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.     

Effect of some glycolytic intermediates and palmitoyl-CoA on alpha-glycerophosphate dehydrogenase in mitochondria isolated from liver of triiodothyronine-treated rats

alpha-Glycerophosphate dehydrogenase (EC 1.1.99.5) in mitochondria from liver of the triiodothyronine-treated rats is competitively inhibited by phosphoenolpyruvate, glyceraldehyde 3-phosphate and 3-phosphoglycerate, the apparent Ki values for phosphoenolpyruvate being 0.76 mM at pH 7.0, 1.7 mM at pH 7.4 and 3.5 mM at pH 7.7. The apparent Ki values for glyceraldehyde 3-phosphate and 3-phosphoglycerate are also pH-dependent. Other glycolytic intermediates, such as 2-phosphoglycerate, 2,3-diphosphoglycerate, pyruvate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate did not alter significantly alpha-glycerophosphate dehydrogenase activity. Palmitoyl-CoA is a competitive inhibitor of this enzyme, with Ki value of about 30 micron.     

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.     

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.     

The contents of adenine nucleotides and glycolytic and tricarboxylic acid cycle intermediates in activated and non-activated plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea)

The steady state content of adenine nucleotides, inorganic phosphate and glycolytic and tricarboxylic acid cycle intermediates were measured in freeze clamped plerocereoids of S. solidus taken from the body cavity of infected fish (non-activated plerocercoids) and the results compared with the metabolite levels in plerocercoids which had been cultured in vitro for 12 h (activated plerocercoids). Of the glycolytic intermediates, the levels of fructose-6-phosphate, fructose-1, 6-diphosphate, 3-phosphoglycerate, phosphoenolpyruvate and lactate all decreased on activation, the remainder of the glycolytic intermediates did not alter significantly. In contrast, the levels of the tricarboxylic acid cycle intermediates all increased 2–3 fold on activation, whilst the adenine nucleotides remained virtually unchanged. The differences in the steady state content in intermediary metabolites in activated and non-activated plerocereoids are discussed in relation to possible mechanisms of metabolic control.     

Alterations in the energy metabolism of the isolated perfused frog heart during calcium depletion and subsequent repletion

Summary The changes in myocardial energy metabolism of isolated perfused Rana ridibunda hearts subjected to prolonged calcium depletion and reperfusion with calcium-containing medium were studied. Calcium-free perfusion resulted in an increase in the concentrations of glucose, glucose-6-phosphate, a-ketoglutarate and malate. The myocardial contents of high-energy phosphates were maintained while concentrations of key amino acids were significantly altered. During the reperfusion period the tissue high-energy phosphate content fell abruptly. A marked increase in glycolytic flux and lactate production was observed. The tissue contents of citric acid cycle intermediates and key amino acids decreased. Examination of the activities of marker enzymes during the calcium-free and reperfusion periods showed that only cytoplasmic enzymes are lost during reperfusion, while the activities of other enzymes remained unchanged. The results suggest that the fluxes of both glycolysis and the citric acid cycle are significantly altered during calcium depletion and following repletion in the amphibian heart. The major characteristics of calcium paradox-induced damage in Rana ridibunda heart are the depletion of high-energy stores, the impairment of mitochondrial oxidative metabolism, and a significant increase in anaerobic metabolism.Abbreviations ADP Adenosine diphosphate - AMP Adenosine monophosphate - ATP Adenosine triphosphate - EDTA Ethylene-diamino-tetraacetic acid - NAD ⁺ Nicotinamide-adeninedinucleotide - NADH Nicotinamide-adenine-dinucleotide (reduced form) - TRA Triethanolamine     

ATP-ADP-dependent phosphorylations of glycolysis metabolites, creatine and glycerol: their compartition and thermodynamic relationship in gastrocnemius muscle cell of exercised guinea pigs

The concentrations of following metabolites were determined in freeze-clamped gastrocnemius muscle samples: glucose 1-phosphate, glucose 6-phosphate, glucose, fructose 1,6-diphosphate, fructose 6-phosphate, D-glyceraldehyde 3-phosphate. dihydroxyacetone phosphate, phosphoenolpyruvate, pyruvate, glycerol 3-phosphate, glycerol, creatine phosphate, creatine, glycerate 3-phosphate, glycerate 2-phosphate, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, inorganic phosphate. The results showed that within the limits of experimental error, concentration homeostasis for this metabolites is founded at least in some cases on equilibria between enzymic transformations. Discrepancies between constant mass ratios measured in this study and equilibrium constants allow the free energy variation delta G to keep creatine phosphate at high concentration to be calculated. For the phosphoglycerate mutase system, the equilibrium constant in controls and trained animals is unchanged and corresponds to that in vitro. Training hindered glycolysis and favoured phosphorylation of creatine by glycerol 3-phosphate. Metabolites of the pyruvate kinase and hexokinase system cannot be homogeneously distributed in one space. The creatine kinase system is also separated from the hexokinase und pyruvate kinase system. A compartition of glycolytic process in gastrocnemius muscle seems to be inferred from these results.     

1H and 51V NMR studies of the interaction of vanadate and 2-vanadio-3-phosphoglycerate with phosphoglycerate mutase.

The formation of complexes of vanadate with 2-phosphoglycerate and 3-phosphoglycerate have been studied using 51V nuclear magnetic resonance spectroscopy. Signals attributed to two 2,3-diphosphoglycerate analogues, 2-vanadio-3-phosphoglycerate and 2-phospho-3-vanadioglycerate, were detected but were not fully resolved from signals of inorganic vanadate and the anhydride formed between vanadate and the phosphate ester moieties of the individual phosphoglycerates. Equilibrium constants for formation of the two 2,3-bisphosphate analogues were estimated as 2.5 M-1 for 2-vanadio-3-phosphoglycerate and 0.2 M-1 for 2-phospho-3-vanadioglycerate. The results of the binding study are fully consistent with non-cooperativity in the binding of vanadiophosphoglycerate to the two active sites of phosphoglycerate mutase (PGM). 2-Vanadio-3-phosphoglycerate was found to bind to the dephospho form of phosphoglycerate mutase with a dissociation constant of about 1 x 10(-11) M at pH 7 and 7 x 10(-11) M at pH 8. Three signals attributed to histidine residues were observed in the 1H NMR spectrum of phosphoglycerate mutase. Two of these signals and also an additional signal, tentatively attributed to a tryptophan, underwent a chemical shift change when the vanadiophosphoglycerate complex was bound to the enzyme. The results obtained here are in accord with these vanadate-phosphoglycerate complexes being much more potent inhibitors of phosphoglycerate mutase than either monomeric or dimeric vanadate. The dissociation constant of 10(-11) M for 2-vanadio-3-phosphoglycerate is about 4 orders of magnitude smaller than the Km for PGM, a result in accordance with the vanadiophosphoglycerates being transition state analogues for the phosphorylation of PGM by 2,3-diphosphoglycerate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurements of glycolytic intermediates during the onset of thermogenesis in the spadix of Arum maculatum

1. This work was done to compare the amounts of glycolytic intermediates in the club of the spadix of Arum maculatum L. at an early stage (α) of development, immediately prior to the increase in glycolysis (pre-thermogenesis), and at the peak of the rapid glycolysis (thermogenesis).2. Glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate and pyruvate were measured. The results indicate that at all the above stages of club development the reactions catalysed by phosphoglucomutase, glucosephosphate isomerase, phosphoglycerate mutase and enolase were close to equilibrium, but those catalysed by phosphofructo-kinase and pyruvate kinase were considerably displaced from equilibrium.3. The amounts of the above compounds per club increased 5-fold between α stage and pre-thermogenesis but the relative amounts remained unchanged. When glycolysis increased by more than 50-fold at thermogenesis, the amount of fructose 1,6-diphosphate per club rose, but no changes were detected in the amounts per club of any of the other compounds listed above. These results are discussed in relation to the control of glycolysis.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

The role of the triose-phosphate shuttle and glycolytic intermediates in fatty-acid and glycerolipid biosynthesis in pea root plastids

The capacity of the triose-phosphate shuttle and various combinations of glycolytic intermediates to substitute for the ATP requirement for fatty-acid and glycerolipid biosynthesis in pea (Pisum sativum L.) root plastids was assessed. In all cases, ATP gave the greatest rates of fatty-acid and glycerolipid biosynthesis. Rates of up to 66 and 27 nmol·(mg protein)^–1·h^–1 were observed for the incorporation of acetate and glycerol-3-phosphate into lipids in the presence of ATP. In the absence of exogenously supplied ATP, the triose-phosphate shuttle gave up to 44 and 33% of the ATP-control activity in promoting fatty-acid and glycerolipid biosynthesis from acetate and glycerol-3-phosphate, respectively. The optimum shuttle components were 2 mM dihydroxyacetonephosphate (DHAP), 2 mM oxaloacetic acid and 4 mM inorganic phosphate (referred to as the DHAP shuttle). Glyceraldehyde-3-phosphate, as a shuttle triose, was approximately 82% as effective as DHAP in promoting fatty-acid synthesis while 2-phosphoglycerate, 3-phosphoglycerate, and phosphoenolpyruvate were only 27–37% as effective as DHAP. When glycolytic intermediates were used as energy sources for fatty-acid synthesis, in the absence of both exogenously supplied ATP and the triose-phosphate shuttle, phosphoenolpyruvate, 2-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate each gave 48%, 17%, 23% and 17%, respectively, of the ATP-control activity. Other triose phosphates tested were much less effective in promoting fatty-acid synthesis. When exogenously supplied ATP was supplemented with the DHAP shuttle or glycolytic intermediates, the complete shuttle increased fatty-acid biosynthesis by 37% while DHAP alone resulted in 24% stimulation. Glucose-6-phosphate, fructose-6-phosphate and glycerol-3-phosphate similarly all improved the rates of fatty-acid synthesis by 20–30%. In contrast, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate all inhibited fatty-acid synthesis by approximately 10% each. The addition of the DHAP shuttle and glycolytic intermediates with or without exogenously supplied ATP caused an increase in the proportion of radioactive oleate and a decrease in the proportion of radioactive palmitate synthesized. The use of these alternative energy sources resulted in higher amounts of free fatty acids and triacylglycerol, and lower amounts of diacylglycerol and phosphatidic acid. The data presented here indicate that ATP is superior in promoting in-vitro fatty-acid biosynthesis in pea root plastids; however, both the triose-phosphate shuttle and glycolytic metabolism can produce some of the ATP required for fatty-acid biosynthesis in these plastids.Abbreviations DHAP dihydroxyacetonephosphate - Fru6P fructose-6-phosphate - G3P glycerol-3-phosphate - Glc6P glucose-6-phosphate - OAA oxaloacetate - PEP phosphoenolpyruvate - 2PGA 2-phosphoglycerate - 3PGA 3-phosphoglycerate - 3PGalde glyceraldehyde-3-phosphate This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada.     

ATP synthesis from 2,3-diphosphoglycerate by cell-free extract of Methanobacterium thermoautotrophicum (strain ΔH)

Cell-free extracts of Methanobacterium thermoautotrophicum were found to catalyze ATP synthesis from an endogeneous substrate. Synthesis was stimulated under hydrogen atmosphere and inhibited by KCL (K _i =150 mM). Comparison of the properties of a number of cell constituents showed the endogeneous substrate to be 2,3-diphosphoglycerate. The compound is converted into 3-phosphoglycerate, and via 2-phosphoglycerate and phosphoenolpyruvate into pyruvate, at which the latter reaction is linked with ATP synthesis.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme m, 2-(methylthio)ethanesulfonate - HS-HTP 7-mercaptoheptanoyl-l-threonine phosphate - CoM-SS-HTP the heterodisulfide of HS-CoM and HS-HTP - BCFE bolled cell-free extract - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid - PEP phosphoenolpyruvate - 2,3-DPG 2,3-diphosphoglycerate - cDPG cyclic 2,3-diphosphoglycerate - 3-PG 3-phosphoglycerate - 2-PG 2-phosphoglycerate     

Ouabain-sensitive interaction between human red cell membrane and glycolytic enzyme complex in cytosol

Binding of 2,3-diphosphoglycerate to monophosphoglycerate mutase, of which it is an obligatory cofactor, causes changes in the resonance positions of the 31P nuclear magnetic resonance spectra of both phosphate groups. It has previously been shown that these resonances shift when other glycolytic enzymes, such as phosphoglycerate kinase, are added to form the 2,3-diphosphoglycerate . monophosphoglycerate mutase . phosphoglycerate kinase complex. In view of this association, we have examined the set of glycolytic enzymes from aldolase to pyruvate kinase and found evidence of direct communication between all of these enzymes. A multi-enzyme complex of 1--2 . 10(6) daltons has been separated from broken cell ghosts by Biogel column filtration and evidence has been presented to show that this complex exhibits aldolase, glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase activity. The glycolytic multi-enzyme complex interacts with the outer face of inside-out vesicles prepared from human red cells and the interaction is suppressed by application of 10(-6) M ouabain to the inner face of these vesicles. These studies show that the conformation of the enzymes comprising the megadalton complex are responsive to the application of ouabain to the outer red cell membrane surface.     

Human erythrocyte 2,3-diphosphoglycerate metabolism. Influence of 1,3-diphosphoglycerate and Pi. In vitro studies at low pH with computer simulations.

The kinetics of 2,3-diphosphoglycerate (2,3-DPG) net breakdown was examined in intact human erythrocytes incubated at pH 7.00 and 37 °C. The concentrations of 2,3-DPG, 1,3-diphosphoglycerate (1,3-DPG), 3-phosphoglycerate, ATP, P_i, glucose, and lactate were determined during 10 to 12 h. Since the concentration of 1,3-DPG has been suggested to be the main regulating factor with respect to the rate of 2,3-DPG net breakdown the interdependence between the concentration of 1,3-DPG and pH was determined in the range of pH 6.9 to 7.4. It was found that the stationary level of 1,3-DPG decreased strongly with decreasing pH within this range. Qualitatively, the net breakdown of 2,3-DPG observed at pH 7.00 can be explained by the lowered level of 1,3-DPG. The influence of the concentration of P_i upon the rate of net degradation of 2,3-DPG at pH 7.00 was studied at low cell volume fraction (0.04), where given concentrations of P_i could be maintained for several hours. A marked increase in the rate of 2,3-DPG net breakdown by P_i was demonstrated. Computer simulations showed that activation of diphosphoglycerate phosphatase by the increasing concentration of P_i and decrease of degree of inhibition of the diphosphoglycerate mutase by the decreasing concentration of 2,3-DPG may well keep the rate of the degradation balanced at the time constant value observed. On the basis of the observed kinetics and a computer simulation, the flux through the phosphoglycerate bypass was estimated to be 10 to 15% of the total glycolytic flux at physiological conditions.     

Methanobacterium thermoautotrophicum (strain ΔH) contains a membrane-bound cyclic 2,3-diphosphoglycerate hydrolase

Cyclic 2,3-diphosphoglycerate (cDPG) hydrolase activity was demonstrated in cofactor-free extract of Methanobacterium thermoautotrophicum (strain H), but not in crude extract. Only after ultrafiltration or dialysis of crude extract cDPG hydrolase activity could be shown. cCPG hydrolysis was optimal at pH 6.0 and 60°C. Hydrolysis of cDPG occurred under nitrogen or hydrogen atmosphere and was completely inhibited by oxygen. Phosphate and potassium chloride were also strong inhibitors: 50% inhibition occurred at 0.6–0.7 mM phosphate or 0.2 M KCl. The enzyme was localized in the membrane fraction and could be solubilized for approximately 60% by treatment with 25 mM of the detergent CHAPS. The K _m and the V _max for cDPG were determined at 60°C and were 59 mM and 216 mU/mg, respectively. Furthermore, cDPG hydrolase was dependent on the presence of Co²⁺. The role of cDPG and cDPG hydrolase is discussed.Abbreviations cDPG cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-diphosphoglycerate - 2-PG 2-phosphoglycerate - 3-PG 3-phosphoglycerate - PG phosphoglycerate - PEP phosphoenolpyruvate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - TRIS tris(hydroxymethyl)-aminomethane - DTT dithiothreitol - CHAPS 3-([3-cholamidopropyl]-dimethylammonio)-1-propanesulfonate - MOPS 3-(N-morpholino) propanesulfonic acid     

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.     

Membrane mediated link between ion transport and metabolism in human red cells

When 10^?6 M oubain is added to human red cells that have been incubated without glucose for two hours, there is a significant shift in the ³¹P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na⁺ + K⁺)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na⁺ + K⁺)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10^?6 M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na⁺ + K⁺)-ATPase and that the interaction is inhibited by 10^?6 M ouabain.     

Vanadium(IV)-stimulated hydrolysis of 2,3-diphosphoglycerate

Vanadium(IV) stimulates the hydrolysis of 2,3-diphosphoglycerate at 23 degrees C. The pH optimum is 5.0. Reactions were analyzed by enzymatic and phosphate release assays. The products of 2,3-diphosphoglycerate hydrolysis are inorganic phosphate and 3-phosphoglycerate. The reaction is inhibited by high concentrations of 2,3-diphosphoglycerate and an equation has been formulated that describes the kinetic constants for this reaction at pH 7. The possible relevance of the reaction to the therapeutic lowering by vanadium(IV) of red cell 2,3-diphosphoglycerate in sickle-cell disease is discussed.     

Inhibition of glycolytic enzymes by 2-phosphotartronate.

2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.