Molecular mechanism of glycolytic flux control intrinsic to human phosphoglycerate kinase

2021 ◽  
Vol 118 (50) ◽  
pp. e2112986118
Author(s):  
Hiromasa Yagi ◽  
Takuma Kasai ◽  
Elisa Rioual ◽  
Teppei Ikeya ◽  
Takanori Kigawa
Keyword(s):  
Ligand Binding ◽  
Bound States ◽  
Adenine Nucleotides ◽  
Phosphoglycerate Kinase ◽  
Adenosine Diphosphate ◽  
Reaction Scheme ◽  
Atomic Scale ◽  
Glycolytic Flux ◽  
Physiological Conditions ◽  
Equilibrium Step

Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP]) ratio controls glycolytic flux; however, the regulatory mechanism underlying reactions catalyzed by individual glycolytic enzymes enabling flux adaptation remains incompletely understood. Phosphoglycerate kinase (PGK) catalyzes the reversible phosphotransfer reaction, which directly produces ATP in a near-equilibrium step of glycolysis. Despite extensive studies on the transcriptional regulation of PGK expression, the mechanism in response to changes in the [ATP]/[ADP] ratio remains obscure. Here, we report a protein-level regulation of human PGK (hPGK) by utilizing the switching ligand-binding cooperativities between adenine nucleotides and 3-phosphoglycerate (3PG). This was revealed by nuclear magnetic resonance (NMR) spectroscopy at physiological salt concentrations. MgADP and 3PG bind to hPGK with negative cooperativity, whereas MgAMPPNP (a nonhydrolyzable ATP analog) and 3PG bind to hPGK with positive cooperativity. These opposite cooperativities enable a shift between different ligand-bound states depending on the intracellular [ATP]/[ADP] ratio. Based on these findings, we present an atomic-scale description of the reaction scheme for hPGK under physiological conditions. Our results indicate that hPGK intrinsically modulates its function via ligand-binding cooperativities that are finely tuned to respond to changes in the [ATP]/[ADP] ratio. The alteration of ligand-binding cooperativities could be one of the self-regulatory mechanisms for enzymes in bidirectional pathways, which enables rapid adaptation to changes in the intracellular environment.

scholarly journals Platelet functions and energy metabolism in a patient with hexokinase deficiency

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 147-153 ◽  
Author(s):  
JW Akkerman ◽  
G Rijksen ◽  
G Gorter ◽  
GE Staal
Keyword(s):  
Adenine Nucleotides ◽  
Energy Charge ◽  
Energy Generation ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Glycolytic Flux ◽  
Adenylate Energy Charge ◽  
Severe Reduction ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract We have studied the regeneration of adenosine triphosphate (ATP) in the glycolytic pathway in platelets with a 75% reduction in hexokinase (HK) activity and have investigated aggregation and Ca2+ secretion. HK- deficient platelets had a normal glycolytic flux in the resting state, but responded insufficiently to stimulation with thrombin (5 U/ml). In contrast, glycogen contents and glycogenolysis were normal. When the metabolic adenine nucleotides were labeled with 14C-adenine, the patient's platelets showed a normal adenylate energy charge and a normal level of 14C-ATP. However, the inhibitor of mitochondrial energy generation, CN-, induced a weaker fall in 14C-ATP in the patient's platelets than in the controls. Analysis of secretion markers revealed decreased amounts of granule-bound ATP and secretable Ca2+, whereas granule-bound adenosine diphosphate (ADP), beta-thromboglobulin, N- acetyl-beta-D-glucosaminidase, and beta-glucuronidase were within the normal range. Aggregation and Ca2+ secretion induced by 5 U/ml thrombin were normal and were not changed in the presence of inhibitors of mitochondrial and glycogenolytic energy generation. Aggregation was also normal at 0.1 U/ml thrombin and was independent of these inhibitors, but Ca2+ secretion was greatly impaired when mitochondrial and glycogenolytic ATP resynthesis was abolished. These findings indicate that a severe reduction in HK activity causes insufficient acceleration of the glycolytic flux during stimulation with thrombin. This leads to impaired dense granule secretion in conditions where secretion depends on concurrent ATP resynthesis and glycolysis is rate limiting.

scholarly journals Platelet functions and energy metabolism in a patient with hexokinase deficiency

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 147-153
Author(s):  
JW Akkerman ◽  
G Rijksen ◽  
G Gorter ◽  
GE Staal
Keyword(s):  
Adenine Nucleotides ◽  
Energy Charge ◽  
Energy Generation ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Glycolytic Flux ◽  
Adenylate Energy Charge ◽  
Severe Reduction ◽  
Dense Granule Secretion ◽  
Granule Secretion

We have studied the regeneration of adenosine triphosphate (ATP) in the glycolytic pathway in platelets with a 75% reduction in hexokinase (HK) activity and have investigated aggregation and Ca2+ secretion. HK- deficient platelets had a normal glycolytic flux in the resting state, but responded insufficiently to stimulation with thrombin (5 U/ml). In contrast, glycogen contents and glycogenolysis were normal. When the metabolic adenine nucleotides were labeled with 14C-adenine, the patient's platelets showed a normal adenylate energy charge and a normal level of 14C-ATP. However, the inhibitor of mitochondrial energy generation, CN-, induced a weaker fall in 14C-ATP in the patient's platelets than in the controls. Analysis of secretion markers revealed decreased amounts of granule-bound ATP and secretable Ca2+, whereas granule-bound adenosine diphosphate (ADP), beta-thromboglobulin, N- acetyl-beta-D-glucosaminidase, and beta-glucuronidase were within the normal range. Aggregation and Ca2+ secretion induced by 5 U/ml thrombin were normal and were not changed in the presence of inhibitors of mitochondrial and glycogenolytic energy generation. Aggregation was also normal at 0.1 U/ml thrombin and was independent of these inhibitors, but Ca2+ secretion was greatly impaired when mitochondrial and glycogenolytic ATP resynthesis was abolished. These findings indicate that a severe reduction in HK activity causes insufficient acceleration of the glycolytic flux during stimulation with thrombin. This leads to impaired dense granule secretion in conditions where secretion depends on concurrent ATP resynthesis and glycolysis is rate limiting.

scholarly journals Stimulation of mitochondrial calcium ion efflux by thiol-specific reagents and by thyroxine. The relationship to adenosine diphosphate retention and to mitochondrial permeability

1979 ◽  
Vol 182 (2) ◽  
pp. 455-464 ◽  
Author(s):  
E J Harris ◽  
M Al-Shaikhaly ◽  
H Baum
Keyword(s):  
Thyroid Hormones ◽  
Calcium Ion ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Ruthenium Red ◽  
Reduced Form ◽  
Factors Affecting ◽  
Ph Values ◽  
Rat Heart Mitochondria ◽  
The Relationship

Respiring rat heart mitochondria were loaded with Ca2+ and then treated with Ruthenium Red. The factors affecting the subsequent Ca2+-efflux were studied. Addition of rotenone or antimycin led to a decline of efflux except at pH values above 7.2, provided the load was less than about 80 nmol per mg of protein. Oligomycin reversed the effect of the respiratory inhibitors. Independently of respiration, efflux was stimulated by the uncoupler trifluoromethyltetrachlorbenzimadazole, by mersalyl and by thyroid hormones. The stimulated efflux could be diminished by ADP, with Mg2+ as cofactor if efflux was rapid. With respiration in progress, efflux could be stimulated by N-ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoate). The effects of mersalyl and of thyroid hormones could be diminished with dithiothreitol. In the absence of stimulating agents, the Ca2+ efflux was proportional to the load up to some critical amount, this critical amount was decreased by the agents. Thyroxine and mersalyl caused not only loss of Ca2+, but also simultaneous, but not necessarily proportional, loss of internal adenine nucleotides. Both efflux rates were kept at a low value by bongkrekic acid added before the stimulating agent. It is concluded that Ca2+ efflux is a measure of a permeability controlled by the binding of ADP (an Mg2+) to the inner membrane, and that this in turn depends on the maintenance of certain thiol gropus in a reduced form by a reaction that uses NADH and ATP and the energy-linked transhydrogenase.

Conformational dynamics and thermodynamics of protein–ligand binding studied by NMR relaxation

2012 ◽  
Vol 40 (2) ◽  
pp. 419-423 ◽  
Author(s):  
Mikael Akke
Keyword(s):  
Ligand Binding ◽  
Bound States ◽  
Conformational Dynamics ◽  
Nmr Relaxation ◽  
Titration Calorimetry ◽  
Conformational Entropy ◽  
Chain Order ◽  
Ligand Interactions ◽  
Galectin 3 ◽  
Relaxation Experiments

Protein conformational dynamics can be critical for ligand binding in two ways that relate to kinetics and thermodynamics respectively. First, conformational transitions between different substates can control access to the binding site (kinetics). Secondly, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics). In the present paper, I focus on the second topic, summarizing our recent results on the role of conformational entropy in ligand binding to Gal3C (the carbohydrate-recognition domain of galectin-3). NMR relaxation experiments provide a unique probe of conformational entropy by characterizing bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side chain order parameters, we have estimated the contributions from conformational entropy to the free energy of binding. Overall, the conformational entropy of Gal3C increases upon ligand binding, thereby contributing favourably to the binding affinity. Comparisons with the results from isothermal titration calorimetry indicate that the conformational entropy is comparable in magnitude to the enthalpy of binding. Furthermore, there are significant differences in the dynamic response to binding of different ligands, despite the fact that the protein structure is virtually identical in the different protein–ligand complexes. Thus both affinity and specificity of ligand binding to Gal3C appear to depend in part on subtle differences in the conformational fluctuations that reflect the complex interplay between structure, dynamics and ligand interactions.

scholarly journals Studies on Human Erythrocyte Nucleotide Metabolism. II. Nonspherocytic Hemolytic Anemia, High Red Cell ATP, and Ribosephosphate Pyrophosphokinase (RPK, E.C. 2.7.6.1) Deficiency

Blood ◽  
1972 ◽  
Vol 39 (5) ◽  
pp. 674-684 ◽  
Author(s):  
William N. Valentine ◽  
Helen M. Anderson ◽  
Donald E. Paglia ◽  
Ernst R. Jaffé ◽  
Patricia N. Konrad ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Reduced Glutathione ◽  
Adenine Nucleotides ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Blood Film ◽  
Nucleotide Metabolism ◽  
Glutathione Metabolism ◽  
Final Decision ◽  
Blood Samples

Abstract A 29-yr-old black woman was found to have a long-standing, nonspherocytic hemolytic disorder associated with a marked reduction in the activity of erythrocyte ribosephosphate pyrophosphokinase (RPK, PRPP synthetase, E.C. 2.7.6.1). Although the patient’s erythrocytes had about 50% of the average RPK activity of normal mature human erythrocytes, this level represented only about 20-30% of the activity in comparable reticulocyte-rich blood samples from patients with other types of hemolytic anemias. The concentrations of adenosine triphosphate adenosine diphosphate, adenosine monophosphate and, therefore, of total adenine nucleotides in her erythrocytes were markedly increased, even well above the levels in extracts of comparable reticulocyte-rich blood samples. ATPase activity was increased three- to fourfold, consistent with the reticulocytosis. Adenylate kinase and adenine phosphoribosyltransferase activities were normal. The activities of all enzymes of the Embden-Meyerhof and hexose monophosphate shunt pathways and enzymes related to glutathione metabolism were normal or increased, consistent with the reticulocytosis. The concentrations of glycolytic intermediates, other than adenine nucleotides, were normal. The conversion of glucose, adenosine, and inosine to lactate was normal or increased. Autohemolysis was of the Dacie Type II. The concentrations of erythrocyte-reduced glutathione were high normal or elevated. The stained blood film showed a striking degree of basophilic stippling of the erythrocytes. Studies of the erythrocytes of the patient’s only known relative, a son, have failed to reveal any hematologic or enzymatic abnormalities. A direct causal relationship between RPK deficiency, high ATP concentrations, and nonspherocytic hemolytic anemia could not be derived from data now available. The final decision as to whether the deficiency is primary and causative or is an epiphenomenon requires investigation of additional cases.

Active GPIIb-IIIa conformations that link ligand interaction with cytoskeletal reorganization

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2487-2495 ◽  
Author(s):  
Traci Heath Mondoro ◽  
Melanie McCabe White ◽  
Lisa K. Jennings
Keyword(s):  
Platelet Aggregation ◽  
Ligand Binding ◽  
Critical Role ◽  
Adenosine Diphosphate ◽  
Full Scale ◽  
Cytoskeletal Reorganization ◽  
Clot Retraction ◽  
Ligand Interaction ◽  
High Affinity ◽  
Combination Treatments

Abstract Glycoprotein (GP) IIb-IIIa plays a critical role in platelet aggregation and platelet-mediated clot retraction. This study examined the intramolecular relationship between GPIIb-IIIa activation and fibrinogen binding, platelet aggregation, and platelet-mediated clot retraction. To distinguish between different high-affinity activation states of GPIIb-IIIa, the properties of an antibody (D3) specific for GPIIIa that induces GPIIb-IIIa binding to adhesive protein molecules and yet completely inhibits clot retraction were used. Clot retraction inhibition by D3 was not due to altered platelet-fibrin interaction; however, combination treatments of D3 and adenosine diphosphate (ADP) inhibited full-scale aggregation and decreased the amounts of GPIIb-IIIa and talin incorporated into the core cytoskeletons. Morphologic evaluation of the D3/ADP aggregates showed platelets that were activated but to a lesser extent when compared to ADP only. ADP addition to platelets caused an increase in the number of D3 binding sites indicating that ligand had bound to the GPIIb-IIIa receptor. These data suggest that high-affinity GPIIb-IIIa– mediated ligand binding can be separated mechanistically from GPIIb-IIIa–mediated clot retraction and that clot retraction requires additional signaling through GPIIb-IIIa after ligand binding. The conformation recognized by D3 represents the expression of a GPIIb-IIIa activation state that participates in full-scale platelet aggregation, cytoskeletal reorganization, and clot retraction.

Complementary role of extracellular ATP and adenosine in ischemic preconditioning in the rat heart

2002 ◽  
Vol 282 (5) ◽  
pp. H1810-H1820 ◽  
Author(s):  
Hideki Ninomiya ◽  
Hajime Otani ◽  
Kejie Lu ◽  
Takamichi Uchiyama ◽  
Masakuni Kido ◽  
...  
Keyword(s):  
Ischemic Preconditioning ◽  
Pyridoxal Phosphate ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Extracellular Atp ◽  
Disulfonic Acid ◽  
Rate Pressure Product ◽  
Adenosine Receptor Antagonist ◽  
Complementary Role

Although adenosine is an important mediator of ischemic preconditioning (IPC), its relative contribution to IPC remains unknown. Because adenosine is formed through the hydrolysis of ATP, the present study investigated the role of ATP and adenosine in IPC. Isolated and buffer-perfused rat hearts underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. The rate-pressure product (RPP) 30 min after reperfusion was taken as an endpoint of functional protection. Interstitial fluid (ISF) adenine nucleotides and adenosine were measured by cardiac microdialysis techniques. Inhibition of IPC-induced recovery of RPP was partial by the adenosine receptor antagonist 8-( p-sulfophenyl)theophylline (SPT; 100 μM) or by the structurally distinct P2Y purinoceptor antagonists suramin (300 μM) or reactive blue (RB; 10 μM) but was additive when SPT was given with suramin or RB. The P2X antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid tetrasodium (50 μM) had no effect on functional protection. The improved functional recovery was not significantly affected by an ecto-5′-nucleotidase inhibitor, α,β-methylene adenosine diphosphate (AMP-CP; 100 μM), alone but was inhibited by AMP-CP plus SPT, suramin, or RB. ISF ATP and adenosine increased temporarily by 10-fold during IPC. AMP-CP augmented the increase in ISF ATP associated with the decrease in ISF adenosine. There was a reciprocal correlation between the ISF concentration of ATP and adenosine in preconditioned hearts. In addition, there was a significant correlation between ISF adenosine and ATP and the inhibitory potency of SPT and suramin or RB against functional protection conferred by IPC. These results suggest that extracellular ATP and adenosine play a complementary role in IPC through P2Y purinoceptors and adenosine receptors, respectively.

Changes of platelets’ function in preeclampsia

Open Medicine ◽  
2011 ◽  
Vol 6 (6) ◽  
pp. 696-700
Author(s):  
Goran Babic ◽  
Slobodan Novokmet ◽  
Slobodan Jankovic
Keyword(s):  
Platelet Aggregation ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Adenosine Diphosphate ◽  
Control Group ◽  
Adenylate Energy Charge ◽  
Cross Sectional ◽  
Blood Samples ◽  
Metabolic Pool ◽  
Aggregation Of Platelets

AbstractIncreased aggregation of platelets during preeclampsia was shown in several studies, yet several others reported no change. The aim of our study was to investigate platelet aggregation in a group of patients suffering from preeclampsia. In a cross-sectional study blood samples were taken from 89 hospitalized patients in the third trimester of pregnancy: 38 were suffering from mild to moderate preeclampsia and 51 patients were without preeclampsia. From the blood samples platelet aggregation, secretion of adenine nucleotides from platelets, concentration of energy-rich adenine compounds and levels of cyclic adenosine-mono-phosphate and cyclic guanosine mono-phosphate in platelets were measured. In the patients with preeclampsia, the adenosine diphosphate threshold for biphasic aggregation [odds ratio (OR):.75; 95% Confidence Interval (CI): 0.55–1.02; p<0.05], total adenine nucleotides concentration in the metabolic pool of platelets (OR:0.99; CI: 0.62–1.57; p<0.01) and cyclic adenosine-mono-phosphate (OR:0.81; CI: 0.57, 1.14; p<0.05) and cyclic guanosine mono-phosphate (OR:.78; CI: 0.55–1.09; p<0.05) levels in platelets were decreased in comparison with the control group, while adenylate energy charge in the metabolic pool of platelets (OR: >100.00; CI: 0.00->100.00; p<0.05) and secretion of adenosine triphosphate (OR:.13; CI: 0.00–14.26; p<0.05) and adenosine diphosphate (OR:.77; CI: 0.08–36.79; p<0.05) were increased. The results of our study show increased activation and aggregation of platelets in pregnant females with preeclampsia.

scholarly journals Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2017-2023 ◽  
Author(s):  
MH Ginsberg ◽  
AL Frelinger ◽  
SC Lam ◽  
J Forsyth ◽  
R McMillan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ligand Binding ◽  
Cell Activation ◽  
Flow Cytometric Analysis ◽  
Adenosine Diphosphate ◽  
Receptor Activation ◽  
Affinity Column ◽  
Primary Defect ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding

Abstract Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPIIb (PMI-1) or IIIa (anti- LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGDSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenic variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to increase the binding of PMI-1 or anti- LIBS1 to the thrombasthenic platelets, suggesting a primary defect in ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient's GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate- stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

scholarly journals The Effect of Chromium on Platelet Function In Vitro

Blood ◽  
1970 ◽  
Vol 35 (5) ◽  
pp. 659-668 ◽  
Author(s):  
HERMAN E. KATTLOVE ◽  
THEODORE H. SPAET
Keyword(s):  
Connective Tissue ◽  
Platelet Function ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Sodium Chromate ◽  
Platelet Factor ◽  
Survival Studies ◽  
Induced Aggregation ◽  
Platelets Function

Abstract Sodium chromate inhibits platelet function in vitro. The primary effect is inhibition of connective tissue-induced aggregation. In addition, the primary wave of epinephrine-induced aggregation is moderately inhibited and adenosine diphosphate-induced aggregation is mildly inhibited. The effect on connective tissue-induced aggregation is due to inhibition of the platelet "release reaction"; chromate inhibited the release of adenine nucleotides, 14C labeled serotonin and the activation of platelet factor III normally caused by connective tissue. The amount of chromium which must be bound to platelets to inhibit aggregation is 10-100 times the amount of radioactive chromium bound to platelets under the usual conditions of labeling for survival studies. However, this does not imply that chromium labeled platelets function normally.

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