaced by a His, showed essentially the same Fru(1,6)P2-independent catalytic activity as the wild-type enzyme, except that the Km and Vmax values were slightly decreased. However, the addition of Fru(1,6)P2 induced significant thermostabilization of the mutant enzyme, as in the case of many allosteric L-LDHs, while Fru(1,6)P2 showed no significant effect on the stability of the wild-type enzyme, indicating that only the single-point mutation, G-->C, sufficiently induces the Fru(1,6)P2-binding ability of L. plantarum L-LDH. The mutant enzyme showed higher thermostability than the wild-type enzyme in the presence of Fru(1,6)P2. In the absence of Fru(1,6)P2, on the other hand, the mutant enzyme was more labile below 65 degrees C but more stable above 70 degrees C.

Allosteric Regulation↗
PubMed1993

Limited proteolysis of yeast phosphofructokinase. Sequence locations of cleavage sites created by the actions of different proteinases.

Purified phosphofructokinase 1 from baker's yeast (Saccharomyces cerevisiae) was subjected to proteolysis by thermolysin, endoproteinase lys-C, trypsin and chymotrypsin under defined solvent conditions. In the absence of substrates and allosteric effectors, the catalytic activity of phosphofructokinase rapidly disappeared in the presence of each proteolytic enzyme. The presence of a saturating concentration of ATP protected phosphofructokinase activity from proteolytic inactivation while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate provided transient activation during proteolysis. Changes in the quaternary structure of phosphofructokinase resulting from proteolysis were estimated by high performance size exclusion chromatography while changes in the primary sequence of the individual alpha and beta polypeptide chains were estimated by polyacrylamide-gel electrophoresis in sodium dodecylsulfate. The site(s) of proteolytic cleavage were identified by N-terminal sequence analysis of resolved electrophoretic components. The presence of ATP protects phosphofructokinase from thermolysin proteolysis, while the collective presence of fructose 6-phosphate, AMP and fructose 2,6-bisphosphate restricts proteolysis to one site in each polypeptide chain involving the peptide bonds preceding Leu199 in the alpha chain and Leu192 in the beta chain. The truncated phosphofructokinase retains its octameric structure. The presence of ATP largely restricts endoproteinase lys-C proteolysis to a single site in the alpha chain involving the peptide bond preceding Val914. This cleavage results in the dissociation of the octameric form of phosphofructokinase into two tetramers. The presence of ATP restricts both trypsin and chymotrypsin proteolysis to the N-terminal and C-terminal regions described above, resulting in the preferential stabilization of the tetrameric form of phosphofructokinase. It would appear that the first 200 and last 80 residues which are unique to the sequence of the yeast phosphofructokinase are not directly involved in catalysis or its allosteric regulation. However, the last 80 residues of the alpha polypeptide chain do appear to stabilize an octameric structure which is unique to yeast phosphofructokinase.

Adenosine Monophosphate↗
PubMed1994

The capacity of reducing-equivalent shuttles limits glycolysis during ethanol oxidation.

The inhibition of glycolysis during ethanol oxidation has been examined in isolated hepatocytes from fasted rats. Glycolytic flux was measured by determining the rate of release of tritium from [6-3H]glucose. During ethanol oxidation, the rate of glycolysis was inhibited 80% in freshly prepared hepatocytes, in which shuttle intermediates are depleted, but was depressed only about 20% in the presence of asparagine, a condition under which activity of the malate/aspartate shuttle was restored to normal levels. The inhibition of glycolysis was also partially released by addition of pyruvate and when alcohol dehydrogenase activity was depressed by 4-methylpyrazole. Titrations with this inhibitor revealed inverse linear relationships between the rates of glycolysis and ethanol oxidation. For any given rate of ethanol oxidation, glycolytic flux was lowest and the [lactate]/[pyruvate] ratio highest in the presence of aminooxyacetate, an inhibitor of the malate/aspartate shuttle, whereas flux was highest and the ratio lowest in the presence of asparagine. During these titrations with 4-methylpyrazole the inhibition of ethanol oxidation and concomitant restoration of glycolysis were accompanied by a decline in the [lactate]/[pyruvate] ratio, a substantial fall in the rate of reducing-equivalent transfer from cytoplasm to mitochondria and an increase in lactate accumulation. These findings imply that the reducing equivalents generated during ethanol oxidation compete with those arising in glycolysis for transfer to the mitochondria. This competition leads to an inhibition of aerobic glycolysis, and at the same time contributes to a rise in cytoplasmic NADH and fall in NAD+ that results in depression of anaerobic glycolysis. Allosteric inhibition of 6-phosphofructo-1-kinase due to a decrease in the concentration of fructose 2,6-bisphosphate did not appear to play a primary role in the inhibition of glycolysis by ethanol. Ethanol oxidation had no effect on glucose phosphorylation as measured with [2-3H]glucose, but induced a substantial increase in cycling between glucose and glucose 6-phosphate.

Animals↗
PubMed1994

Enhancement of the reductive activation of chloroplast fructose-1,6-bisphosphatase by modulators and protein perturbants.

To characterize the mechanism of chloroplast fructose-1,6-bisphosphatase activation, we have examined kinetic and structural changes elicited by protein perturbants and reductants. At variance with its well-known capacity for enzyme inactivation, 150 mM sodium trichloroacetate yielded an activatable chloroplast fructose-1,6-bisphosphatase in the presence of 1.0 mM fructose 1,6-bisphosphate and 0.1 mM Ca2+. Other sugar bisphosphates did not replace fructose 1,6-bisphosphate whereas Mg2+ and Mn2+ were functional in place of Ca2+. Variations of the emission fluorescence of intrinsic fluorophores and a noncovalently bound extrinsic probe [2-(p-toluidinyl)naphthalene-6-sulfonate] indicated the presence of conformations different from the native form. A similar conclusion was drawn from the analysis of absorption spectra by means of fourth-derivative spectrophotometry. The effect of these conformational changes on the reductive process was studied by subsequently incubating the enzyme with dithiothreitol. The reaction of chloroplast fructose-1,6-bisphosphatase with dithiothreitol was accelerated 13-fold by the chaotropic anion: second-order rate constants were 48.1 M-1.min-1 and 3.7 M-1.min-1 in the presence and in the absence of trichloroacetate, respectively. Thus, the enhancement of the reductive activation by compounds devoid of redox activity illustrated that the modification of intramolecular noncovalent interactions of chloroplast fructose-1,6-bisphosphatase plays an essential role in the conversion of enzyme disulfide bonds to sulfhydryl groups. In consequence, a conformational change would operate concertedly with the reduction of disulfide bridges in the light-dependent activation mediated by the ferredoxin-thioredoxin system.

Cations, Divalent↗
PubMed1994

Glucokinase expression in rat hepatoma cells induces glucose uptake and is rate limiting in glucose utilization.

In contrast to hepatocytes, hepatoma cells lack glucokinase activity and show increased aerobic glycolysis. FTO-2B and H4IIE rat hepatoma cell lines were obtained in which the rat glucokinase gene was expressed (FTOGK and H4GK). These lines were generated by infection of the hepatoma cells with a retroviral vector carrying the phosphoenolpyruvate carboxykinase (PEPCK)-glucokinase chimeric gene. Both the FTOGK and H4GK cells expressed the chimeric gene in a regulated manner, like the endogenous PEPCK gene. Glucokinase activity was detected in both FTOGK and H4GK. These cells lines showed a marked increase in glucose uptake with 18.5 mM glucose in the incubation medium. FTOGK and H4GK showed an increase in the content of glucose 6-phosphate, and were able to accumulate high levels of glycogen, in contrast to FTO-2B cells, which were unable to store the polysaccharide. In addition, cells expressing glucokinase showed high concentration of fructose 2,6-bisphosphate and substantial lactate production, which was related to the glucose concentration in the medium and the time of incubation. These results suggest that glucose phosphorylation is rate limiting for glucose uptake and utilization in FTO-2B and H4IIE cells.

Animals↗
PubMed1995

Involvement of phosphorylase kinase inhibition in the effect of resorcinol and proglycosyn on glycogen metabolism in the liver.

The purpose of this study was to identify the mechanism by which proglycosyn and resorcinol decrease the phosphorylase a content and the fructose 2,6-bisphosphate concentration in isolated hepatocytes. The intracellular concentrations of the glucuronide derivatives of proglycosyn and resorcinol have been measured by HPLC in hepatocytes incubated for 5 min or 30 min with different concentrations of these agents. At both times, there was a reciprocal relationship between the phosphorylase a content and the intracellular concentration of the glucuronidated metabolites, half-maximal inactivation being observed at about 2 mumol/g protein and 0.25 mumol/g protein for resorcinylglucuronide and proglycosyn-glucuronide, respectively. Glycogen synthase was not significantly activated by these agents after 5 min but was well activated after 30 min. Preincubation of hepatocytes with 1 mM resorcinol or with 100 microM proglycosyn resulted in a decrease in the rate at which phosphorylase was activated following the addition of glucagon, vasopressin, the protein phosphatase inhibitor calyculin A or the calcium ionophore A 23187, but did not reduce the rate of synthase inactivation. Proglycosynglucuronide and resorcinylglucuronide inhibited phosphorylase kinase in liver Sephadex filtrates, with Ki values of about 0.75 mM and 4 mM, respectively. Preincubation of the filtrates with ATP and cAMP decreased the sensitivity of phosphorylase kinase to resorcinylglucuronide by about fourfold. It is concluded that the effect of resorcinol and proglycosyn on the phosphorylase a content is due, at least partly, to an inhibition of phosphorylase kinase by their glucuronidated metabolites. Resorcinol and proglycosyn caused a parallel decrease in the concentration of fructose 2,6-bisphosphate and of hexose 6-phosphates, without significantly changing the activity of 6-phosphofructo-2-kinase. The decrease in the fructose 2,6-bisphosphate concentration appears therefore to be secondary to the decrease in the hexose 6-phosphate concentration.

Animals↗
PubMedDate not supplied

Inhibition of glyceraldehyde 3-phosphate dehydrogenase by adenine nucleotides in pachytene primary spermatocytes from rat testes.

The effect of adenine nucleotides on the catalytic activity of spermatocyte glyceraldehyde 3-phosphate dehydrogenase (GA3PDH) was studied for its significance to metabolic regulation. In the presence of glucose (10 mM), the ATP level declined whereas the ADP and AMP levels remained unchanged. During incubation with glucose, fructose 1,6-bisphosphate (fruc 1,6-P2) and dihydroxyacetone phosphate (DHAP) were accumulated markedly. GA3PDH was inhibited by ATP (Ki = 2.27 mM), ADP (Ki = 1.21 mM) and AMP (Ki = 0.73 mM) competitively with NAD (Km = 0.24 mM). The results suggest that glycolysis in spermatocytes is regulated by GA3PDH.

Adenine Nucleotides↗
PubMedDate not supplied

Metabolism of round spermatids: kinetic properties of pyruvate kinase.

Round spermatids (steps 1-8) were isolated from rat testes and kinetic properties of pyruvate (PK) in their extract were examined. A plot of PK activity against phosphoenolpyruvate (PEP) or ADP concentration appeared sigmoidal. Km values for PEP and ADP were 0.12 and 0.29 mM, respectively. However, fructose 1,6-bisphosphate (FBP) stimulated the enzyme markedly by increasing its affinity for PEP. FBP (0.35 microM) was required for 50% activation of PK, when the PK activity was measured at 25 microM PEP and 0.2 mM ADP. In contrast, ATP (Ki = 6.5 mM) inhibited the PK activity. On the other hand, in the presence of 5 mM glucose, the level of FBP in spermatids increased markedly, while that of ATP declined rapidly. The level of ADP remained constant. When the activity of PK in spermatid extract was measured at intracellular levels of FBP, ADP and ATP, it was maximum. The results suggest that PK becomes probably fully activated when glucose is metabolized in the glycolytic pathway of spermatids. It seems unlikely that PK is the rate-limiting step in glycolysis of spermatids.

Adenosine Diphosphate↗
PubMedDate not supplied

Metabolism of round spermatids in the rat: effect of gossypol on the glucose transport.

Low dose of gossypol (5 microM) completely inhibited uptake of 2-deoxy-D-glucose by rat spermatids. With gossypol 2-deoxy-D-glucose transport into spermatids increased curvelinearly for 10 minutes after the start of incubation, but reached a plateau afterwards, whereas without gossypol it continuously increased. The levels of fructose-1,6-bis-phosphate (FBP) and ATP were inversely correlated when the cells were exposed to glucose. However, in the presence of gossypol the levels of ATP and FBP decreased simultaneously. In addition, gossypol at 10 microM stimulated oxygen consumption when lactate was present. These results suggest that gossypol probably inhibits glucose transport into spermatids by lowering the ATP level by uncoupling respiratory chain-linked phosphorylation.

Adenosine Triphosphate↗
PubMed1981

Regionally selective metabolic effects of hypoglycemia in brain.

Regional CNS levels of glucose reserves, glycolytic intermediates, and high-energy phosphate reserves were measured in insulin-treated, hypoglycemic rats and correlated with EEG activity. Intravenous administration of insulin to paralyzed, ventilated animals causes concomitant reduction of blood glucose levels and progressive abnormality and eventual loss of EEG activity. In all regions of brain examined, glucose and glycogen levels decrease until they are essentially depleted, and glucose-6-phosphate and fructose-1,6-biphosphate fall approximately 80%. Pyruvate levels decrease 50% in cerebral cortex and brain stem and a lesser amount in striatum, hippocampus, thalamus, and cerebellum. Lactate levels fall 50-60% in all regions except cerebellum, where no change is observed. ATP and phosphocreatine levels remain normal until the EEG is isoelectric, and then decrease in all regions except cerebellum. These results demonstrate that hypoglycemia does not have a uniform effect on brain glucose and energy metabolism, and cerebellum seems to be relatively protected.

Adenosine Triphosphate↗
PubMed1990

Fructose 2,6-bisphosphate changes in rat brain during ischemia.

Brain ischemia was produced by bilateral ligation of the common carotid arteries of spontaneously hypertensive rats. The concentrations of fructose 2,6-bisphosphate and other glycolytic intermediates as well as of pyridine and adenine nucleotides were measured in frozen brain samples. In contrast to the decrease reported in hepatocytes under anoxic conditions, the fructose 2,6-bisphosphate content was increased by 20-30% during the early stages of ischemia. Elevation in fructose 1,6-bisphosphate level and lactate formation followed the rise in fructose 2,6-bisphosphate content, a finding suggesting that this compound plays a key role in the compensatory acceleration of glycolysis under ischemic conditions in vivo.

Animals↗
PubMed1991

Fructose 2,6-bisphosphate in hypoglycemic rat brain.

Fructose 2,6-bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6-bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6-bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6-bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6-phosphofructo-2-kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6-phosphate). These results suggest that fructose 2,6-bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6-phosphofructo-1-kinase and glycolysis.

Animals↗
PubMed1995

Glucose metabolism and internal pH of Lactococcus lactis subsp. lactis cells utilizing NMR spectroscopy.

The metabolism of glucose was studied in Lactococcus lactis subsp. CNRZ 125 by 13C NMR. The initial rate of glucose utilization was higher for exponential phase cells than for stationary phase cells [150 vs 85 nmol g (dry wt)-1 s -1]. 31P NMR was used to determine changes in glycolytic phosphorylated intermediates (fructose-1,6-diphosphate, dihydroxyacetone phosphate and phosphoglycerate). The internal pHs of L. lactis subsp. lactis CNRZ 141 and CNRZ 125 were also measured by 31P NMR as a function of the external pH during growth. When the external pH was 6.8, the internal pHs of strain CNRZ 141 and CNRZ 125 were similar, 7.4. After the external pH had decreased to 5.5, the internal pH of strain CNRZ 141 had declined by 0.6 unit, whereas that of strain CNRZ 125 had decreased by only 0.2 unit of pH.

Dihydroxyacetone Phosphate↗
PubMed1996

Lactate and acetate production in Listeria innocua.

Listeria innocua NCTC 11289 was grown aerobically in continuous culture in defined media at 30 degrees C. Both acetate and lactate were produced, the proportion of acetate decreased with increasing dilution rate. Enzymatic analysis showed lactate dehydrogenase was activated 10-fold by fructose-1, 6-bisphosphate. The presence of phosphate acetyltransferase and acetate kinase but not pyruvate oxidase was detected, suggesting the sequential action of phosphate acetyltransferase and acetate kinase to produce acetate from acetyl CoA via acetylphosphate.

Acetate Kinase↗
PubMed1994

Effects of metabolic blockers on Ca(2+)-dependent currents in cultured sensory neurones from neonatal rats.

1. The whole cell variant of the patch clamp technique was used to record high voltage-activated Ca2+ currents and Ca(2+)-activated Cl- tail currents from cultured neonatal rat dorsal root ganglion neurones. The aim of the project was to use these currents as physiological indices of intracellular Ca2+ regulation under control conditions and in the presence of metabolic inhibitors. 2. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (5 microM) and sodium cyanide (1 microM) inhibited Ca2+ currents within 20 s, even when ATP was present in the patch pipette solution, suggesting a direct action on Ca2+ channels. These metabolic inhibitors did not affect Ca2+ current 'run down' or inactivation kinetics. 3. Cultured neonatal dorsal root ganglion neurones of the rat were relatively insensitive to the removal of glucose and ATP from the recording solutions for up to 3 h. These data suggest that the Ca2+ homeostatic mechanisms in these cells are highly resistant to metabolic insult. 4. However 2-deoxy-D-glucose (5 mM) in the extracellular recording medium with no ATP or glucose present did prolong the deactivation time of Ca(2+)-activated Cl- tail currents and increase the total charge flow following activation of a 500 ms voltage-activated Ca2+ current. This effect was prevented by inclusion of D-fructose 1,6-diphosphate (500 microM) in the patch pipette solution. 5. We conclude that some agents used to induce chemical hypoxia, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone and sodium cyanide, may interact directly with voltage-activated Ca2+ channels and are therefore not appropriate for use in studying disturbed neuronal Ca2+ homeostasis. However, the use of 2-deoxy-D-glucose in the absence of glucose and ATP does represent a model of disturbed Ca2+ homeostasis in cultured dorsal root ganglion neurones. In this study we have combined the whole cell recording technique with cultured neurones under conditions which produce a degree of metabolic stress as reflected by prolonged Ca(2+)-activated Cl- tail currents. The reduced efficiency of handling of intracellular Ca2+ loads may be an important factor contributing to the onset of neuronal damage during hypoxia and ischaemia.

Adenosine Triphosphate↗