Enzymes Of Embden-Meyerhof Pathway Research Articles

Sickle hemoglobin disturbs normal coupling between erythrocyte O2 content, glycolysis and antioxidant capacity

Energy metabolism in red blood cells (RBCs) is characterized by oxygen (O2)‐responsive variation in flux through the Embden Meyerhof Pathway (EMP) or the Hexose Monophosphate Pathway (HMP). As such, generation of ATP, NADH, and 2,3‐DPG (EMP) or NADPH (HMP) shifts with RBC O2 content, due to competition between deoxy‐hemoglobin (Hb) and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxy RBCs favors HMP flux and NADPH generation (maximizing glutathione (GSH)‐based antioxidant systems). We hypothesized that sickle hemoglobin (HbS) disrupts cdB3‐based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia (SCA), we demonstrate constrained NADPH and GSH recycling and reduced resilience to oxidative stress. We further illustrate abnormal association of HbS to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. These findings are confirmed by EMP/HMP 1H NMR glucose flux analysis in addition to RBC immunofluorescent imaging during O2 loading/unloading. Moreover, selective inhibition of inappropriately dispersed GAPDH rescues antioxidant capacity. Such disturbance of cdB3‐based linkage between O2 gradients and RBC metabolism suggests a novel mechanism by which hypoxia may influence SCA phenotype.

Sickle hemoglobin disturbs normal coupling among erythrocyte O2 content, glycolysis, and antioxidant capacity

AbstractEnergy metabolism in RBCs is characterized by O2-responsive variations in flux through the Embden Meyerhof pathway (EMP) or the hexose monophosphate pathway (HMP). Therefore, the generation of ATP, NADH, and 2,3-DPG (EMP) or NADPH (HMP) shift with RBC O2 content because of competition between deoxyhemoglobin and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxygenated RBCs favors HMP flux and NADPH generation (maximizing glutathione-based antioxidant systems). We tested the hypothesis that sickle hemoglobin disrupts cdB3-based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia, we demonstrate in the present study constrained HMP flux, NADPH, and glutathione recycling and reduced resilience to oxidative stress manifested by membrane protein oxidation and membrane fragility. Using a novel, inverted membrane-on-bead model, we illustrate abnormal (O2-dependent) association of sickle hemoglobin to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. This finding was confirmed by immunofluorescent imaging during RBC O2 loading/unloading. Moreover, selective inhibition of inappropriately dispersed GAPDH rescues antioxidant capacity. Such disturbance of cdB3-based linkage between O2 gradients and RBC metabolism suggests a novel mechanism by which hypoxia may influence the sickle cell anemia phenotype.

Enzyme co-localization in the pea leaf cytosol: 3-P-glycerate kinase, glyceraldehyde-3-P dehydrogenase, triose-P isomerase and aldolase

Nearest neighbor analysis of immunocytolocalization experiments indicates that the Embden–Meyerhof pathway enzymes aldolase, triose-P isomerase, glyceraldehyde-3-P dehydrogenase and P-glycerate kinase are located close to one another in the pea leaf cytosol. Direct transfer of the triose phosphates between aldolase, glyceraldehyde-3-P dehydrogenase and triose-P isomerase, direct transfer of glyceraldehyde-3-P between glyceraldehyde-3-P dehydrogenase and triose-P isomerase, and direct transfer of 1,3-P 2-glycerate between glyceraldehyde-3-P dehydrogenase and P-glycerate kinase is then a possibility in the plant cytosol. In contrast, there is no indication that fructose bisphosphatase is co-localized with aldolase, and it unlikely that fructose bisphosphate is channeled from aldolase to fructose bisphosphatase in this system.

Regulation of Glycolytic Flux in Ischemic Preconditioning: A STUDY EMPLOYING METABOLIC CONTROL ANALYSIS

Exact adjustment of the Embden-Meyerhof pathway (EMP) is an important issue in ischemic preconditioning (IP) because an attenuated ischemic lactate accumulation contributes to myocardial protection. However, precise mechanisms of glycolytic flux and its regulation in IP remain to be elucidated. In open chest pigs, IP was achieved by two cycles of 10-min coronary artery occlusion and 30-min reperfusion prior to a 45-min index ischemia and 120-min reperfusion. Myocardial contents in glycolytic intermediates were assessed by high performance liquid chromatographic analysis of serial myocardial biopsies under control conditions and IP. Detailed time courses of metabolite contents allow an in-depth description of EMP regulation during index ischemia using metabolic control analysis. IP reduced myocardial infarct size (control, 90.0 +/- 3.1 versus 5.05 +/- 2.1%; p < 0.001) and attenuated myocardial lactate accumulation (end-ischemic contents, 31.9 +/- 4.47 versus 10.3 +/- 1.26 micromol/wet weight, p < 0.0001), whereby a decrease in anaerobic glycolytic flux by at least 70% could constantly be observed throughout index ischemia. By calculation of flux:metabolite co-responses, the mechanisms of glycolytic regulation were investigated. The continuous deceleration of EMP flux in control myocadium could neither be explained on the basis of substrate availability nor be attributed to regulatory "key enzymes," as multisite regulation was employed for flux adjustment. In myocardium subjected to IP, an even pronounced deceleration of EMP flux during index ischemia was observed. Again, the adjustment of EMP flux was because of multisite modulation without any evidence for flux limitation by substrate availability or a key enzyme. However, IP changed the regulatory properties of most EMP enzymes, and some of these patterns could not be explained on the basis of substrate kinetics. Instead, other regulatory mechanisms, which have previously not yet been described for EMP enzymes, must be considered. These altered biochemical properties of the EMP enzymes have not yet been described.

Occurrence of Metacercariae (Trematoda: Gymnophallidae) on Amphitrite ornata (Annelida: Terebellidae)

gotes can catabolize glucose and proline. Thymidine and uridine are also taken up and incorporated into TCA precipitable products (Fig. 2). Thus no metabolic lesion is apparent as measured by the incorporation of glucose, proline, uridine and thymidine into TCA precipitable products. These data coupled with our earlier demonstration of all enzymes of Embden-Meyerhof pathway, tricarboxylic acid cycle and pentose shunt in the amastigote form (Meade et al., 1984, loc. cit.) show that the cytozoic stage of the parasite is qualitatively comparable to the culture form. Metabolic lesions or limitations may, however, be present in other pathways which could not be detected by the types of ex(I)

Heterogeneity of rabbit platelets: Effect of chronic blood loss on glycolytic and related enzyme activity

Rabbits were chronically bled, with Fe replacement, every 3–4 days for 21 days. Their platelet count and megathrombocyte number (large-heavy, young platelets) increased 1.7-and 2.3-fold, respectively. Eight of the 11 enzymes of the Embden-Meyerhof pathway increased 2–5-fold in activity per g wet weight during the period of blood letting. The five related enzymes (phosphoglucomutase, glucose 6- P dehydrogenase, 6- P gluconic dehydrogenase, glutathione reductase, and α-glycerol- P dehydrogenase) as well as the three Embden-Meyerhof pathway enzymes (aldolase, enolase, and pyruvate kinase) did not increase in activity over basal values. It is concluded that chronic blood loss with Fe replacement leads to a specific increase in enzyme activity of 8 of the 11 Embden-Meyerhof pathway enzymes.

Glycolysis in the adult dog heartworm, Dirofilaria immitis

1. 1. The activities of Embden-Meyerhof pathway enzymes and related enzymes of carbohydrate metabolism, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, hexokinase, glucosephosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, alcohol dehydrogenase and lactic dehydrogenase, were determined in the adult dog heartworm, Dirofilaria immitis. 2. 2. The presence of significant amounts of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicates the possibility of an operative pentose phosphate pathway.

The glycolytic pathway in adult liver fluke, Fasciola hepatica

1. 1. The activities of Embden-Meyerhof pathway enzymes and related enzymes of carbohydrate metabolism; hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, phosphoglucomutase, phosphoglucoisomerase, phosphomannoisomerase, phosphofructokinase, aldolase, triosephosphate isomerase, α-glycerophosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase and lactic dehydrogenase were determined in adult F. hepatica and rat liver. 2. 2. Glycolysis in F. hepatica proceeds only to the formation of phosphoenolpyruvate; pyruvate kinase activity could not be detected. 3. 3. Lactic dehydrogenase was much less active in fluke than in rat liver. 4. 4. Phosphofructokinase appears to be a rate-limiting step in that part of the Embden-Meyerhof pathway which is present. 5. 5. The possible fate of phosphoenolpyruvate in relation to the apparent absence of pyruvate kinase is discussed.

Glycolysis in young and mature normal human erythrocytes.

CONSIDERABLE data exist concerning the decline of metabolic activity which occurs during the normal ageing of human erythrocytes. In particular, several of the glycolytic enzymes are present in higher concentration in younger cells (that is, reticulocytes). This is true of phosphohexoisomerase1,2, triosephosphate dehydrogenase3, aldolase2, glucose-6-phosphate dehydrogenase and 6-phosphogluconic dehydrogenase1,4. However, in the assay methods used the maximum activity of these enzymes is measured at their optimum pH in dilute haemolysates containing added excess of substrate, coenzyme and activator; these are conditions which do not obtain in intact erythrocytes. Indeed, the results of Chapman et al.5 demonstrate that glucose consumption in normal intact erythrocytes proceeds at a rate less than that calculable from the concentrations of Embden–Meyerhof pathway enzymes that are present. It does not follow, therefore, that the presence of higher concentrations of glycolytic enzymes, as in reticulocytes, produces an increased rate of glycolysis. However, Bernstein2 showed that glucose consumption and lactate production in intact erythrocytes were, in fact, increased in proportion to the reticulocyte concentration of the samples.