Metabolism of 3-phosphoglyceroyl phosphate in phosphoenolpyruvate-enriched human erythrocytes

The concentration of 3-phosphoglyceroyl phosphate in erythrocytes was increased by more than 100-fold when red cells were incubated with extracellular phosphoenolpyruvate at 37 degrees C. Since these elevated levels were maintained for 60 min, the metabolism of 3-phosphoglyceroyl phosphate and related compounds could be investigated in phosphoenolpyruvate-treated erythrocytes. 2,3-Bisphosphoglycerate synthesis was not affected by intracellular pH when the 3-phosphoglyceroyl phosphate level was constant but did vary with 3-phosphoglyceroyl phosphate concentration. On the other hand, the relationship between the rate of 2,3-bisphosphoglycerate synthesis and 3-phosphoglyceroyl phosphate concentration was not straightforward. At relatively low concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis agreed with a rate calculated from a formula incorporating kinetic parameters of purified 2,3-bisphosphoglycerate synthase (Rose, Z.B. (1973) Arch. Biochem. Biophys. 158, 903-910). However, at high concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis was lower than the calculated value. The concentration of glucose 1,6-bisphosphate did not increase even when 3-phosphoglyceroyl phosphate was elevated to 200 microM. Elevated levels of intracellular 2,3-bisphosphoglycerate did not inhibit glycolytic activity in these erythrocytes. These results suggest that incubation of erythrocytes with phosphoenolpyruvate is a useful technique to investigate the effect of metabolic perturbations at the intermediate stages of glycolysis.     

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.     

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.     

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.     

2,3-Bisphosphoglycerate inhibits ATP-stimulated proteolysis

Intracellular protein breakdown could be regulated at the substrate level by changes in the environment. Under in vitro conditions, ATP increases the proteolytic susceptibility of several mitochondrial and cytosolic proteins, while 2,3-bisphosphoglycerate not only has the opposite effect but also prevents the ATP-stimulated proteolysis. ATP and 2,3-bisphosphoglycerate, present at relatively high levels in many tissues, provide a good model of environmental components that may influence intracellular proteolysis.     

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.     

Magnetic resonance studies of the interaction of divalent metal cations with 2,3-bisphosphoglycerate

The binding of Mg²⁺ to intracellular 2,3-bisphosphoglycerate in the human red blood cell is significant to the function of the cell. We have studied interactions of Mg²⁺ and Mn²⁺ with 2,3-bisphosphoglycerate by magnetic resonance spectroscopy. The results of this study reveal the presence of two independent divalent metal cation binding sites of similar affinity (K_D = 3.0 ± 0.5 mM) in the 2,3-bisphosphoglycerate molecule, one on each phosphoryl group, contrary to the assumption of one metal ion binding site made in the previous literature. Over the range of their intracellular concentrations, ATP and ADP, however, possess only one metal ion site in spite of the presence of multiple phosphoryl groups. These results are consistent with the chemistry of metal-chelation which requires the formation of 5- or 6-membered rings for the stability of chelate structures.     

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.     

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 breakdown of adenine nucleotides in glucose-depleted human red cells.

1) The rate of 2,3-bisphosphoglycerate breakdown is independent of pH value. 2) The adenine nucleotide pattern at alkaline pH values with its characteristic lowering of ATP and the accompanying accumulation of fructose-1,6-bisphosphate is caused by a relative excess of the activity of the hexokinase-phosphofructokinase system as compared wity pyruvate kinase. 3) The breakdown of adenine nucleotides proceeds via AMP mainly through phosphatase and not via AMP deaminase. 4) The constancy of the sum of nucleotides as long as glucose is present is postulated to be due to resynthesis via adenosine kinase which competes successfully with adenosine deaminase. 5) A procedure is given to calculate ATPase activity of glucose-depleted red cells. The results indicate that the ATPase activity is less at lower pH values and declines with time. An ATPase with a high Km for ATP is postulated. 6) During glucose depletion ATP production is mostly derived from the breakdown of 2,3-bisphosphoglycerate and the supply from the pentose phosphate pool both of which proceed at a constant rate. The contribution of pentose phosphate from the breakdown of adenine nucleotides amounts to 40% of the lactate formed at pH 6.8 and is about twice the lactate at pH 8.1.     

Hemoglobins, XXVIII. Phosphate-protein-interaction, gene expression and function: the genetic and allosteric control of the oxygen affinity of the fetal blood (author's transl)

This work describes possible molecular mechanisms concerning the control of oxygen affinity in fetal blood of mammalia. There is a genetic control of oxygen affinity through a fetal gene: at constant phosphate concentration (Hb less than P2-glycerate) in humans there is a hemoglobin with only five binding sites to 2,3-bisphosphoglycerate, resulting in an increased oxygen affinity. In several species (sheep, cattle, goat) with Met-Leu as the N-terminal group of the beta-chains, the 2,3-bisphosphoglycerate binding sites are deleted in positions beta 1 and beta 2, so that the regulation is phosphate-independent and thus providing a fetal hemoglobin with an increased oxygen affinity. The allosteric control is observed in pigs. In the postembryonal development "adult" hemoglobin with seven contacts (beta-chains) is demonstrated. The increased oxygen affinity is achieved here by a reduced biosynthesis of 2,3-bisphosphoglycerate (Hb greater than P2-glycerate) (Rapoport-Luebering-cycle). The functional control is discussed with respect to the ontogeny of the hemoglobins.     

Role of cytoplasmic ATP in the restoration and maintenance of a membrane permeability barrier in transformed mammalian cells

Addition of ATP to medium surrounding intact, transformed 3T3 cells activates the formation of aqueous channels in the plasma membrane. This results in efflux of nucleotide pools and ions and entry into the cytosol of charged, phosphorylated species. In such permeabilized cells, glycolysis is totally dependent on the external addition of glucose, inorganic phosphate, ADP, K+, Mg2+ and NAD+ which restore lactic acid formation to levels found in untreated cells. As expected, such reconstitution of glycolytic activity is found to restore intracellular ATP levels. This is accompanied by sealing of the membrane channels so that efflux of nucleotide pools ceases. Pyruvate, a substrate for mitochondrial ATP synthesis, when provided along with ADP and inorganic phosphate also produces sealing of the membrane channels. On the other hand, reactivation of pentose phosphate shunt activity, which does not lead to ATP synthesis, does not induce restoration of the membrane permeability barrier. Furthermore, compounds which lower the internal ATP pool prevent sealing, and also render the plasma membrane more sensitive to external ATP (Rozengurt and Heppel, '79). Sealing of aqueous channels following restoration of the internal ATP pool is associated with phosphorylation of the inner membrane surface, and is unaffected by inhibitors of protein synthesis, microfilament or microtubular assembly. These results indicate the probable role of intracellular ATP in the restoration and/or maintenance of an active membrane barrier against efflux of small molecules and ions in transformed 3T3 cells.     

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.     

A procedure for decreasing the level of 2,3-bisphosphoglycerate in red cells in vitro

The physiological adaptation to anemia and other hypoxic states includes an increase in the level of 2,3-bisphosphoglycerate (2,3-DPG) in the red cell. We suggest that the high level of 2,3-DPG may have adverse effects in vivo. It has been found that red cells incubated with glycolate lose 2,3-DPG at a rapid rate relative to controls. ATP is stable. Net 2,3-DPG synthesis is observed after the glycolate is removed from the cells suggesting that they are not harmed. The effect appears to be specific for glycolate since lactate, glyoxylate, glycerate, acetate, and citrate were without effect. This procedure could be used to assess the effects of decreasing the 2,3-DPG level to normal in the erythrocytes of sickle cell and other anemias.     

Functional homology of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, phosphoglycerate mutase, and 2,3-bisphosphoglycerate mutase

The bisphosphatase domain of the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase has been shown to exhibit a structural similarity to yeast phosphoglycerate mutase and human red blood cell 2,3-bisphosphoglycerate mutase including very similar active site sequences with a histidyl residue being involved in phospho group transfer. The liver bifunctional enzyme was found to catalyze the hydrolysis of glycerate 1,3-bisphosphate to glycerate 3-phosphate and inorganic phosphate. The Km for glycerate 1,3-bisphosphate was 320 microM and the Vmax was 11.5 milliunits/mg. Incubation of the rat liver enzyme with [1-32P]glycerate 1,3-bisphosphate resulted in the formation of a phosphoenzyme intermediate, and the labeled amino acid was identified as 3-phosphohistidine. Tryptic and endoproteinase Lys-C peptide maps of the 32P-phosphoenzyme labeled either with [2-32P]fructose 2,6-bisphosphate or [1-32P]glycerate 1,3-bisphosphate revealed that 32P-radioactivity was found in the same peptide, proving that the same histidyl group accepts phosphate from both substrates. Fructose 2,6-bisphosphate inhibited competitively the formation of phosphoenzyme from [1-32P]glycerate 1,3-bisphosphate. Effectors of fructose-2,6-bisphosphatase also inhibited phosphoenzyme formation. Substrates and products of phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase also modulated the activities of the bifunctional enzyme. These results demonstrate that, in addition to a structural homology, the bisphosphatase domain of the bifunctional enzyme has a functional similarity to phosphoglycerate mutase and 2,3-bisphosphoglycerate mutase and support the concept of an evolutionary relationship between the three enzyme activities.     

Factors that regulate the activity of the phosphatidylinositol kinase present in oocyte membranes of Xenopus laevis

1. Phosphatidylinositol kinase present in the membranes of Xenopus laevis oocytes was characterized. 2. The enzyme requires Mg2+ or Mn2+ at 10 mM and exogenous phosphatidylinositol (50 microM) increases the formation of phosphatidylinositol-4-phosphate. 3. The oocyte phosphatidylinositol kinase cannot use GTP as a phosphate donor but this compound inhibits competitively the utilization of ATP. 4. Addition of phosphatidylserine and phosphatidylinositol-4,5-bisphosphate stimulates the phosphorylation of phosphatidylinositol but 2,3-bisphosphoglycerate at 5 mM concentration is a strong inhibitor of the reaction.     

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.     

Fatty acid synthesis by isolated leucoplasts from developingBrassica seeds: Role of nucleoside triphosphates and DHAP-shuttle as the source of energy

Fatty acid synthesis in leucoplasts isolated from developing seeds ofBrassica campestris was absolutely dependent on external source of ATP. None of the other nucleoside triphosphates could replace ATP in the reaction mixture. Use of ADP alone also resulted in reduced rates of fatty acid synthesis. However, in combination with inorganic phosphate or inorganic pyrophosphate, it improved the rate of fatty acid synthesis, giving up to 50% of the ATP-control activity. Inorganic phosphate or inorganic pyrophosphate alone again did not serve as an energy source for fatty acid synthesis. AMP, alongwith inorganic pyrophosphate could promote fatty acid synthesis to up to 42% of the activity obtained with ATP. The three components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate of dihydroxy acetone phosphate-shuttle together could restore 50% of the activity obtained with ATP. Omission of any one of the components of this shuttle drastically reduced the rate of fatty acid synthesis to 15–24% of the ATP-control activity. Inclusion of ATP in reaction mixtures containing shuttle components enhanced the rate of synthesis over control. The optimum ratio of shuttle components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate determined was 1:1:2. Maximum rates of fatty acid synthesis were obtained when dihydroxy acetate phosphate was used as the shuttle triose. Glyceraldehyde-3-P, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate as shuttle trioses were around 35–60% as effective as dihydroxy acetone phosphate in promoting fatty acid synthesis. The results presented here indicate that although the isolated leucoplasts readily utilize exogenously supplied ATP for fatty acid synthesis, intraplastidic ATP could also arise from dihydroxy acetone phosphate shuttle components or other appropriate metabolites     

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.     

Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.