The role of red cell membrane in the regulation of glycolysis and the 2,3-bisphosphoglycerate-cycle

Pyruvate and K-ferricyanide stimulation of net ATP and 2,3-bisphosphoglycerate synthesis is very probably due to enhancement of glyceraldehyde 3-phosphate dehydrogenase activity. Significant peculiarities in the K-ferricyanide effect and its depression by non-penetrating-SH inhibitors at low concentrations were noted and suggested that membrane-bound enzymes play a substantial part in the synthesis of ATP and 2,3-bisphosphoglycerate. Experiments with isolated ghosts showed their ATP-and 2,3-bis-phosphogylcerate-building capacity. Pulse-labeling with 32P-Pi and determination of specific radioactive in intracellular inorganic phosphate and ATP-gamma-P demonstrated that the ferricyanide-stimulated compartment utilizes only intracellular inorganic phosphate for ATP (and 2,3-bisphosphoglycerate) synthesis, and does so only when extracellular inorganic phosphate is present.     

Activation of human erythrocyte 2,3-bisphosphoglycerate phosphatase at physiological concentrations of substrate

In human erythrocytes the reactions of the 2,3-bisphosphoglycerate shunt are catalyzed primarily by one protein, 2,3-bisphosphoglycerate synthase-phosphatase. At low concentrations of 2,3-bisphosphoglycerate the phosphatase is activated by several anions including inorganic phosphate and sulfite, and the phosphate activation is inhibited by low concentrations of 3-phosphoglycerate [Z. B. Rose and J. Liebowitz (1970) J. Biol. Chem. 245, 3232-3241]. Phosphate and sulfite also activate at high but physiological concentrations of 2,3-bisphosphoglycerate (5 mM), but the inhibition by 3-phosphoglycerate is much weaker. The basal activity (without added phosphate or sulfite) was also found to be higher and to be 3-phosphoglycerate sensitive; this is attributed to activation either by 2,3-bisphosphoglycerate itself or by a contaminant in it. These results allow previous observations of 2,3-bisphosphoglycerate hydrolysis in intact erythrocytes to be reconciled with the properties of the purified enzyme under near-physiological conditions.     

Inosine/pyruvate/phosphate medium but not adenosine/pyruvate/phosphate medium introduces millimolar amounts of 5-phosphoribosyl 1-pyrophosphate in human erythrocytes. A 31P-n.m.r. study.

Incubation of human erythrocytes in medium containing inosine (10 mM), pyruvate (10 mM), phosphate (50 mM) and NaCl (75 mM) at pH 6.6 leads to a more than 1000-fold increase in the concentration of 5-phosphoribosyl 1-pyrophosphate (PRPP), as identified and quantified by 31P-n.m.r. spectroscopy. The accumulation is highly pH-dependent, with a maximum at extracellular pH 6.60, and the maximum value of 1.3-1.6 mmol/l of erythrocytes is attained within 1 h at 37 degrees C. PRPP was accumulated despite high concentrations of 2,3-bisphosphoglycerate (2,3-BPG), an inhibitor of PRPP synthetase. The concentration of PRPP correlated with the intracellular concentration of inorganic phosphate (Pi). Substitution of either adenosine or adenosine plus inosine for inosine in the medium did not lead to 31P-n.m.r.-detectable accumulation of PRPP. These results show that neither 2,3-BPG nor PRPP itself inhibits the synthesis of PRPP in the human erythrocyte. Adenosine, however, prevents the inosine-stimulated accumulation of PRPP.     

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate levels in erythrocytes with high and low 2,3-bisphosphoglycerate content during postnatal development

In rabbit and sheep erythrocytes the concentrations of 2,3-bisphosphoglycerate, fructose 2,6-bisphosphate and glucose 1,6-bisphosphate suffer important changes after birth, which differ in both species. The changes of fructose 2,6-bisphosphate and glucose 1,6-bisphosphate correlate with the changes in the levels of the enzymatic activities involved in their synthesis. The change of 2,3-bisphosphoglycerate levels in rabbit but not in sheep erythrocytes could be explained by the changes of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios.     

Roles of hemoglobin Allostery in hypoxia-induced metabolic alterations in erythrocytes: simulation and its verification by metabolome analysis

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [(13)C]glucose to [(13)C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O(2)-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O(2) from the cells.     

Interrelationship between energy metabolism from various substrates and the 2,3-bisphosphoglycerate bypass in human erythrocytes

Metabolism of the substrates D-ribose, xylitol, D-Xylulose, D-fructose, D-glucose and mixtures of these compounds were studied in human erythrocytes. The metabolic rates obtained with the various substrates affected the intracellular levels of ATP and 2,3-bisphosphoglycerate. Small amounts of substrate utilization resulted in a decrease of the ATP and more pronounced of the 2,3-bisphosphoglycerate concentration while carbon utilization rates beyound 14 microgram atom C/ml packed cells/120 min yielded constant levels of ATP and 2,3-bisphosphoglycerate. From these results it can be concluded that a carbon utilization rate of 14 microgram atom C/ml cells/120 min is able to cover the ATP requirement of the red cells under steady state conditions. Based on the carbon utilization rates obtained with the various substrates and the rates of 2,3-bisphosphoglycerate decomposition an attempt is made to calculate the contribution of the 2,3-bisphosphoglycerate bypass to substrate metabolism. In case of xylitol as substrate the decrease in the 2,3-bisphosphoglycerate content provides the regeneration of NAD thus facilitating uptake and metabolism of xylitol.     

Regulation of protein synthesis in rabbit reticulocyte lysates by 2,3-bisphosphoglycerate.

2,3-Bisphosphoglycerate was the most potent effector of glycolytic intermediates tested for their effects on protein synthesis in gel-filtered lysates from rabbit reticulocytes. 2,3-Bisphosphoglycerate at low levels was stimulatory but became inhibitory at high levels. Both effects were dependent on Mg²⁺ concentrations. The higher the concentration of Mg²⁺, the higher the concentration of 2,3-bisphosphoglycerate required for maximal activation. 2,3-Bisphosphoglycerate concentrations required to exhibit an inhibitory effect increased as Mg²⁺ concentration increased. Both effects of 2,3-bisphosphoglycerate are discussed in terms of regulation of hemoglobin synthesis during maturation of erythroid cells.     

Carbon and hydrogen metabolism of xylitol and various sugars in human erythrocytes.

1) Erythrocytes are able to metabolize D-ribose, D-xylitol, D-xylulose, D-fructose and D-glucose; the rates of metabolism increase in that order from 2430 to 26200 ng atom C/ml packed cells per 120 min of incubation. 2) The utilization of the carbon of these substrates and its recovery in the products were found to be in balance, when the change in the 2,3-bisphosphoglycerate concentration was taken into account. 3) The metabolic rates strongly affected the 2,3-bisphosphoglycerate level. Without addition of substrate the decomposition rate of this intermediate was found to be 1030 nmol/ml packed cells per 120 min. 4) The net decrease of the 2,3-bisphosphoglycerate concentration and the conversion of this compound into lactate provides a NAD regeneration system which enables the red blood cells to utilize xylitol. 5) The rate of carbon metabolism via the pentose phosphate cycle is determined by the NADPH requirement of the erythrocytes which was found to be 160 nmol/ml packed cells per 120 min under the experimental conditions employed.     

Effect of 2,3-diphosphoglycerate concentration on the alkaline fragility of phosphofructokinase-deficient canine erythrocytes

1. Erythrocytes in whole blood samples from dogs with phosphofructokinase (PFK) deficiency had lower 2,3-diphosphoglycerate (2,3-DPG) concentrations, higher ATP concentrations, and were more alkaline fragile than normal canine erythrocytes. 2. Reticulocytes from a PFK-deficient dog contained nearly three times the ATP concentration of normal canine erythrocytes, and had 2,3-DPG concentrations similar to normal canine erythrocytes. 3. PFK-deficient reticulocytes are not alkaline fragile. 4. The erythrocyte 2,3-DPG concentration in whole blood samples from PFK-deficient dogs was increased to normal by in vitro incubation with dihydroxyacetone, pyruvate and phosphate. This incubation resulted in only a slight increase in ATP concentration. 5. The alkaline fragility of these 2,3-DPG replenished PFK-deficient erythrocytes was normal. 6. Findings in this study indicate that the increased alkaline fragility of canine PFK-deficient erythrocytes is the result of decreased intracellular 2,3-DPG concentration.     

2,3-Bisphosphoglycerate,fructose, 2,6-bisphosphate and glucose 1,6-bisphosphate during maturation of reticulocytes with low 2,3-bisphosphoglycerate content

In contrast to the species with erythrocytes of high 2,3-bisphosphoglycerate content, in the sheep the concentration of 2,3-bisphosphoglycerate decreases during maturation of reticulocytes. The decrease can be explained by the drop of the phosphofructokinase/pyruvate kinase and 2,3-bisphosphoglycerate synthase/2,3-bisphosphoglycerate phosphatase activity ratios that result from the decline of phosphofructokinase, pyruvate kinase, phosphoglycerate mutase and the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase. The concentrations of fructose 2,6-bisphosphate and aldohexose 1,6-bisphosphates also decrease during sheep reticulocyte maturation in parallel to the 6-phosphofructo 2-kinase and the glucose 1,6-bisphosphate synthase activities.     

Interactions of Na+ with haemoglobin-organic phosphate complexes

Longitudinal and transverse relaxation times were measured in aqueous solutions containing haemoglobin and 2,3-bisphosphoglycerate and in dilute lysates of human erythrocytes. Analysis of the data in terms of calculated excess relaxation rates shows that Na+ interacts with the protein-organic phosphate complex. The comparable magnitude of the effect in the model system and in dilute lysate suggests that intracellular Na+ binds to the haemoglobin-bisphosphate complex. Results obtained with adenosine triphosphate and D-glucose indicate that there is also interaction between Na+ and haemoglobin complexes of these molecules.     

Bisphosphoglycerate synthase activity in cells of the erythrocytic line of rat

The specific activity of bisphosphoglycerate synthase (EC 2.7.5.4) is significantly higher in rat mature erythrocytes than in reticulocytes from phenylhydrazine treated animals, while 2,3-bisphosphoglycerate levels do not differ between them. The enzyme specific activity and the concentration of 2,3-bisphosphoglycerate are both negligible in the erythroid cell pool from the bone marrow of anaemic animals. Thus, a cellular specialization in the last stages of erythropoiesis is confirmed. Human bisphosphoglycerate synthase shows a lower specific activity than that of rat without a parallel decrease in 2,3-bisphosphoglycerate levels.     

Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.     

Phosphorus-31 nuclear magnetic resonance studies on intact erythrocytes. Determination of intracellular pH and time course changes in phosphorus metabolites

A method to determine the intracellular pH of intact erythrocytes using phosphorus-31 nuclear magnetic resonance spectroscopy is described. Changes in phosphorus metabolites due to the alkalization of intracellular pH were also examined. The normal erythrocytes gave signals of phosphate groups corresponding to 2,3-bisphosphoglycerate, inorganic phosphate, ATP, and NAD. Among them, the separation between alpha and gamma peaks of ATP was shown to be a good indicator of the intracellular pH free from the perturbation caused by hemoglobin. This method enabled us to determine the intracellular pH of the erythrocytes without any pretreatment. The separation between alpha and gamma peaks of ATP was also dependent on the degree of complexation with Mg2+, and was consistent with approximately 80% of total ATP complexing with Mg2+ in the samples investigated here. The pKa value of ATP in the erythrocytes was estimated to be 6.1 at 23 degrees C, which is lower than the value of 6.5 obtained for the Mg2+-free ATP solution. In the alkalized erythrocytes, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were observed in addition to the metabolites found in the normal erythrocytes. Time course changes in these phosphorus metabolites were followed along with the intracellular pH monitored from ATP peaks.     

Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis.

Culture of hamster embryos with 0.35 mM inorganic phosphate results in developmental arrest at the 2-cell stage. These arrested 2-cell embryos were found to have significantly elevated levels of both intracellular pH and intracellular free calcium. Culture of 2-cell embryos with both glucose and phosphate did not further alter intracellular ionic homeostasis. Developmental arrest of 2-cell embryos was dependent on the concentration of phosphate used. Culture with 1.25 microM phosphate did not alter development, while concentrations of 2.5 microM and 5.0 microM resulted in a percentage of embryos arresting development at the 2-cell stage. Analysis of intracellular levels of pH and calcium after culture with different phosphate concentrations revealed a significant negative correlation between intracellular calcium levels and development beyond the 2-cell stage. There was no correlation between the increase in intracellular pH and embryo development in the presence of phosphate. The increase in intracellular calcium levels after culture with phosphate appears to be derived from intracellular pools, as preventing the influx of extracellular calcium did not alter development beyond the 2-cell stage. Therefore, it is apparent that a disruption in ionic homeostasis is associated with developmental arrest of hamster embryos cultured with phosphate.     

Metabolism of glycerate 2,3-P2--XII. Characterization of the 2,3-bisphosphoglycerate synthase-phosphatase and of the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase from pig brain

2,3-Bisphosphoglycerate synthase-phosphatase and the hybrid phosphoglycerate mutase/2,3-bisphosphoglycerate synthase-phosphatase have been partially purified from pig brain. Their 2,3-bisphosphoglycerate synthase, 2,3-bisphosphoglycerate phosphatase and phosphoglycerate mutase activities are concurrently lost upon heating and treatment with reagents specific for histidyl, arginyl and lysyl residues. The two enzymes differ in their thermal stability and sensitivity to tetrathionate. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. The synthase activity of both enzymes shows a nonhyperbolic pattern which fits to a second degree polynomial. The Km, Ki and optimum pH values are similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase from erythrocytes and the hybrid enzyme from skeletal muscle. The synthase activity is inhibited by inorganic phosphate and it is stimulated by glycolyate 2-P.     

Red cells of newborn rats have low bisphosphoglyceromutase and high pyruvate kinase activities in association with low 2,3-bisphosphoglycerate

2,3-Bisphosphoglycerate is a physiologically important regulator of red cell oxygen affinity during mammalian development. The rat has no fetal hemoglobin, but the newborn red cell has low 2,3-bisphosphoglycerate and high ATP concentrations, and high oxygen affinity. This report shows that red cell bisphosphoglyceromutase activity increases from near zero in the newborn rat to very high levels by four weeks of age. This increase roughly parallels the increase in red cell 2,3-bisphosphoglycerate concentration. Red cell pyruvate kinase activity declines ten-fold from birth to four weeks of age. This decrease is associated with a changeover in red cell populations from larger to smaller cells. The glycolytic rate is at least 50% higher in newborn than adult rat red cells. The data suggest that high pyruvate kinase activity and glycolytic rate contribute to the high ATP concentration in newborn rat red cells, but that their low 2,3-bisphosphoglycerate concentration is due primarily to low bisphosphoglyceromutase activity.     

The basis of echinocytosis of the erythrocyte by glucose depletion

Echinocytosis of erythrocytes by glucose depletion is attributed to adenosine triphosphate depletion, but its process still remains unknown. A mechanism of control of the erythrocyte shape has been previously proposed in which the anion exchanger Band 3, linked to flexible membrane skeleton, has a pivotal role. Recruitments of its inward facing (Band 3(i) ) and outward facing (Band 3(o) ) conformations contract and relax the membrane skeleton, thus promoting echinocytosis and stomatocytosis, respectively. The Band 3(o) /Band 3(i) equilibrium ratio increases with the increase of the Donnan equilibrium ratio, and preferential inward and outward transport by Band 3 of substrates slowly transported are echinocytogenic and stomatocytogenic, respectively. The mechanism suggests the following process. The major organic phosphate 2,3-bisphosphoglycerate is catabolized to lactate to form inorganic phosphate, 3-phosphoglycerate, and adenosine triphosphate. The last two products can be reversibly transformed into 1,3-bisphosphoglycerate and adenosine diphosphate by the glycolytic enzyme phosphoglycerate kinase, thus allowing 2,3-bisphosphoglycerate formation by 2,3-bisphosphoglycerate synthase/phosphatase. The catabolic and cyclic processes initially oppose echinocytosis by increasing the Donnan ratio and outward transport of slowly transported inorganic phosphate by Band 3 (its basic form is transported with a hydrogen ion). Echinocytosis occurs when inward transport of this product becomes predominant. This process can rationalize direct and indirect observations.     

Factors affecting the rate of purine ribonucleotide dephosphorylation in human erythrocytes.

Purine ribonucleotide dephosphorylation was measured in intact human erythrocytes in vitro to evaluate those factors which might regulate this process in vivo. It was found that purine nucleotides which exist predominantly in the triphosphate form (e.g. ATP and GTP) are protected from dephosphorylation while those nucleotides normally present as the monophosphate (e.g. IMP) are susceptible to dephosphorylation. This point was emphasised by studying an individual whose erythrocytes accumulated ITP rather than IMP; erythrocytes from this individual has a more stable pool of inosine phosphates than did erythrocytes from normal individuals. The concentration of intracellular phosphate was also shown to affect the rate of dephosphorylation. The dephosphorylation of IMP was inhibited at intracellular phosphate concentrations above approx. 3 mM. AMP dephosphorylation (in cells whose AMP concentration was increased by incubating them in the presence of 2-deoxyglucose) was inhibited by phosphate more strongly than was found for IMP. In contrast, the dephosphorylation of GMP did not appear to be affected by phosphate concentration. High oxygen tension was a powerful stimulator of IMP dephosphorylation while low oxygen tension protected IMP from dephosphorylation. This finding shows that human erythrocytes are similar to those of other mammals in this regard and points to a possible physiological determinant of purine turnover in these cells.     

The regulatory principles of glycolysis in erythrocytes in vivo and in vitro. A minimal comprehensive model describing steady states, quasi-steady states and time-dependent processes.

A simple mathematical model for glycolysis in erythrocytes is presented which takes into account ATP synthesis and consumption. The system is described by four ordinary differential equations. Conditions in vivo are described by a stable steady state. The model predicts correctly the metabolite concentrations found in vivo. The parameters involved are in agreement with data on the separate steps. The metabolite changes found in pyruvate kinase-deficient erythrocytes and the species variations among erythrocytes from different animals are described satisfactorily. The roles of the enzymes in the control of metabolites and glycolytic flux are expressed in the form of a control matrix and control strengths [R. Heinrich & T.A. Rapoport (1974) Eur. J. Biochem. 42, 89-95] respectively. Erythrocytes from various species are shown to be adapted to a maximal ATP-consumption rate. The calculated eigenvalues reveal the pronounced time-hierarchy of the glycolytic reactions. Owing to the slowness of the 2,3-bisphospho-glycerate phosphatase reaction, quasi-steady states occur during the time-interval of about 0.5-2h incubation, which are defined by perturbed 2,3-bisphosphoglycerate concentrations. The theoretical predictions agree with experimental data. In the quasi-steady state the flux control is exerted almost entirely by the hexokinase-phosphofructokinase system. The model describes satisfactorily the time-dependent changes after addition of glucose to starved erythrocytes. The theoretical consequences are discussed of the conditions in vitro with lactate accumulation and the existence of a time-independent conservation quantity for the oxidized metabolites. Even in this closed system quasi-steady states occur which are characterized by approximately constant concentrations of all glycolytic metabolites except for the accumulation of lactate, fructose 1,6-bisphosphate and triose phosphate.