Activating effect of adenosine on rat erythrocyte glycolysis.

1. Adenosine increases the adenine nucleotide pool in rat erythrocytes. Hence, we tested the effect of the nucleoside on the glycolytic pathway in red blood cells. 2. A 2.5-fold increase in the level of fructose-1,6-bisphosphate and a 34% augmentation in lactate pool were observed in rat erythrocytes, 30 min after adenosine treatment. 3. Under conditions preventing adenosine metabolism, 1 microM nucleoside addition to isolated erythrocytes induced an 89% increase in lactate production and an increase in glucose consumption. 4. Activation of red cell phosphofructokinase (PFK) is produced by addition of microM concentrations of adenosine. Our data suggest a role for adenosine in the glycolysis flux regulation through PFK activation.     

A futile cycle in erythrocyte glycolysis

The storage lesion which limits the shelf life of human blood in blood banking is associated with a metabolic loss of 2,3-diphosphoglycerate and ATP. This metabolic loss is driven by intracellular ATPases which are usually considered to include the ion pumps and the reactions which maintain the discoid shape of the human erythrocyte. Under the acidic conditions of blood storage, the energy-yielding reactions of the glycolytic pathway are restricted at the hexokinase and phosphofructokinase steps. We show here that under such circumstances the enzyme of the diphosphoglycerate shunt, diphosphoglycerate mutase/phosphatase and the glycolytic enzyme phosphoglycerate kinase can form a futile cycle with ATPase activity. This ATPase activity responds to 2-phosphoglycolate which is known to activate both diphosphoglycerate mutase and diphosphoglycerate phosphatase reactions. When the enzymes of the futile cycle are combined with the enzymes of the lower glycolytic pathway in a reconstitution experiment designed to represent conditions within the stored erythrocyte, the futile cycle does provide an ATPase activity which results in the metabolic loss of 2,3-diphosphoglycerate. An isotope incorporation experiment demonstrates that the futile cycle is active in glucose-depleted erythrocytes.     

Calorimetric study on human erythrocyte glycolysis. Heat production in various metabolic conditions.

The heat production of human erythrocytes was measured on a flow microcalorimeter with simultaneous analyses of lactate and other metabolites. The heat production connected with the lactate formation was about 17 kcal (71 kJ) per mol lactate formed which corresponded to the sum of heat production due to the formation of lactate from glucose and the heat production due to neutralization. The heat production rate increased as the pH of the suspension increased, corresponding to the increase in lactate formation. Glycolytic inhibitors such as fluoride and monoiodoacetate caused a decrease in the rate of heat production, whereas arsenate induced a large transient increase in heat production associated with a transient increase in lactate formation. Decrease in pyruvate concentration was usually associated with increase in heat production, although the decreased pyruvate concentration was coupled with formation of 2,3-bisphosphoglycerate. When inosine, dihydroxyacetone or D-glyceraldehyde was used as a substrate, an increase in the heat production rate was observed. Addition of methylene blue caused an oxygen uptake which was accompanied by a remarkable increase in heat production rate corresponding to about 160 kcal (670 kJ) per mol oxygen consumed. The value for heat production in red cells in the above-mentioned metabolic conditions was considered in relation to earlier known data on free energy and enthalpy changes of the different metabolic steps in the glycolytic pathway.     

A comparative analysis of kinetic models of erythrocyte glycolysis

Since the 1970s, with Heinrich as a pioneer in the field, numerous kinetic models of erythrocyte glycolysis have been constructed. A functional comparison of eight of these models indicates that the production of ATP and GSH in the red blood cell is largely controlled by the demand reactions. The rate characteristics for the supply and demand blocks indicate a good homeostatic control of ATP and GSH concentrations at different work loads for the pathway, while the production rates of ATP and GSH can be adjusted as needed by the demand reactions.     

Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis.

Previous studies demonstrated that the in vitro tyrosine phosphorylation of the human erythrocyte anion transporter, band 3, prevented the binding of various glycolytic enzymes to the N terminus of the cytoplasmic tail. Since these enzymes are inhibited in their bound state, the functional consequences of band 3 tyrosine phosphorylation in the red cell should be to activate the enzymes and elevate glycolysis. We searched for various enhancers of band 3 tyrosine phosphorylation using a novel assay designed to measure the phosphotyrosine levels at the band 3 tyrosine phosphorylation/glycolytic enzyme-binding site. This assay measures the extent of phosphorylation of a synthetic band 3 peptide entrapped within resealed red cells. Using this assay, three distinct compounds, all mild oxidants, were found to stimulate the tyrosine phosphorylation of band 3. All three compounds were also found to elevate glycolytic rates in intact erythrocytes. Moreover, the antitumor drug adriamycin was found to coordinately prevent these agents from stimulating both band 3 tyrosine phosphorylation and erythrocyte glycolysis. These results suggest a possible function for a protein tyrosine kinase in human erythrocytes, to regulate glycolysis through the tyrosine phosphorylation of band 3.     

Comparative activation by AMP and cyclic-AMP of rat erythrocyte and reticulocyte glycolysis.

Incubation of raty erythrocytes and reticulocytes in Tris-Ringer's medium with 5 mM cyclic-AMP or AMP increased lactate formation and glucose utilization. The glycolysis-stimulating effect of cyclic-AMP is very similar to that of AMP and, in both cases, it seems to be higher in reticulocytes than in erythrocytes. 0.5 mM norepinephrine produced a much higher lactate formation in reticulocytes than in erythrocytes, suggesting a greater adenylate cyclase activity in younger cells. 300 micrometer cyclic-AMP and AMP reverse inhibition produced by ATP (up to 1.5 micrometer) on phosphofructokinase from rat reticulocyte haemolysates. Both nucleotides are positive allosteric effectors of the enzyme as shown by displacement of F6P-saturation curve to hyperbolic kinetics. Similar results were previously obtained with rat erythrocytes. This deinhibitory effect is suggested to be responsible of the above glycolysis-stimulating effect.     

A modelling study of feedforward activation in human erythrocyte glycolysis

Though feedforward activation (FA) is a little known principle of control in metabolic networks, there is one well-known example; namely, the activation of pyruvate kinase (PK) by fructose-1,6-biphosphate (FBP) in glycolysis. The effects of this activation on the enzyme's kinetics are well characterised, but its possible role in glycolytic control has not been determined, and, experimentally, there is as yet no direct way of modifying the enzyme to remove just the FBP activation without affecting other aspects of the enzyme's kinetics. Given this limitation, we used a detailed numerical simulation of human erythrocyte glycolysis to simulate the effects of selective removal of the activation of PK by FBP on steady-state metabolite concentrations and on the dynamic response of glycolytic flux to a sudden increase of the cell's demand for ATP. Our modelling results predict that in the absence of FA steady-state levels of metabolites within the activation loop, i.e. from FBP to phosphoenolpyruvate, would be four- to thirteen-fold higher than normal, whereas levels of ATP and metabolites outside the loop, i.e. glucose-6-phosphate, fructose-6-phosphate and pyruvate, would be lower than normal. Existing clinical evidence in a patient with haemolytic anaemia, correlated with a lack of activation of PK by FBP (Paglia D.E., Valentine W.N., Holbrook C.T., Brockway R., Blood (1983) 62 972-979), is consistent with this prediction. In response to changing demand for ATP, the model predicts that the corresponding change of glycolytic flux would entail changes of metabolite concentrations in the absence of FA, but that in its presence the levels of metabolites within the activation loop remain essentially unperturbed. Thus, our results suggest that by stabilising metabolite pools in the face of variable glycolytic flux, FA may serve to avoid perturbations of the oxygen affinity of haemoglobin (sensitive to the levels of 2,3-phosphoglycerate) and of cell osmolality that would otherwise occur during variations in the cell's demand for ATP. In addition, by significantly raising the steady-state setpoint of intermediate metabolite pools, the productivity index (ratio of glycolytic flux to total metabolites in the pathway) of glycolysis would fall almost four-fold in the absence of forward activation.