A model study of the fructose diphosphatase-phosphofructokinase substrate cycle.
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ABSTRACT:
SUBMITTER: Bloxham DP
PROVIDER: S-EPMC1177845 | biostudies-other | 1973 Jun
REPOSITORIES: biostudies-other
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Project description:1. Substrate cycling of fructose 6-phosphate through reactions catalysed by phosphofructokinase and fructose diphosphatase was estimated in bumble-bee (Bombus affinis) flight muscle in vivo. 2. Estimations of substrate cycling of fructose 6-phosphate and of glycolysis were made from the equilibrium value of the (3)H/(14)C ratio in glucose 6-phosphate as well as the rate of (3)H release to water after the metabolism of [5-(3)H,U-(14)C]glucose. 3. In flight, the metabolism of glucose proceeded exclusively through glycolysis (20.4mumol/min per g fresh wt.) and there was no evidence for substrate cycling. 4. In the resting bumble-bee exposed to low temperatures (5 degrees C), the pattern of glucose metabolism in the flight muscle was altered so that substrate cycling was high (10.4mumol/min per g fresh wt.) and glycolysis was decreased (5.8mumol/min per g fresh wt.). 5. The rate of substrate cycling in the resting bumble-bee flight muscle was inversely related to the ambient temperature, since at 27 degrees , 21 degrees and 5 degrees C the rates of substrate cycling were 0, 0.48 and 10.4mumol/min per g fresh wt. respectively. 6. Calcium ions inhibited fructose diphosphatase of the bumble-bee flight muscle at concentrations that were without effect on phosphofructokinase. The inhibition was reversed by the presence of a Ca(2+)-chelating compound. It is proposed that the rate of fructose 6-phosphate substrate cycling could be regulated by changes in the sarcoplasmic Ca(2+) concentration associated with the contractile process.
| S-EPMC1177847 | biostudies-other
Project description:1. The activities of fructose 1,6-diphosphatase were measured in extracts of muscles of various physiological function, and compared with the activities of other enzymes including phosphofructokinase, phosphoenolpyruvate carboxykinase and the lactate-dehydrogenase isoenzymes. 2. The activity of phosphofructokinase greatly exceeded that of fructose diphosphatase in all muscles tested, and it is concluded that fructose diphosphatase could not play any significant role in the regulation of fructose 6-phosphate phosphorylation in muscle. 3. Fructose-diphosphatase activity was highest in white muscle and low in red muscle. No activity was detected in heart or a deep-red skeletal muscle, rabbit semitendinosus. 4. The lactate-dehydrogenase isoenzyme ratio (activities at high and low substrate concentration) was measured in various muscles because a low ratio is characteristic of muscles that are more dependent on glycolysis for their energy production. As the ratio decreased the activity of fructose diphosphatase increased, which suggests that highest fructose-diphosphatase activity is found in muscles that depend most on glycolysis. 5. There was a good correlation between the activities of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle, where the activities of these enzymes were similar to those of liver and kidney cortex. However, the activities of pyruvate carboxylase and glucose 6-phosphatase were very low in white muscle, thereby excluding the possibility of gluconeogenesis from pyruvate and lactate. 6. It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the alpha-glycerophosphate-dihydroxyacetone phosphate and malate-oxaloacetate cycles in this tissue.
| S-EPMC1270420 | biostudies-other
Project description:In healthy individuals, plasma glucose levels are maintained within a normal range. During fasting, endogenous glucose is released either through glycogenolysis or gluconeogenesis. Gluconeogenesis involves the formation of glucose-6-phosphate from a variety of precursors followed by its subsequent hydrolysis to glucose. Gluconeogenesis occurs in the liver and the kidney. In order to compare gluconeogenesis in canine liver and kidney, the activity and expression of the rate limiting enzymes that catalyze the fructose-6-phosphate and fructose 1,6-bisphosphate steps, namely, phosphofructokinase-1 (PFK-1) (glycolysis) and fructose bisphosphatase-1 (FBP-1) (gluconeogenesis), were examined. Healthy male and female beagle dogs aged 1-2 years were euthanized humanely, and samples of their liver and kidney were obtained for analysis. The levels of PFK-1 and FBP-1 in canine liver and kidney were assessed by enzymatic assays, Western blotting, and RT-qPCR. Enzyme assays showed that, in dogs, the kidney had higher specific activity of PFK-1 and FBP-1 than the liver. Western blotting and RT-qPCR data demonstrated that of the three different subunits (PFK-M, PFK-L, and PFK-P) the PFK-1 in canine liver mainly comprised PFK-L, whereas the PFK-1 in the canine kidney comprised all three subunits. As a result of these differences in the subunit composition of PFK-1, glucose metabolism might be regulated differently in the liver and kidney.
| S-EPMC6863710 | biostudies-literature
Project description:1. The effects of Ca(2+) on the activities and regulatory properties of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from vertebrate red and white muscle and insect fibrillar and non-fibrillar muscle have been investigated. These muscles were selected because of the possible difference in the role of glycolysis in energy production in the vertebrate muscles, and the possible difference in the role of Ca(2+) in the control of contraction in the two types of insect muscle. An increase in Ca(2+) concentration from 0.001mum to 10mum did not modify the activities nor did it modify the regulatory properties of these enzymes from these various muscles. 2. Concentrations of Ca(2+) above 0.1mm inhibited the activities of hexokinase and phosphofructokinase from the different muscles. It has been suggested that this inhibition may provide the basis for a theory of regulation of glycolysis (Margreth et al., 1967). If phosphofructokinase is located within the sarcoplasmic reticulum, its activity will be inhibited when the muscle is at rest, but the release of Ca(2+) from the reticulum during contraction will lead to a stimulation of its activity and hence an increase in glycolytic flux. The distribution of hexokinase and phosphofructokinase in the various cell fractions of these muscles was very variable. In particular, both enzymes were present almost exclusively in the 100000g supernatant fraction in the extracts of insect flight muscles. Thus there is no correlation between the properties of the enzymes and their distribution in muscle. 3. It is concluded that Ca(2+) does not control the activities of the important regulatory enzymes of glycolysis in muscle. It is suggested that in some muscles the sensitivity of the control mechanism at the level of phosphofructokinase to changes in the concentration of AMP may be increased by a process known as ;substrate-cycling'.
| S-EPMC1177617 | biostudies-other
Project description:1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 mumoles of substrate were split/g. fresh wt./hr. at 22 degrees . 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0.1 mm, by AMP and by trace quantities of Zn(2+), Fe(2+) and Fe(3+); it is ;activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C(3) compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C(3) intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. alpha-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of alpha-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed.
| S-EPMC1206525 | biostudies-other
Project description:Mammalian phosphofructokinase originated by duplication, fusion, and divergence of a primitive prokaryotic gene, with the duplicated fructose 6-phosphate catalytic site in the C-terminal half becoming an allosteric site for the activator fructose 2,6-bisphosphate. It has been suggested that both sites are shared across the interface between subunits aligned in an antiparallel orientation, the N-terminal half of one subunit facing the C-terminal half of the other. The composition of these binding sites and the way in which subunits interact to form the dimer within the tetrameric enzyme have been reexamined by systematic point mutations to alanine of key amino acid residues of human muscle phosphofructokinase. We found that residues His-199, His-298, Arg-201, and Arg-292 contribute to the catalytic site and not to the allosteric site, because their mutation decreased the affinity for fructose 6-phosphate without affecting the activation by fructose 2,6-bisphosphate or its binding affinity. In contrast, residues Arg-566, Arg-655, and His-661 were critical components of the fructose bisphosphate allosteric site, because their mutation strongly reduced the action and affinity of the activator, with no alteration of substrate binding to the active site. Our results suggest that mammalian phosphofructokinase subunits associate with the N-terminal halves facing each other to form the two catalytic sites/dimer and the C-terminal halves forming the allosteric sites. Additionally, mutation of certain residues eliminated activation by fructose 1,6-bisphosphate, but not its binding, with little effect on activation by fructose 2,6-bisphosphate, indicating a divergence in the signal transduction route despite their binding to the same site.
| S-EPMC2666562 | biostudies-literature
Project description:Background:High glycolytic rate is a hallmark of cancer (Warburg effect). Glycolytic ATP is required for fuelling plasma membrane calcium ATPases (PMCAs), responsible for extrusion of cytosolic calcium, in pancreatic ductal adenocarcinoma (PDAC). Phosphofructokinase-fructose-bisphosphatase-3 (PFKFB3) is a glycolytic driver that activates key rate-limiting enzyme Phosphofructokinase-1; we investigated whether PFKFB3 is required for PMCA function in PDAC cells. Methods:PDAC cell-lines, MIA PaCa-2, BxPC-3, PANC1 and non-cancerous human pancreatic stellate cells (HPSCs) were used. Cell growth, death and metabolism were assessed using sulforhodamine-B/tetrazolium-based assays, poly-ADP-ribose-polymerase (PARP1) cleavage and seahorse XF analysis, respectively. ATP was measured using a luciferase-based assay, membrane proteins were isolated using a kit and intracellular calcium concentration and PMCA activity were measured using Fura-2 fluorescence imaging. Results:PFKFB3 was highly expressed in PDAC cells but not HPSCs. In MIA PaCa-2, a pool of PFKFB3 was identified at the plasma membrane. PFKFB3 inhibitor, PFK15, caused reduced cell growth and PMCA activity, leading to calcium overload and apoptosis in PDAC cells. PFK15 reduced glycolysis but had no effect on steady-state ATP concentration in MIA PaCa-2. Conclusions:PFKFB3 is important for maintaining PMCA function in PDAC, independently of cytosolic ATP levels and may be involved in providing a localised ATP supply at the plasma membrane.
| S-EPMC7114799 | biostudies-literature
Project description:1. The present study extends the finding of Krebs & Woodford (1965) that muscle fructose diphosphatase is more sensitive to AMP inhibition than liver fructose diphosphatase. 2. Hen breast fructose diphosphatase has a K(i) for AMP of 0.1mum; the plot of percentage inhibition is non-sigmoid and the reciprocal plot of activity against AMP concentration is sometimes linear. 3. Percentage inhibition plots for other muscle fructose diphosphatases are sigmoid curves which exhibit different threshold responses to the AMP concentration. 4. The intracellular content of AMP in all muscles tested exceeds the inhibition concentration range of AMP. 5. The sensitivity of muscle fructose diphosphatase to AMP inhibition is decreased by the presence of Mg(2+) or Mn(2+) ions; in the presence of Mn(2+) the inhibition curve for hen breast fructose diphosphatase becomes sigmoid. 6. From the formation constants for the Mg(2+) and Mn(2+) chelates, the effect of these ions in chelation of AMP can be calculated. Although chelation of AMP can explain the Mg(2+) effect, it cannot explain the marked relief of AMP inhibition by Mn(2+). 7. It is suggested that Mn(2+) has a specific effect on this enzyme which reduces the sensitivity to AMP inhibition.
| S-EPMC1270594 | biostudies-other
Project description:Myeloproliferative neoplasms are characterized by overproduction of myeloid lineage cells with frequent acquisition of oncogenic JAK2V617F kinase mutations. The molecular mechanisms that regulate energy requirements in these diseases are poorly understood. Transformed cells tend to rely on fermentation instead of more efficient oxidative phosphorylation for energy production. Our data in JAK2V617F-transformed cells show that growth and metabolic activity were strictly dependent on the presence of glucose. Uptake of glucose and cell surface expression of the glucose transporter Glut1 required the oncogenic tyrosine kinase. Importantly, JAK2V617F as well as active STAT5 increased the expression of the inducible rate-limiting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), which controls glycolytic flux through 6-phosphofructo-1-kinase. PFKFB3 was required for JAK2V617F-dependent lactate production, oxidative metabolic activity and glucose uptake. Targeted knockdown of PFKFB3 also limited cell growth under normoxic and hypoxic conditions and blocked in vivo tumor formation in mice. Overall, these data suggest that inducible PFKFB3 is required for increased growth, metabolic activity and is regulated through active JAK2 and STAT5. Novel therapies that specifically block PFKFB3 activity or expression would, therefore, be expected to inhibit JAK2/STAT5-dependent malignancies and related cancers.
| S-EPMC3227767 | biostudies-literature
Project description:1. The calculated energy charge of the liver cell from migrating salmon is very low (0.464), in keeping with the extended starvation and high rates of muscular and biosynthetic activity of these organisms. 2. Affinity of fructose 1,6-diphosphatase for substrate increases with a decrease in temperature. 3. Arrhenius plots of the saturation kinetics are complex and suggest an interconversion of one or more forms of the enzyme; this interconversion is dependent on the identity of the cofactor. 4. Affinity of salmon fructose 1,6-diphosphatase for its allosteric inhibitor (AMP) is lower than in other fructose 1,6-diphosphatases and this enzyme-AMP interaction is largely insensitive to temperature. The functional significance of diminished AMP-sensitivity is that it allows normal or high fructose 1,6-diphosphatase activity during a low energy charge. 5. These findings suggest mechanisms for the maintenance of high rates of gluconeogenesis in salmon during spawning migration.
| S-EPMC1185194 | biostudies-other