Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum.

2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum delta H. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50 degrees C (below the optimum growth temperature of 62 degrees C). Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13CO2-pulse/12CO2-chase experiments at 50 degrees C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O2. The enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl2, and dithiothreitol. Both Km and Vmax have been determined at 37 degrees C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens.     

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     

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     

Cyclic 2,3-diphosphoglycerate metabolism in Methanobacterium thermoautotrophicum (strain ΔH): characterization of the synthetase reaction

The levels of cyclic 2,3-diphosphoglycerate (cDPG) in methanogenic bacteria are governed by the antagonistic activities of cDPG synthetase and cDPG hydrolase. In this paper we focus on the synthetase from Methanobacterium thermoautotrophicum. The cytoplasmic 150 kDa enzyme catalyzed cDPG synthesis from 2,3-diphosphoglycerate (apparent K_m=21 mM), Mg²⁺ (K_m=3.1 mM) and ATP (K_m=1–2 mM). In batch-fed cultures, the enzyme was constitutively present (6–6.5 nmol per min per mg protein) during the different growth phases. In continuous cultures, activity decreased in response to phosphate limitation. The synthetase reaction proceeded with maximal rate at pH 6 and at 65° C and was specifically dependent on high (>0.3M) K⁺ concentrations. The reaction conditions remarkably contrasted to those of cDPG degradation catalyzed by the previously described membrane-bound cDPG hydrolase.Abbreviations cDPG Cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-Diphosphoglycerate - 2-PG 2-Phosphoglycerate - 3-PG 3-Phosphoglycerate     

Malaria parasite-infected erythrocytes inhibit glucose utilization in uninfected red cells

The erythrocytic stages of the malaria parasite depend on anaerobic glycolysis for energy. Using [2-13C]glucose and nuclear magnetic resonance, the glucose utilization rate and 2,3-diphosphoglycerate (2,3-DPG) level produced in normal RBCs and Plasmodium falciparum infected red blood cell populations (IRBCs, with <4% parasite infected red cells), were measured. The glucose flux in IRBCs was several-folds greater, was proportional to parasitemia, and maximal at trophozoite stage. The 2,3-DPG levels were disproportionately lower in IRBCs, indicating a downregulation of 2,3-DPG flux in non-parasitized RBCs. This may be due to lowered pH leading to selective differential inhibition of the regulatory glycolytic enzyme phosphofructokinase. This downregulation of the glucose utilization rate in the majority (>96%) of uninfected RBCs in an IRBC population may have physiological implications in malaria patients.     

Diurnal changes of the 2,3-diphosphoglycerate concentration in human red cells and the influence of posture.

The 2,3-diphosphoglycerate (2,3-DPG) concentration, oxygen half saturation pressure at pH 7.4 (P50), pH in plasma and red cells, and mean corpuscular hemoglobin concentration (MCHC) of venous blood were determined during unrestricted daily activity (series I) throughout 24 hrs as well as during prolonged bed rest until noon (series II). In series I almost synchronous dirunal behavior of P50 2,3-DPG, and plasma pH as well as red cell pH became significantly apparent with highest values in the afternoon. The [2,3-DPG] yielded most pronounced alterations, which made up to 13.5% of the average day value. During prolonged recumbency the [2,3-DPG] showed a nonsignificant tendency to decline; the P50 remained unchanged throughout that period. The possible reason for the missing [2,3-DPG] increase is a reduced change of red cell pH in series II. An influence of a posture dependent aldosterone secretion either directly on the 2,3-DPG metabloism of indirectly via mediating the red cell pH and thus ruling the formation of this organic PHOSPHORIS COMPOUND IS DISCUSSED.     

Acetylation of human hemoglobin by methyl acetylphosphate. Evidence of broad regio-selectivity revealed by NMR studies.

The development of chemical modification agents that reduce the tendency of sickle hemoglobin (HbS) to aggregate represents an important chemotherapeutic goal. Methyl acetylphosphate (MAP) has been reported to bind to the 2,3-diphosphoglycerate (2,3-DPG) binding site of hemoglobin, where it selectively acetylates residues, resulting in increased solubility of HbS. We have prepared [1-(13)C]MAP and evaluated the adduct formation with hemoglobin using (1)H-(13)C HMQC and HSQC NMR studies. These spectra of the acetylated hemoglobin adducts showed 10-11 well resolved adduct peaks, indicating that the acetylation was not highly residue specific. The chemical shift pattern observed is in general similar to that obtained recently using [1'-(13)C]aspirin as the acetylating agent (Xu, A. S. L., Macdonald, J. M., Labotka, R. J., and London, R. E. (1999) Biochim. Biophys. Acta 1432, 333-349). Blocking the 2, 3-DPG binding site with inositol hexaphosphate (IHP) resulted in a selective reduction in intensity of adduct resonances, presumably corresponding to residues located in the 2,3-DPG binding cleft. The pattern of residue protection appeared to be identical to that observed in our previous study using IHP and labeled aspirin. Pre-acetylation of hemoglobin using unlabeled MAP, followed by acetylation with [1'-(13)C]aspirin indicated a general protective effect, with the greatest reduction of intensity for resonances corresponding to acetylated residues in the 2,3-DPG binding site. These studies indicated that both MAP and aspirin exhibit similar, although not identical, acetylation profiles and target primarily the betaLys-82 residue in the 2,3-DPG binding site, as well as sites such as betaLys-59 and alphaLys-90, which are not located in the beta-cleft of hemoglobin.     

Pathway of propionate oxidation by a syntrophic culture of Smithella propionica and Methanospirillum hungatei

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by (13)C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-(13)C]propionate was converted to [2-(13)C]acetate, with no [1-(13)C]acetate formed. Butyrate from [3-(13)C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-(13)C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-(13)C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-(13)C-labeled propionate yielded both [1-(13)C]acetate and [2-(13)C]acetate. When (13)C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, (13)C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.     

Biosynthesis of cyclic 2,3-diphosphoglycerate. Isolation and characterization of 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus

Starting from 2-phosphoglycerate the biosynthesis of cDPG comprises two steps: (i) the phosphorylation of 2-phosphoglycerate to 2,3-diphosphoglycerate and (ii) the intramolecular cyclization to cyclic 2,3-diphosphoglycerate. The involved enzymes, 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase, were purified form Methanothermus fervidus. Their molecular and catalytic properties were characterized.     

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.     

2,3-DPG-Hb complex: a hypothesis for an asymmetric binding

This study was undertaken to test the symmetry of 2,3-diphosphoglycerate (2,3-DPG) binding site in hemoglobin (Hb). From Arnone's study [A. Arnone, Nature (London) 237 (1972) 146] the 2,3-DPG binding site is located at the top of the cavity, that runs through the center of the deoxy-Hb molecule. However, it is possible that this symmetry reported by Arnone, for crystals of 2,3-DPG-Hb complex, might not be conserved in solution. In this paper, we report the 31P nuclear magnetic resonances of the 2,3-DPG interaction with Hb. The 2,3-DPG chemical shifts of the P2 and P3 resonance are both pH- and hemoglobin-dependent [protein from man, polar bear (Ursus maritimus), Arctic fox (Alopex lagopus) and bovine]. 2,3-DPG binds tightly to deoxyhemoglobin and weakly, nevertheless significantly, to oxyhemoglobin. In particular, our results suggest similar spatial position of the binding site of 2,3-DPG in both forms of Hb in solutions. However, the most unexpected result was the apparent loss of symmetry in the binding site, which might correlate with the ability of the hemoglobin to modulate its functional behavior. The different interactions of the phosphate groups indicate small differences in the quaternary structure of the different deoxy forms of hemoglobin. Given the above structural perturbation an asymmetric binding in the complex could justify, at least in part, different physiological properties of Hb. Regardless, functionally relevant effects of 2,3-DPG seem to be measured and best elucidated through solution studies.     

Glucose metabolism of adult Schistosoma japonicum as revealed by nuclear magnetic resonance spectroscopy with D-[13C6]glucose

Excretory end-products of adult Schistosoma japonicum, fed D-[13C6]glucose in vitro under aerobic and anaerobic conditions, were studied using 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy. The glucose in the medium is degraded to produce lactate and alanine aerobically and succinate and acetate as well as lactate and alanine anaerobically. Succinate and acetate have not been previously recorded as excretory products resulting from the metabolism of glucose for schistosomes. The presence of [13C3] and [2,3-13C2]lactate, and [1,2,2'-13C3] and [2,2'-13C2]succinate as end-products suggests that a partial reversed tricarboxylic acid (TCA) cycle is active in adult Schistosoma japonicum under anaerobic conditions. The physiological role of this pathway in adult schistosomes remains obscure.     

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.     

2,3-Cyclopyrophosphoglycerate in methanogens: evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism

The novel compound 2,3-cyclopyrophosphoglycerate (CPP) is the major small molecule carbon pool in Methanobacterium thermoautotrophicum. High-field 13C NMR 13CO2 pulse/unenriched CO2 chase experiments have shown that the labeled CPP rapidly loses its 13C to an insoluble pool, while the CPP steady-state concentration is maintained (as monitored by 31P NMR spectroscopy). The biosynthesis of CPP from CO2, acetyl coenzyme A, and pyruvate as precursors has been established by a 13C NMR study of ethanol extracts of Mb. thermoautotrophicum fed with 13CO2, [1-13C]- and [2-13C]acetate, and [1-13C]pyruvate. That CPP is a post-phosphoenolpyruvate metabolite has been confirmed by in vitro experiments with cell extracts. A role for CPP in carbohydrate metabolism was established when [1-13C]glucose fed to cells resulted in the formation of [3-13C]CPP exclusively. Possible functions of CPP within the cell are discussed.     

Effect of 2,3-diphosphoglycerate on the phosphorylation of protein 4.1 by protein kinase C.

We have previously shown that 2,3-diphosphoglycerate (2,3-DPG) inhibits the phosphorylation of erythrocyte membrane cytoskeletal proteins by endogenous casein kinases. Here, we report that 2,3-DPG stimulates the phosphorylation of protein 4.1 by protein kinase C. Studies with red cell membrane preparations showed that while the phosphorylation of most of the membrane proteins by endogenous membrane-bound kinases and purified kinase C was inhibited by 2,3-DPG, the phosphorylation of protein 4.1 was slightly enhanced by the metabolite. The effect of 2,3-DPG was further examined using purified protein 4.1 preparations. Our results indicate that 2,3-DPG stimulates both the rate and the extent of phosphorylation of purified protein 4.1 by kinase C. The amount of phosphate incorporated was found to double to 2 mol of phosphate per mole of protein 4.1 in the presence of 10 mM 2,3-DPG. The increase in phosphorylation was distributed over all phosphorylation sites as revealed by an analysis of the labeling patterns of phosphopeptides resolved by high performance liquid chromatography, but a significantly higher incorporation was detected in two of the phosphopeptides. The stimulatory effect of 2,3-DPG on the phosphorylation of protein 4.1 was observed only with kinase C. Phosphorylation by the cytosolic erythrocyte casein kinase and the cyclic AMP-dependent protein kinase was inhibited by 2,3-DPG. Moreover, the stimulatory effect of 2,3-DPG seemed to be unique to the phosphorylation of protein 4.1 since a similar effect had not been observed with other protein kinase C substrates. Our results suggest that 2,3-DPG may play an important role in the regulation of cytoskeletal interactions.     

Effect of long-term training and acute physical exercise on red cell 2,3-diphosphoglycerate

A statistically significant 10% increase (p less than 0.005) in mean red cell 2,3-diphosphoglycerate (2,3-DPG) concentration, concomitantly with a mean 16% increase (p less than 0.001) in the predicted maximal oxygen uptake (VO2max) was observed in 29 recruits, who were studied during 6 months of physical training in military service. The increase in 2,3-DPG was higher, the lower the initial 2,3-DPG and VO2max levels. The mean initial 2,3-DPG level was higher in the subjects with a higher initial VO2max. A strenuous but highly aerobic 21-km marching exercise elicited a mean 9% increase (p less than 0.005) in red cell 2,3-DPG concentration. A significantly greater response of 2,3-DPG to marching exercise was observed in subjects with a lower pre-test VO2max than in those with a higher pre-test VO2max. During another more competitive march 2,3-DPG remained almost unchanged and was associated with a tendency towards a negative correlation with the acccompanying lactate response (r = -0.60, p less than 0.05). Red cell 2,3-DPG response to a standardized exercise is considered to be a suitable indicator for evaluating the effect of training on an individual.     

N.m.r. studies of red cells

Recent n.m.r. studies of intact red cells are described. With 1H n.m.r. the normal high resolution spectra of red cells, even at high fields, are relatively uninformative because the very large number of resonances from the cells merge into a broad envelope. If a simple 90-tau-180 degree spin echo pulse sequence is used, however, many resonances can all be resolved. These include signals from haemoglobin histidines, glutathione, lactate and pyruvate. 13C and 31P signals have also been seen with a spectrometer converted to observe these nuclei essentially simultaneously. N.m.r. is well suited to monitor the time course of events after a perturbation of the cell system. Lactate increase, glutathione recovery after oxidation and alkylation of glutathione by iodoacetate can all be observed directly in red cell suspensions by means of 1H spin echo n.m.r. This method has also been used to measure isotope exchange (1H-2H) of lactate and of pyruvate at both the C-3 and the C-2 positions, and some of these exchange rates can be interpreted in terms of the activity of specific enzymes in the cells. 1H spin echo n.m.r. has also been used to obtain information about the transport rates of small molecules into cells. By means of the 13C/31P spectrometer and [13C-1] glucose, the 13C enrichment of 2,3-diphosphoglycerate (2,3-DPG) can be monitored at the same time as the levels of 2,3-DPG, ATP and inorganic phosphate are observed by 31P n.m.r.     

Glutamate racemization and catabolism in Fusobacterium varium

The pathways of glutamate catabolism in the anaerobic bacterium Fusobacterium varium, grown on complex, undefined medium and chemically defined, minimal medium, were investigated using specifically labelled (13)C-glutamate. The metabolic end-products acetate and butyrate were isolated from culture fluids and derivatized for analysis by nuclear magnetic resonance and mass spectrometry. On complex medium, labels from L-[1-(13)C]glutamate and L-[4-(13)C]glutamate were incorporated into C1 of acetate and equally into C1/C3 of butyrate, while label derived from L-[5-(13)C]glutamate was not incorporated. The isotopic incorporation results and the detection of glutamate mutase and 3-methylaspartate ammonia lyase in cell extracts are most consistent with the methylaspartate pathway, the best known route of glutamate catabolism in Clostridium species. When F. varium was grown on defined medium, label from L-[4-(13)C]glutamate was incorporated mainly into C4 of butyrate, demonstrating a major role for the hydroxyglutarate pathway. Upon addition of coenzyme B(12) or cobalt ion to the defined medium in replicate experiments, isotope was located equally at C1/C3 of butyrate in accord with the methylaspartate pathway. Racemization of D-glutamate and subsequent degradation of L-glutamate via the methylaspartate pathway are supported by incorporation of label into C2 of acetate and equally into C2/C4 of butyrate from D-[3-(13)C]glutamate and the detection of a cofactor-independent glutamate racemase in cell extracts. Together the results demonstrate a major role for the methylaspartate pathway of glutamate catabolism in F. varium and substantial participation of the hydroxyglutarate pathway when coenzyme B(12) is not available.     

Comparative effects of phosphoenolpyruvate on selected mammalian erythrocytes

1. Incubation of human, rat, cow, sheep, dog, rabbit and monkey erythrocytes with phosphoenolpyruvate (PEP) resulted in increased intracellular 2,3-diphosphoglycerate (2,3-DPG). 2. Physiologic temperature (37 degrees C) and a pH less than 6.5 were required for transport and metabolism of PEP in rat and monkey erythrocytes. 3. Although erythrocytes from all species (except pig) exhibited PEP transport and metabolism, hemoglobin oxygen affinity (HOA) was affected only in species whose hemoglobins are sensitive to 2,3-DPG. 4. These results suggest that the effect of PEP incubation on HOA is mediated through 2,3-DPG.     

Effects of training on erythrocyte 2,3-diphosphoglycerate in normal men

The erythrocyte 2,3-diphosphoglycerate concentration (2,3-DPG) and the activity of red cell hexokinase, pyruvate kinase, glucose-6 phosphate dehydrogenase and glutathione reductase were studied in 27 normal volunteers before and after 2 and 4 months of physical endurance training. The 4 months of training increased maximal oxygen uptake and physical working capacity (PWC130) by 16% (p less than 0.001) and 29% (p less than 0.001) respectively. Resting heart rate was decreased (p less than 0.001) by 11 beats.min-1. With 2 months of training the erythrocyte 2,3-DPG concentration increased by 9% (p less than 0.001); with 4 months training the increase was only 4% (p less than 0.05). The training-induced increase in red cell 2,3-DPG was not accompanied by enhanced activity of erythrocyte hexokinase, pyruvate kinase, glucose-6 phosphate dehydrogenase or glutathione reductase. It is concluded that the rise in red cell 2,3-DPG induced by physical endurance training is not due to activation of red cell glycolytic enzymes or the enzymes involved in the pentose-phosphate cycle.