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PubMed1982

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase.

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.

Animals↗
PubMed1983

Studies on the hysteretic properties of chloroplast fructose-1,6-bisphosphatase.

Chloroplast fructose-1,6-bisphosphatase hysteresis in response to modifiers was uncovered by carrying out the enzyme assays in two consecutive steps. The activity of chloroplast fructose-1,6-bisphosphatase, assayed at low concentrations of both fructose-1,6-bisphosphatase and Mg2+, was enhanced by preincubating the enzyme with dithiothreitol, thioredoxin f, fructose 1,6-bisphosphate, and Ca2+. In the time-dependent activation process, fructose 1,6-bisphosphate and Ca2+ could be replaced by other sugar biphosphates and Mn2+, respectively. Once activated, chloroplast fructose-1,6-bisphosphatase hydrolyzed fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate in the presence of Mg2+, Mn2+, or Fe2+. The A0.5 for fructose 1,6-bisphosphate (activator) was lowered by reduced thioredoxin f and remained unchanged when Mg2+ was varied during the assay of activity. On the contrary, the S0.5 for fructose 1,6-bisphosphate (substrate) was unaffected by reduced thioredoxin f and depended on the concentration of Mg2+. Ca2+ played a dual role on the activity of chloroplast fructose-1,6-bisphosphatase; it was a component of the concerted activation and an inhibitor in the catalytic step. Provided dithiothreitol was present, the activating effectors were not required to maintain the enzyme in the active form. Considered together these results strongly suggest that the regulation of fructose-1,6-bisphosphatase in chloroplast occurs at two different levels, the activation of the enzyme and the catalysis.

Calcium↗
PubMed1983

The action of insulin on hepatic fructose 2,6-bisphosphate metabolism.

The inhibition of hepatocyte 6-phosphofructo-1-kinase by glucagon was suppressed by insulin when the enzyme was measured in crude extracts. However, no effect of either hormone was observed after the removal of allosteric effectors from the enzyme, suggesting that the alterations in activity may be due to changes in the level of fructose 2,6-bisphosphate, a potent allosteric activator of the enzyme. Insulin opposed the action of both glucagon and exogenous cyclic AMP to lower fructose 2,6-bisphosphate levels. The concentration of glucagon and of cyclic AMP that gave a half-maximal decrease in fructose 2,6-bisphosphate levels was increased in the presence of 10 nM insulin from 0.03 to 0.09 nM and from 12 to 36 microM, respectively. Insulin also counteracted the effect of maximal concentrations of epinephrine on fructose 2,6-bisphosphate levels. In the presence of 0.02 nM glucagon or 10 microM epinephrine, 10 nM insulin enhanced 6-phosphofructo-2-kinase and decreased fructose 2,6-bisphosphatase activity in (NH4)2SO4-treated hepatocyte extracts. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was shown to be a substrate for the cAMP-dependent protein kinase but not for phosphorylase kinase. It was concluded that insulin opposed the action of glucagon and epinephrine by affecting the phosphorylation state of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Fructose 2,6-bisphosphate levels were decreased in liver cells from diabetic rats. Addition of 30 mM glucose elevated fructose 2,6-bisphosphate levels in cells from fed and 24-h-starved rats but not in cells from diabetic rats. This was probably due to decreases in both 6-phosphofructo-2-kinase and glucokinase activity in the diabetic state. These results show that insulin has both short and long term effects on fructose 2,6-bisphosphate metabolism in liver.

Animals↗
PubMed1982

[Activity of gluconeogenetic enzymes in the kidney of pigs during pre- and post-natal development].

In pig fetuses (19 of 8 dams) developed by Caesarean section the dry matter and protein content of the kidneys and their PEPCK activity remain constant during the last third (from 80th to 112th day) of gestation. After birth the dry matter content of the kidneys rises slowly, but their protein content remarkably. In the kidneys of suckling piglets (17 animals of 3 offsprings) the FDPase activity remains at the same level from birth to the 9th day of life, while in the same time the G6Pase activity rises 1.5-2 times. In the kidneys of newborn piglets the total PEPCK activity increases 3-4 times and the activity of the cytosolic enzyme 2-3 times during the first 12 hours of life. At the end of the first week of life the total PEPCK activity decreases by one-third, while the activity of the cytosolic enzyme remains stable. In the kidneys of slaughter pigs (n = 7) the dry matter content and the FDPase activity are significantly higher, the protein content and the G6Pase activity are the same as in the kidneys of piglets. The total PEPCK activity is one-half, the activity of the cytosolic enzyme one-third lower than in the kidneys of piglets. In the kidneys of adult pigs the PEPCK activity is localized to equal parts in the cytosol and in the mitochondria, but in some development stages the mitochondrial part exceeds that of the cytosol. In adult pigs the PEPCK activity of the renal cortex is 2.5-3 times higher than that of the renal medulla.

Animals↗
PubMed1983

Fructose 2,6-bisphosphate and fructose-6-P 2-kinase in Saccharomyces cerevisiae in relation to metabolic state in wild type and fructose-6-P 1-kinase mutant strains.

In wild type Saccharomyces cerevisiae, fructose-6-P is known to be in much lower amounts than needed to saturate fructose-6-P 1-kinase in vitro, and the same is true for a mutant with reduced affinity for fructose-6-P, even though its in vivo fructose-6-P concentration is much higher than normal. Both the wild type and mutant fructose-6-P 1-kinases were activated in vitro by fructose-2,6-P2 in the 0.1 microM concentration range, and the effector was present in more than adequate amounts. Hence, it is likely to be necessary for sufficient flux through the fructose-6-P 1-kinase reaction in vivo, and the data also fit with fructose-2,6-P2 acting at different sites on the enzyme from fructose-6-P. In growth on glucose, a variety of wild type strains contained 5-10 microM fructose-2,6-P2, and various fructose-6-P 1-kinase mutant strains had levels of up to 150 microM in the presence of glucose. Fructose-2,6-P2 was also found (0.5-10 microM) in derepressed cultures after glucose exhaustion and in growth on pyruvate. Activities of fructose-6-P 2-kinase in the various strains and situations are also presented. The data generally indicate a correlation between levels of fructose-2,6-P2 and fructose-6-P and suggest that fructose-2,6-P2 is not rapidly degraded after glucose exhaustion.

Enzyme Activation↗
PubMed1984

Evidence for two catalytic sites on 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Dynamics of substrate exchange and phosphoryl enzyme formation.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase appears to be the only enzyme catalyzing the formation and hydrolysis of Fru-2,6-P2. The enzyme as we isolate it, contains a trace of tightly bound Fru-6-P. In this condition, it exhibited an ATPase activity comparable to its kinase activity. Inorganic phosphate stimulated all of its activities, by increasing the affinity for all substrates and increasing the Vmax of ATP and Fru-2,6-P2 hydrolysis. The enzyme catalyzed ADP/ATP and Fru-6-P/Fru-2,6-P2 exchanges at rates comparable to net reaction rates. It was phosphorylated by both [gamma-32P]ATP and [2-32P] Fru-2,6-P2, and the label from either donor was chased by either unlabeled donor, showing that the bound phosphate is hydrolyzed if not transferred to an acceptor ligand. The rate of labeling of the enzyme by [2-32P]Fru-2,6-P2 was 2 orders of magnitude greater than the maximal velocity of the bisphosphatase and therefore sufficiently fast to be a step in the hydrolysis. Both inorganic phosphate and Fru-6-P increased the rate and steady state of enzyme phosphorylation by ATP. Fru-2,6-P2 inhibited the ATPase and kinase reactions and Fru-6-P inhibited the Fru-2,6 bisphosphatase reaction while ATP and ADP had no effect. Removal of the trace of Fru-6-P by Glu-6-P isomerase and Glu-6-P dehydrogenase reduced enzyme phosphorylation by ATP to very low levels, greatly inhibited the ATPase, and rendered it insensitive to Pi, but did not affect ADP/ATP exchange. (alpha + beta)Methylfructofuranoside-6-P did not increase the rate or steady state labeling by ATP. These results suggest that labeling of the enzyme by ATP involved the production of [2-32P]Fru-2,6-P2 from the trace Fru-6-P. The 6-phosphofructo-2-kinase, fructose 2,6-bisphosphatase, and ATP/ADP exchange were all inhibited by diethylpyrocarbonate, suggesting the involvement of histidine residues in all three reactions. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a Fru-2,6 bisphosphatase site which is readily phosphorylated by Fru-2,6-P2.

Adenosine Diphosphate↗
PubMed1984

Multiple forms of pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase from wheat seedlings. Regulation by fructose 2,6-bisphosphate.

Two forms of pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase have been isolated from wheat seedlings. One of these enzymes, termed PFP-1, has been purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of two different polypeptide chains of Mr = 67,000 (alpha) and 60,000 (beta). PFP-1 has been assigned a molecular structure consisting of alpha 2 beta 2 based on an estimated Mr of 234,000 for the native enzyme. PFP-2, the other form of phosphotransferase, has also been purified extensively. Preliminary data suggest that the active form of PFP-2 is probably a dimer of a polypeptide chain of Mr = 60,000. Immunological studies indicate that the two enzyme preparations share common antigenic determinants. The two forms of enzyme have very similar kinetic properties. The phosphotransferases are activated by fructose 2,6-bisphosphate (Fru-2,6-P2) which lowers the Km of the enzymes for fructose 6-phosphate but not that for PPi. Interestingly, PFP-1 is significantly more active than PFP-2 in the absence of Fru-2,6-P2. Also, PFP-1 exhibits a greater affinity (Ka = 7 nM) than PFP-2 (Ka = 26 nM) for the activator. Based on kinetic, immunological, and physicochemical parameters, it is suggested that the two enzymic forms are related in that they share the same catalytic moiety, i.e. the 60,000-dalton or beta subunit. The beta subunit when in complex formation with the alpha subunit, as in PFP-1, becomes more active in the absence of Fru-2,6-P2 as well as exhibits a greater sensitivity toward the effector.

Enzyme Activation↗
PubMed1984

Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes. Effects of phosphorylated metabolites on enzymes of carbohydrate metabolism.

Isolated rat hepatocytes convert 2,5-anhydromannitol to 2,5-anhydromannitol-1-P and 2,5-anhydromannitol-1,6-P2. Cellular concentrations of the monophosphate and bisphosphate are proportional to the concentration of 2,5-anhydromannitol and are decreased by gluconeogenic substrates but not by glucose. Rat liver phosphofructokinase-1 phosphorylates 2,5-anhydromannitol-1-P; the rate is less than that for fructose-6-P but is stimulated by fructose-2,6-P2. At 1 mM fructose-6-P, bisphosphate compounds activate rat liver phosphofructokinase-1 in the following order of effectiveness: fructose-2,6-P2 much greater than 2,5-anhydromannitol-1,6-P2 greater than fructose-1,6-P2 greater than 2,5-anhydroglucitol-1,6-P2. High concentrations of fructose-1,6-P2 or 2,5-anhydromannitol-1,6-P2 inhibit phosphofructokinase-1. Rat liver fructose 1,6-bisphosphatase is inhibited competitively by 2,5-anhydromannitol-1,6-P2 and noncompetitively by 2,5-anhydroglucitol-1,6-P2. The AMP inhibition of fructose 1,6-bisphosphatase is potentiated by 2,5-anhydroglucitol-1,6-P2 but not by 2,5-anhydromannitol-1,6-P2. Rat liver pyruvate kinase is stimulated by micromolar concentrations of 2,5-anhydromannitol-1,6-P2; the maximal activation is the same as for fructose-1,6-P2. 2,5-Anhydroglucitol-1,6-P2 is a weak activator. 2,5-Anhydromannitol-1-P stimulates pyruvate kinase more effectively than fructose-1-P. Effects of glucagon on pyruvate kinase are not altered by prior treatment of hepatocytes with 2,5-anhydromannitol. Pyruvate kinase from glucagon-treated hepatocytes has the same activity as the control pyruvate kinase at saturating concentrations of 2,5-anhydromannitol-1,6-P2 but has a decreased affinity for 2,5-anhydromannitol-1,6-P2 and is not stimulated by 2,5-anhydromannitol-1-P. The inhibition of gluconeogenesis and enhancement of glycolysis from gluconeogenic precursors in hepatocytes treated with 2,5-anhydromannitol can be explained by an inhibition of fructose 1,6-bisphosphatase, an activation of pyruvate kinase, and an abolition of the influence of phosphorylation on pyruvate kinase.

Animals↗
PubMed1984

Characterization of fructose 1,6-bisphosphatase from bakers' yeast.

Active nonphosphorylated fructose bisphosphatase (EC 3.1.3.11) was purified from bakers' yeast. After chromatography on phosphocellulose, the enzyme appeared as a homogeneous protein as deduced from polyacrylamide gel electrophoresis, gel filtration, and isoelectric focusing. A Stokes radius of 44.5 A and molecular weight of 116,000 was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate resulted in three protein bands of Mr = 57,000, 40,000, and 31,000. Only one band of Mr = 57,000 was observed, when the single band of the enzyme obtained after polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate was eluted and then resubmitted to electrophoresis in the presence of sodium dodecyl sulfate. Amino acid analysis indicated 1030 residues/mol of enzyme including 12 cysteine moieties. The isoelectric point of the enzyme was estimated by gel electrofocusing to be around pH 5.5. The catalytic activity showed a maximum at pH 8.0; the specific activity at the standard pH of 7.0 was 46 units/mg of protein. Fructose 1,6-bisphosphatase b, the less active phosphorylated form of the enzyme, was purified from glucose inactivated yeast. This enzyme exhibited maximal activity at pH greater than or equal to 9.5; the specific activity measured at pH 7.0 was 25 units/mg of protein. The activity ratio, with 10 mM Mg2+ relative to 2 mM Mn2+, was 4.3 and 1.8 for fructose 1,6-bisphosphatase a and fructose 1,6-bisphosphatase b, respectively. Activity of fructose 1,6-bisphosphatase a was 50% inhibited by 0.2 microM fructose 2,6-bisphosphate or 50 microM AMP. Inhibition by fructose 2,6-bisphosphate as well as by AMP decreased with a more alkaline pH in a range between pH 6.5 and 9.0. The inhibition exerted by combinations of the two metabolites at pH 7.0 was synergistic.

Adenosine Monophosphate↗
PubMed1984

Effect of enzyme concentrations on sustained oscillations in the fructose 6-phosphate/fructose 1,6-bisphosphate-cycle.

Sustained oscillations were investigated in an open and homogeneous enzyme system reconstituted from phosphofructokinase, fructose 1,6- biphosphatase , pyruvate kinase, adenylate kinase, and glucose 6-phosphate isomerase. The generation of oscillations if mainly due to antagonistic kinetic effects of AMP on phosphofructokinase and fructose 1,6- biphosphatase under the experimental conditions applied. Sustained oscillations were obtained in a broad range of maximum activities of phosphofructokinase or fructose 1,6- biphosphatase are increased. At low maximum activities of the two enzymes oscillations arise, which form a folded limit cycle, while by increasing concentrations of phosphofructokinase or fructose 1,6-bisphosphatase sustained oscillations are introduced, which are restricted to two dimensions.

Adenylate Kinase↗
PubMedDate not supplied

[Multiplicity of auto-oscillations and steady states in an open reaction catalyzed by E. coli phosphofructokinase. Quantitative model].

A quantitative mathematical model of two-substrate reaction catalized by phosphofructokinase from E. coli has been investigated. The model takes into account the tetrameric enzyme structure and resulting kinetic peculiarities of the reaction catalized, in particular, iso-and alosteric effects of ADP on enzyme activity. Fitting of the model parameters to experimental data allowed to approximate these data with good accuracy. The ranges of admitted rates of fructose-6-phosphate input and ATP regeneration at which autooscillations and multiple steady-states occur in the system have been calculated. A minimal open system with three enzymes has been proposed for experimental demonstration of the autooscillatory behaviour.

Adenosine Diphosphate↗
PubMedDate not supplied

[Analysis of allosteric mechanisms of suppressing parasitic recirculation in the futile cycle fructose-6-P--fructose-1,6-P2].

The uncontrollable substrate recirculation in the central futile cycle (FC) in the carbohydrate energy metabolism fructose-6-P (F6P) in equilibrium or formed from fructose-1,6-P2 (FBP), makes it impossible to maintain a stable level of ATP because of its wasteful expenditure in the cycle reactions which are equivalent to the ATPase reaction and also because of the diversion of FBP from glycolytic phosphorylation of ADP. It follows from the analysis of a mathematical model of the carbohydrate energy metabolism that the allosteric inhibition of fructosebisphosphatase (FBPase) by FBP and AMP leads to suppression of the recirculation in the FC and recovery of the ability of glycolysis to stabilize the level of ATP with high accuracy. The allosteric activation of phosphofrucktokinase (PFK) by AMP couples the expenditure of ATP and F6P in the FC with ATP consumption by a load.

Adenosine Monophosphate↗
PubMedDate not supplied

[Role of stationary activation of glycolysis in the heart during compensatory hyperfunction and aging].

Stationary stimulation of glycolysis was observed in rat heart muscle which produced the compensatory hyperfunction after aorta coarctation. The general character of alterations in energy balance of the hypertrophic heart muscle corresponded to the rearrangements, which occurred during ageing; therefore the coarctation of aorta caused more distinct stimulation of glycolysis in old animals than in young ones. Stimulation of glycolysis in ;the hypertrophic heart muscle was carried out via phosphofructokinase activation as G-PPK was increased by 87% during the heart compensatory hyperfunction. Stimulation of glycolysis, which developed at the beginning of the heart compensatory hyperfunction and continued up to its final steps, was apparently the adaptational mechanism providing the additional intake of ATP into the contractile apparatus, and so this stimulation prevented the decrease of oxygen consumption, produced in the hypertrophic heart muscle.

Adenosine Diphosphate↗