Participation of glyceraldehyde-3-phosphate dehydrogenase in the regulation of 2,3-diphosphoglycerate level in erythrocytes

Data are presented concerning the possible participation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in regulation of the glycolytic pathway and the level of 2,3-diphosphoglycerate in erythrocytes. Experimental support has been obtained for the hypothesis according to which a mild oxidation of GAPDH must result in acceleration of glycolysis and in decrease in the level of 2, 3-diphosphoglycerate due to the acyl phosphatase activity of the mildly oxidized enzyme. Incubation of erythrocytes in the presence of 1 mM hydrogen peroxide decreases 2,3-diphosphoglycerate concentration and causes accumulation of 3-phosphoglycerate. It is assumed that the acceleration of glycolysis in the presence of oxidative agents described previously by a number of authors could be attributed to the acyl phosphatase activity of GAPDH. A pH-dependent complexing of GAPDH and 3-phosphoglycerate kinase or 2, 3-diphosphoglycerate mutase is found to determine the fate of 1,3-diphosphoglycerate that serves as a substrate for the synthesis of 2,3-diphosphoglycerate as well as for the 3-phosphoglycerate kinase reaction in glycolysis. A withdrawal of the two-enzyme complexes from the erythrocyte lysates using Sepharose-bound anti-GAPDH antibodies prevents the pH-dependent accumulation of the metabolites. The role of GAPDH in the regulation of glycolysis and the level of 2,3-diphosphoglycerate in erythrocytes is discussed.     

The effect of pH and pO2 changes in the blood on the energy metabolism of human erythrocytes in old age

Changes in pH and pO2 of the blood have been studied for age peculiarities of their effect on the glycolysis rate and the content of ATP and 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes (in vitro). The fresh venous blood of practically healthy young (aged 20-29) and old (aged 75-85) people was used. Acidosis was shown to induce inhibition of glycolysis and decrease of the ATP and 2.3-DPG concentrations in erythrocytes, while alkalosis and hypoxemia-an increase of the glycolysis rate and 2.3-DPG content. In the both cases changes in the indices studied were considerably lower in old people as compared to young ones.     

Age-related changes in glycolysis and levels of modulators of oxygen transport function of hemoglobin in human erythrocytes

The glycolysis rate and the amount of the acid-transport hemoglobin function modulators (diphosphoglycerate (2,3-DPG), ATP, glutathione, chlorides) in human erythrocytes are studied under the effect of age changes. It is shown that the glycolysis rate and the content of ATP decrease under ageing by 16% and 21%, respectively. The concentration of 2,3-DPG lowers insignificantly (by 8%). At the same time it is established that the amount of reduced glutathione in erythrocytes of middle-aged and old people enhances by 25-29% and that of chlorine ions by 23-24%. The revealed changes in the concentration of modulators of the hemoglobin affinity to oxygen with ageing are, probably, one of reasons of the oxyhemoglobin dissociation increase.     

Oxygen-transport function and carbohydrate metabolism in rat erythrocytes during hypoxic hypoxia and treatment with insulin

Hemoglobin affinity to oxygen, enzyme activity and metabolite concentration of carbohydrate metabolism were determined in erythrocytes of rats which were administered insulin solution. A valid decrease of the hemoglobin value P50 (pressure of hemoglobin half-saturation with oxygen), as well as a decrease of the enzyme activity of 2,3-diphosphoglycerate shunt and increase of the activity of regulatory glycolysis enzymes--hexokinase and pyruvate kinase in erythrocytes with multiple introduction of hormones to animals have been established. Such changes in rat erythrocytes were registered with the simultaneous effect of insulin and hypoxic hypoxia evoked by the "lift" of rats in the altitude chamber to the conditional altitude of 9000 m. It is found out that preliminary injection of insulin considerably increases survivability of rats under hypoxic hypoxia at great altitudes.     

Effects of dibutyryl-cANP on glycolysis in washed human erythrocytes

The experiments were carried out with washed human erythrocytes in order to study the effects of dibutyryl-cAMP (DB-cAMP) on glycolysis. 5 mM DB-cAMP significantly increases glucose consumption and lactate production in incubated erythrocytes. The cross-over plot of glycolytic intermediates shows that increased glycolysis is probably the result of activation of phosphofructokinase by DB-cAMP. Under the same condition DB-cAMP significantly protects the 2,3-diphosphoglycerate level in incubated erythrocytes.     

A reassessment of the phosphoglycerate bypass enzymes in human erythrocytes.

Dissociation of the human erythrocyte into cytoplasmic and membranous components, shows that all of the cell's intrinsic 2,3-diphosphoglycerate phosphatase activity is associated with the soluble component. Further fractionaction of the cytoplasm on DEAE cellulose illustrates that both 1,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase activities occur coincidently within one peak. Thermal denaturation of the peak proteins at 60° results in a parallel loss in phosphatase and mutase activity. The identical phenomenon is observed in the presence of the 2,3-diphosphoglycerate phosphatase activator, 2-phosphoglycolate. Homogeneous 1,3-diphosphoglycerate mutase, which quantitatively accounts for all of the intrinsic 2,3-diphosphoglycerate phosphatase within the red cell, also exhibits thermal instability at 60°. These findings suggest that the phosphoglycerate bypass in erythrocytes is under the control of a single, bifunctional enzyme.     

Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes

Summary Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.     

An incubation medium for the elevation of adenosine triphosphate and 2,3-diphosphoglycerate in fresh and long-preserved human erythrocytes.

The levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in freshly drawn human erythrocytes can be tripled by a 2 h incubation at 37 degrees C in a medium containing 21 mM glucose, 1.8 mM adenine, 5 mM pyruvate, 10 mM inosine, and 96 mM phosphate. Similar incubation conditions will restore the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood levels preserved for 12 and 15 weeks, respectively, to those of fresh cells. Omission of pyruvate from the incubation medium further increases the level of ATP slightly, but there is little elevation of 2,3-diphosphoglycerate. Under these conditions labelled pyruvate and lactate production from [14-C]glucose or [14-C]inosine is not diminished, but labelled fructose 1,6-diphosphate, rather than 2,3-diphosphoglycerate, accumulates. In addition, omission of pyruvate from the incubation medium, with a concomitant decrease in accumulation of 2,3-diphosphoglycerate, diminishes the concentration of inorganic phosphate required for optimal ATP elevation. A 5 h incubation in the glucose-adenine-pyruvate-inosine-phosphate medium elevates the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood preserved in the cold for 15 weeks to twice that of fresh cells, indicating that the cells retain their metabolic potential even after prolonged storage at 2 degrees C. The medium may provide a method of rejuvenating 10-12 week cold-preserved erythrocytes for transfusion purposes, by a 1 h incubation at 37 degrees C.     

Variations of Some Enzyne and Coenzyme Concentrations in Nornal Human Erythrocytes Fractionated by Velocity Sedimentation

Abstract

The concentrations of glucose-6-phosphate dehydrogenase, adenosine triphosphate and 2,3-diphosphoglycerate were measured in various fractions of normal human erythrocytes separated by velocity sedimentation in isotonic saline and sucrose buffer based on their tendency to aggregate. The levels of 2,3-diphosphoglycerate varied most drastically in fresh erythrocytes, being lowest in fast sedimenting cells and highest in slowly sedimenting cells. This result implies that the 2,3-diphosphoglycerate concentrations is not constant in all normal human erythrocytes and that 2,3-diphosphoglycerate may be involved in the mechanism of red cell clumping in vitro.     

Erythrocyte 2,3-diphosphoglycerate and hemoglobin electrophoretic characteristics in rodent adaptation to high mountains

Adaptation of the erythropoetic system to hypoxia was shown to be different in different populations of montaine rodents of the Central Caucasus. One type of adaptation includes the increase in 2,3-diphosphoglycerate content of erythrocytes, the hemoglobin structure remaining apparently unchanged. This pattern was found in two groups of animals -- in one population of the ground squirrel Citellus suslicus whose ancestors were introduced to the mountains some 25 years ago, and in the wood mouse Apodemus sylvaticus which is a usual inhabitant of highland, although it does not form there any isolated populations. The other type of adaptation was found in the ground squirrel Citellus pygmaeus musicus, which is native to high altitude. The content of 2,3-diphosphoglycerate in the erythrocytes in this animal is the same as in the sea-level subspecies C. p. planicola, whereas the electrophoretic picture of hemoglobin in these two subspecies was found to be different.     

Evidence for three enzymatic activities in one electrophoretic band of 3-phosphoglycerate mutase from red cells.

Electrophoresis of 3-phosphoglycerate mutase from erythrocytes of man and several animal species has been performed on cellulose acetate strips. In most cases the electrophoretic pattern of this enzymatic activity shows three bands. 2,3-diphosphoglycerate phosphatase and diphosphoglycerate mutase from erythrocytes of the same species have been revealed after migration during the same electrophoresis. We found that the band of 2,3-diphosphoglycerate phosphatase and the band of diphosphoglycerate mutase activities migrate at the same level as one of the bands corresponding to 3-phosphoglycerate mutase. Here, we discuss the possible existence of a single molecule carrying three enzymatic activities.     

A metabolic osmotic model of human erythrocytes

A metabolic osmotic model of red blood cells is presented which takes into account the main reaction steps of glycolysis and the passive and active fluxes of ions across the cell membrane. Cellular energy metabolism and osmotic behaviour are linked by the ATP consumption for the active transport of cations as well as by the osmotic action of the glycolytic intermediate 2,3-diphosphoglycerate (2,3-DPG). The model is based on a system of differential equations describing the metabolic reactions and transport processes. Further, two algebraic conditions for the osmotic equilibrium and the electroneutrality of the cell are considered. Using realistic system parameters the model allows the calculation of a great number of dependent variables, among them the cell volume, the concentrations of metabolites and ions and the transmembrane potential. Only stationary states are considered.The parameter dependence of important model variables is characterized by control coefficients. The main results are: (a) The volume of erythrocytes is mainly determined by the permeabilities of the leak fluxes of cations, the content of hemoglobin and the activity of the hexokinase-phosphofructokinase system of glycolysis; (b) Changes of volume affect the glycolytic rate mainly by changing the concentration of ATP which is a regulator of glycolysis; (c) A change in the membrane area may affect the other cell properties only if it is connected with variations of the number of active and leak sites of the membrane.     

The absence of 2,3-diphosphoglycerate from myocytes,hepatocytes and adipocytes

Myocytes, hepatocytes and adipocytes were prepared from heart, liver and epididymal fat pad of the rat. No detectable level of 2,3-diphosphoglycerate was found. Evidence is also present which indicates the absence from these cells of 2,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase. Previous findings by others of the presence of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase probably resulted from erythrocytes sequestered in the tissue.     

Glycolysis in human erythrocytes containing elevated concentrations of 2,3-P2-glycerate

In studies on the mechanism of the inhibitory effect of 2,3-diphosphoglycerate on glycolysis in human erythrocytes, the following results were obtained:1) Glucose consumption and lactate production are reduced by 70 and 40% relative to normal erythrocytes in red blood cells containing five times the normal amount of 2,3,-P₂-glycerate (“high-diphosphoglycerate” cells) at an extracellular pH of 7.4. The marked dependency of glycolysis on the extracellular pH observed in normal erythrocytes is almost completely lost in the “high-diphosphoglycerate” cells.2) About 50% of the inhibition of glycolysis in “high-diphosphoglycerate” cells can be accounted for by the 2,3-P₂-glycerate-induced decrease of the red-cell pH. This fall of the red-cell pH which occurs as a consequence of the Donnan effect of the non-penetrating 2,3-P₂-glycerate anion leads to a reduction of the glycolytic rate due to the properties of the enzyme phosphofructokinvse.3) The remaining part of the inhibitory effect must be attributed to an inhibition by 2,3-P₂-glycerate of glycolytic enzymes. From measurements of glycolytic rates and of the concentrations of glycolytic intermediates in the absence and presence of methylene blue it is concluded that the hexokinase reaction is inhibited by an elevation of 2,3-P₂-glycerate concentration. A marked increase of 3-P-glycerate concentration in “high-diphosphoglycerate” cells suggests that also the enzyme pyruvate kinase is inhibited by 2,3-P₂-glycerate.4) The dependencies of net-change of 2,3-P₂-glycerate concentration on the red-cell pH are identical in normal and “high-diphosphoglycerate” cells indicating that the balance between formation and decomposition of 2,3-P₂-glycerate is the same in erythrocytes with normal and very high concentrations 2,3-P₂-glycerate.     

The absence of 2,3-diphosphoglycerate from myocytes, hepatocytes and adipocytes.

Myocytes, hepatocytes and adipocytes were prepared from heart, liver and epididymal fat pad of the rat. No detectable level of 2,3-diphosphoglycerate was found. Evidence is also presented which indicates the absence from these cells of 2,3-diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase. Previous findings by others of the presence of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase probably resulted from erythrocytes sequestered in the tissue.     

Studies on the energy metabolism ofRana ridibunda erythrocytes

Summary Rana ridibunda erythrocytes have a complete sequence of glycolytic enzymes but not the tricarboxylic acid cycle enzymes.The steady state contents of the glycolytic intermediates were measured in quick frozenRana ridibunda erythrocytes. A comparison of the mass action ratios with the equilibrium constants for the glycolytic reactions showed that phosphoglucomutase, phosphoglucose isomerase, aldolase, triosephosphate isomerase, phosphoglycerate mutase and enolase reactions are all near equilibrium whilst hexokinase, phosphofructokinase and pyruvate kinase are displaced from equilibrium.The steady state contents of glycolytic intermediates, lactate, adenine nucleotides, inorganic phosphate have been measured during various periods up to 4 h of incubation of erythrocytes in the presence of glucose. In the incubation experiment glycolysis had been stimulated by the high pH-value of the medium. After 4 h of incubation 3 patterns of changes can be distinguished. One group of intermediates (glucose, glucose 6-phosphate, 2-phosphoglycerate and inorganic phosphate) in which the concentration of metabolites was lower than the zero time values. A second group of metabolites (fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and AMP) in which the concentration was about the same at zero time and after 4 h of incubation. The metabolites of the third group (dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-diphosphoglycerate, 2,3-diphosphoglycerate, 3-phosphoglycerate, pyruvate, lactate, ADP, ATP and glucose 1-phosphate) all increased their content during the 4 h of incubation in comparison to the zero time values.From the results it appears that in the amphibian erythrocyte glycolysis seems to be similar to that of mammalian erythrocytes as far its control and organisation is concerned down to the level of PEP, with the exception of the low concentration of phosphoglycerate compounds.Abbreviations 2,3DPG 2,3-diphosphoglycerate - EDTA [ethylene dinitrilo]-tetra-acetic acid - P _i inorganic phosphate - DTNB 5,5-dithio-bis-(2-nitrobenzoic acid) - PEP phosphoenolpyruvate - RBC red blood cells     

Red blood cell glucose metabolism in Down's syndrome

The specific activity of red blood cell glycolytic enzymes was determined in 20 Down's syndrome patients and compared with 20 normal controls. According to previous evidence, a 50% increase of phosphofructokinase and a 30% increase of glucose-6-phosphate dehydrogenase and glutathione peroxidase activity was found. Metabolic studies of the patients' erythrocytes revealed a decrease in fructose-6-phosphate and 2, 3-diphosphoglycerate concentrations, while fructose-1, 6-diphosphate and ADP both increased. Glucose utilization by intact erythrocytes from Down's syndrome patients did not differ from that of normal controls. However, addition of methylene blue or inorganic phosphate produced a higher stimulation of erythrocyte glycolysis in patients with Down's syndrome compared to controls. These metabolic abnormalities could be, at least in part, ascribed to the increased phosphofructokinase activity which is due to a gene-dosage effect.     

Diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase activities of red cells: comparative electrophoretic study

Erythrocyte diphosphoglycerate mutase (EC 2.7.5.4.) and 2,3-diphosphoglycerate phosphatase (EC 3.1.3.13.) activities of normal human adults, and DPG mutase deficient subject as well as of several animal species were subjected to electrophoretic study on starch gel. In U.V. light 2,3-diphosphoglycerate phosphatase activity was revealed as a band of fluorescence decrease on a fluorescent background, by the oxydation of NADH, whereas diphosphoglycerate mutase appeared as a fluorescent zone. It was found that the electrophoretic pattern of both DPG mutase and 2,3-DPG phosphatase activities was different from one species to the other, but that, in each species, 2,3-DPG phosphatase activity showed the same electrophoretic pattern as DPG mutase activity.     

The effects of ionophore A23187 on erythrocytes relationship of ATP and 2,3-diphosphoglycerate to calcium-binding capacity

The divalent cation ionophore, A23187, was employed as a means to load fresh human erythrocytes with calcium, and the capacity for accumulation was characterized. Erythrocytes exposed to A23187 in calcium-containing media rapidly accumulated calcium in millimolar quantities. The final cellular concentration was dependent upon medium calcium concentration and the size of the cellular organophosphate pool. When ATP and 2,3-diphosphoglycerate contents were depleted or repleted, the cellular calcium content changed proportionally. Calcium loading of fresh erythrocytes produced no discernible change in the cellular concentrations of ATP or 2,3-diphosphoglycerate. Calcium accumulation was also accompanied by loss of cellular potassium and H₂O, deterioration of cell filterability, and spheroechinocytic transformation.