Role of hexokinase in the regulation of glucose metabolism in human erythrocytes

Red blood cell glucose metabolism was studied in erythrocytes from a patient with trisomy 10 p which resulted in + 50% hexokinase specific activity, in normal controls and in cases of heterozygous hexokinase deficiency. The results obtained show that the hexokinase activity level is an important factor in the control of the erythrocyte's glycolytic rate while having no appreciable effect on the hexose monophosphate pathway under resting conditions. No clear conclusion could be drawn when an oxidative stress was present.     

Relationship between the rate of erythrocyte hexose monophosphate pathway and the glucose 6-phosphate concentration

Erythrocytes of individuals with increased (+ 50%) or reduced (-35%) hexokinase activity contain respectively 70 and 17 nmole/ml RBC of glucose-6-phosphate (normal concentration 30 +/- 5nmole/ml RBC) and show comparable rates of the HMP (60 +/- 5nmole/hr/ml RBC). Similarly, in RBC of different ages, obtained by density gradient ultracentrifugation, the glucose-6-phosphate concentration range from 57 (young cells) to 18 (old cells) nmole/ml RBC but the rate at which glucose is utilized in the HMP is unchanged. These data exclude a regulatory role of glucose 6-phosphate in the HMP even if its concentration is under that required for maximal G6PD activity.     

Regulation of the human-erythrocyte hexose-monophosphate shunt under conditions of oxidative stress. A study using NMR spectroscopy, a kinetic isotope effect, a reconstituted system and computer simulation

The regulation of the hexose monophosphate shunt of human erythrocytes under conditions of oxidative stress has been investigated by monitoring the reduction of oxidised glutathione (GSSG) to reduced glutathione (GSH) in erythrocytes containing high levels of GSSG; 1H NMR and a biochemical assay were used to measure the changes. A reconstituted metabolic system prepared with the purified erythrocyte enzymes was used in conjunction with studies of intact cells and haemolysates to determine the dependence of the rate of GSH production on the activities of hexokinase and glucose-6-phosphate dehydrogenase. Both of these enzymes have previously been claimed to be the rate-limiting step of oxidatively stimulated flux through the hexose monophosphate shunt. The absence of a kinetic isotope effect on the rate of GSH production in these systems, when [1-2H]glucose replaced glucose as the source of reducing equivalents, showed that glucose-6-phosphate dehydrogenase activity was not a strong determinant of the flux. The dependence of the rate of GSH production on the concentration of the hexokinase inhibitors glucose 1,6-bisphosphate and glycerate 2,3-bisphosphate showed that, under conditions of oxidative stress, hexokinase was the principal determinant of flux through the shunt. Glucose 1,6-bisphosphate at the concentration present in vivo appears to be more important in limiting hexokinase activity, and thus the rate of glucose utilisation, than was previously assumed. A detailed computer model of the system was developed based on the reported kinetic parameters of the enzymes involved. A sensitivity analysis of this model predicted that the hexokinase reaction would have a sensitivity coefficient of 0.995 with respect to the maximal rate of GSH production.     

Increased glucose metabolism by enzyme-loaded erythrocytes in vitro and in vivo normalization of hyperglycemia in diabetic mice.

The main metabolic properties of human red blood cells (RBC) overloaded with glucose catabolizing enzymes such as hexokinase and glucose oxidase were evaluated. Human erythrocytes loaded with human hexokinase metabolized 3.1 +/- 0.2 mumol/h/ml RBC of glucose, an amount double that consumed by normal and unloaded cells (1.46 +/- 0.16 mumol/h/ml RBC), while glucose oxidase-loaded erythrocytes consumed up to 5.5 +/- 0.5 mumol/h/ml RBC of glucose but with a time-dependent increase in methemoglobin formation due to the H2O2 produced in the glucose oxidase reaction. This methemoglobin production was greatly reduced while glucose consumption was increased (8.1 +/- 0.4 mumol/h/ml RBC) by coentrapment of hexokinase and glucose oxidase. Similar results were obtained in mouse red blood cells, although the role of hexokinase was less pronounced due to a higher basal level of this enzyme. When administered to diabetic mice the hexokinase/glucose oxidase-overloaded erythrocytes had a circulating half-life of 5 days and were able to regulate blood glucose at near physiological levels. A single intraperitoneal administration of 500 microliters of enzyme-loaded cells maintained a near-normal blood glucose concentration for 7 +/- 1 days, while repeated administrations at 10-day intervals were effective in the regulation of blood glucose levels for several weeks. These results suggest that enzyme-loaded erythrocytes can behave as circulating bioreactors and can provide a new way to reduce abnormally elevated blood glucose.     

Pig red blood cell hexokinase: Regulatory characteristics and possible physiological role

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with n_H = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mm). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (K_i = 120 μm). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 μmol of glucose/h/ml red blood cells (RBC) compared with values of 0.56–2.85 μmol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mm against a plasma level of 5 mm). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation.     

Malarial parasite hexokinase and hexokinase-dependent glutathione reduction in the Plasmodium falciparum-infected human erythrocyte

The metabolism of glucose in Plasmodium falciparum-infected human erythrocytes is increased 50- to 100-fold. This is accomplished in part by parasite-directed synthesis of a protozoan hexokinase with unique kinetic, electrophoretic, and heat stability properties. The total hexokinase activity is increased approximately 25-fold over that of control uninfected erythrocytes of the same age from the same donor. The parasite hexokinase has a lower affinity for glucose than the mammalian enzyme (Km = 431 microM +/- 21 S.D. for the parasite enzyme versus 98 microM +/- 10 for the erythrocyte enzyme), but the Km for ATP and the Vmax for both glucose and ATP are similar. The NADPH-dependent reduction of oxidized glutathione (GSSG) requires the formation of glucose 6-phosphate which in turn is metabolized by the pentose shunt pathway in which NADPH is generated. Using glucose as the substrate, lysates of P. falciparum-infected normal erythrocytes demonstrated enhanced ability to reduce GSSG. The rate of GSSG reduction was proportional both to the parasitemia and the hexokinase activity of the lysates. However, infected glucose-6-phosphate dehydrogenase-deficient red cell lysates displayed a severely restricted ability to reduce GSSG under the same conditions. In conclusion, P. falciparum-infected red cells contain a parasite-encoded hexokinase with unique properties which initiates the large increase in glucose consumption. In normal infected red cells, reduction of GSSG is also dependent upon hexokinase activity, but in infected glucose-6-phosphate dehydrogenase-deficient red cells, the absence of this pentose shunt enzyme remains the rate-limiting step in GSSG reduction.     

Interrelations between glycolysis and the hexose monophosphate shunt in erythrocytes as studied on the basis of a mathematical model

A mathematical model is presented which comprises the reactions of glycolysis, the hexose monophosphate shunt (HMS) and the glutathione system in erythrocytes. The model is used to calculate stationary and time-dependent metabolic states of the cell in vitro and in vivo. The model properly accounts for the following metabolic features observed in vitro: (a) stimulation of the oxidative pentose pathway after addition of pyruvate due to a NADP-dependent lactate dehydrogenase as coupling enzyme between glycolysis and the oxidative pentose pathway, (b) relative share of the oxidative pentose pathway in the total consumption of glucose amounting to approximately 10% in the normal case and to approximately 90% under conditions of oxidative stress excreted by methylene blue. From the application of the model to in vivo conditions it is predicted that (c) under normal conditions glycolysis and the HMS are independently regulated by the energetic and oxidative load, respectively, (d) under conditions of enhanced energetic or oxidative load both glycolysis and the HMS are mainly controlled by the hexokinase; in this situation the highest possible values of the energetic and oxidative load which are compatible with cell integrity are strongly coupled and considerably restricted in comparison with the normal case, (e) the stationary states possess bifurcation points at high and low values of the energetic load.     

Amelioration of glucose induced hemolysis of human erythrocytes by vitamin E

Cells under aerobic condition are always threatened with the insult of reactive oxygen species, which are efficiently taken care of by the highly powerful antioxidant systems of the cell. The erythrocytes (RBCs) are constantly exposed to oxygen and oxidative stress but their metabolic activity is capable of reversing the injury under normal conditions. In vitro hemolysis of RBCs induced by 5, 10 and 20 mM glucose was used as a model to study the free radical induced damage of biological membranes in hyperglycemic conditions and the protection rendered by vitamin E on the same. RBCs are susceptible to oxidative damage, peroxidation of the membrane lipids, release of hemoglobin (hemolysis) and alteration in activity of antioxidant enzymes catalase and superoxide dismutase. The glucose induced oxidative stress and the protective effect of vitamin E on cellular membrane of human RBCs manifested as inhibition of membrane peroxidation and protein oxidation and restoration of activities of superoxide dismutase and catalase, was investigated.Thiobarbituric acid reactive substances are generated from decomposition of lipid peroxides and their determination gives a reliable estimate of the amount of lipid peroxides present in the membrane. Vitamin E at 18 μg/ml (normal serum level) strongly enhanced the RBC resistance to oxidative lysis leading to only 50–55% hemolysis in 24 h, whereas RBCs treated with 10 and 20 mM glucose without vitamin E leads to 70–80% hemolysis in 24 h. Levels of enzymic antioxidants catalase, superoxide dismutase and nonenzymic antioxidants glutathione showed restoration to normal levels in presence of vitamin E. The study shows that vitamin E can protect the erythrocyte membrane exposed to hyperglycemic conditions and so a superior antioxidant status of a diabetic patient may be helpful in retarding the progressive tissue damage seen in chronic diabetic patients.     

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

Electrophoretic characterization and subcellular distribution of hexokinase isoenzymes in red blood cells of rabbits

The isoenzyme pattern of hexokinase in rabbit red cells (erythrocytes, fetal erythrocytes and reticulocytes) were determined by means of agarose gel and disc electrophoresis. One duplicated hexokinase (4a and 4b according to the IUPAC-nomenclature) was detected in rabbit erythrocytes as also described for human erythrocytes. Besides the isoenzymes 4a and 4b reticulocytes also contain hexokinase 2 and 3 like rabbit and rat liver. The high KM glucose phosphorylating enzyme, hexokinase 1 could be demonstrated only under specific conditions in the reticulocytes during the initial stage of the anemia. After the fractionation of reticulocyte homogenates the total hexokinase activity was recovered in the mitochondria and cytosol to nearly equal amounts as revealed by the distribution of markers. Hexokinase 2 and 3 were detectable in reticulocytes and in isolated mitochondria only after the addition of certain dissociating agents. In contrast to the tightly bound mitochondrial hexokinases 2 and 3 the type 4a and 4b are more loosely bound and exhibit a bilocal distribution between mitochondria and cytosol of reticulocytes.     

Preeclampsia inactivates glucose-6-phosphate dehydrogenase and impairs the redox status of erythrocytes and fetal endothelial cells

Inactivation of glucose-6-phosphate dehydrogenase (G6PD) may contribute to vascular dysfunction in preeclampsia, and oxidative stress has been implicated in the pathogenesis of this disease. We have compared the susceptibility of erythrocytes and human umbilical vein endothelial cells (HUVEC) to oxidative stress in women with normotensive or preeclamptic pregnancies. The redox status of erythrocytes was also correlated with neutrophil-mediated superoxide (O₂⁻) production in women recruited to the “Vitamins in Preeclampsia” (VIP) trial. Erythrocytes and HUVEC from women with preeclampsia demonstrated impaired redox regulation and diminished response to glucose, detectable at 14–20 weeks gestation prior to onset of the clinical disease. Hexokinase and G6PD activities were decreased in erythrocytes and G6PD activity was decreased in HUVEC from preeclamptic pregnancies. Phorbol-ester-stimulated O₂⁻ was enhanced in preeclamptic neutrophils. Impaired redox regulation in erythrocytes and HUVEC in preeclampsia may be due to diminished hexokinase and G6PD activities resulting from increased release of reactive oxygen species from activated neutrophils. Our findings provide the first evidence that decreased G6PD activity in preeclampsia is associated with impaired redox regulation in erythrocytes and fetal endothelial cells. The deficiency in G6PD in preeclampsia potentially accounts for the lack of protection against oxidative stress afforded by antioxidant vitamin C/E supplementation in the VIP trial.     

Flavohemoglobin: structure and reactivity

Flavohemoglobins (flavoHbs) are made of a globin domain fused with a ferredoxin reductaselike FAD- and NAD-binding modules. These proteins are widely represented among bacteria and yeasts and represent a most challenging research subject in view of their high reactivity both as reductases and as oxidases. The functional annotations of flavoHbs are still controversial, and different physiological roles that are linked to cell responses to oxidative and/or nitrosative stress have been proposed. The flavoHb from Escherichia coli (HMP) has been the object of a large number of investigations to unveil its physiological role in the framework of bacterial resistance to nitrosative stress. HMP expression has been demonstrated to respond to the presence of NO in the culture medium, and an explicit mechanism has been proposed that involves NO scavenging and its reduction to N(2)O under anaerobic conditions. In contrast to (or together with) the anaerobic NO-reductase activity, HMP has also been shown to be able to catalyze the oxidation of NO to NO(3) (-) (NO-dioxygenase activity) both in vivo and in vitro in the presence of O(2) and NADH. HMP has also been shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. The alkylhydroperoxide reductase activity taken together with the unique lipid-binding properties of HMP suggests that this flavoHb may be involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress.     

Absent effect of zinc deficiency on the oxidative stress of erythrocytes in chronic uremic rats

Both anemia and zinc deficiency are commonly observed in patients with chronic uremia. Oxidative stress of red blood cells (RBC) has been suggested to participate in the development of anemia in these patients with chronic uremia due to reduced life span of RBC. Whether zinc deficiency aggravates the effect of oxidative stress on RBC of chronic uremia is still not understood. We thus performed the study to determine the influence of zinc deficiency on the oxidative stress of RBC in uremic rats. Zinc deficiency was induced by long-term dietary zinc deficiency. Five-sixth nephrectomy (5/6 Nx) was used to produce chronic uremia. Experiment was carried out in the following five groups: normal control (NL), chronic uremia (Nx), chronic uremia + dietary zinc deficiency (Nx-D), Nx-D + zinc supplement (Nx-DZ) and Chronic uremia + pair-fed (Nx-PF). Osmotic fragility and lipid peroxidation of RBC were used to evaluate the oxidative stress of RBC. Five weeks after 5/6 nephrectomy (Nx), 5/6 Nx rats present a syndrome of uremia to elevate the levels of plasma creatinine and urea, and reduce the level of plasma zinc (1.12 +/- 0.08 vs 1.35 +/- 0.05 ug/ml). But they does not find to produce anemia and to increase osmotic fragility and lipid peroxidation in RBC. Dietary zinc deficiency in Nx-D group produced severe anorexia and reduced plasma zinc and selenium levels and the activity of RBC-GPX. Yet in Nx-D rats, osmotic fragility and susceptibility of lipid peroxidation in red cells did not increase, because of the increase of plasma copper level (1.85 +/- 0.3 vs 1.41 +/- 0.05 microg/ml) and RBC-SOD activity (1.95 +/- 0.27 vs 0.78 +/- 0.05 unit/g Hb). Zinc supplement in Nx-D rats (Nx-DZ group) recovered the appetite and normalized the levels of plasma zinc, copper and selenium. Food restriction in 5/6 Nx rats (Nx-PF group) decreased plasma copper level and increased osmotic fragility of RBC and elevated the susceptibility of lipid peroxidation after stressing RBC with H2O2 Because Nx-PF rats presented a lower RBC-SOD activity (0.44 +/- 0.11 vs 0.78 +/- 0.05 unit/g Hb) and a lower plasma copper level. We further found a positive relationship (r=0. 802,p<0.01) between plasma copper level and RBC-SOD activity in normal and uremic rats. This study suggests that RBC-SOD activity may play an important role in preventing RBC oxidative stress. Plasma copper level may be a marker of RBC-SOD activity. We conclude, in chronic uremia, zinc deficiency doses not result in RBC oxidative stress as plasma copper level is normal, but may affect the absorption of intestinal nutrition.     

Augmented erythrocyte band-3 phosphorylation in septic mice

Infection-induced RBC dysfunction has been shown to play a role in the modulation of host response to injury and infection. The underlying biochemical mechanisms are not known. This study investigated alterations in RBC band-3 phosphorylation status and its relationship to anion exchange activity in vitro as well as under in vivo septic conditions induced by cecal ligation and puncture (CLP) in mice. Pervanadate treatment in vitro increased band-3 tyrosine phosphorylation that was accompanied by decreased RBC deformability and anion exchange activity. Following sepsis, band-3 tyrosine phosphorylation in whole RBC ghosts as well as in cytoskeleton-bound or soluble RBC protein fractions were elevated as compared to controls. Although anion exchange activity was similar in RBCs from septic and control animals, band-3 interaction with eosin-5-maleimide (EMA), which binds to band-3 lysine moieties, was increased in cells from septic animals as compared to controls, indicating that sepsis altered band 3 organization within the RBC membrane. Since glucose-6-phosphate dehydrogenase is a major antioxidant enzyme in RBC, in order to assess the potential role of oxidative stress in band-3 tyrosine phosphorylation, sepsis-induced RBC responses were also compared between WT and (G6PD) mutant animals (20% of normal G6PD activity). Band-3 membrane content and EMA staining were elevated in G6PD mutant mice compared to WT under control non-septic conditions. Following sepsis, G6PD mutant animals showed lessened responses in band-3 tyrosine phosphorylation and EMA staining compared to WT. RBC anion exchange activity was similar between mutant and WT animals under all tested conditions. In summary, these studies indicate that sepsis results in elevated band-3 tyrosine phosphorylation and alters band-3 membrane organization without grossly affecting RBC anion exchange activity. The observations also suggest that factors other than oxidative stress are responsible for the sepsis-induced increase in RBC band-3 tyrosine phosphorylation.     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Correlation of enzymatic,metabolic, and behavioral deficits in thiamin deficiency and its reversal

To clarify the enzymatic mechanisms of brain damage inthiamin deficiency, glucose oxidation, acetylcholine synthesis, and the activities of the three major thiamin pyrophosphate (TPP) dependent brain enzymes were compared in untreated controls, in symptomatic pyrithiamin-induced thiamin-deficient rats, and in animals in which the symptoms had been reversed by treatment with thiamin. Although brain slices from symptomatic animals produced¹⁴CO₂ and¹⁴C-acetylcholine from [U-¹⁴C]glucose at rates similar to controls under resting conditions, their K⁺-induced-increase declined by 50 and 75%, respectively. In brain homogenates from these same animals, the activities of two TPP-dependent enzymes transketolase (EC 2.2.1.1) and 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3) decreased 60–65% and 36%, respectively. The activity of the third TPP-dependent enzyme, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3.) did not change nor did the activity of its activator pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43). Although treatment with thiamin for seven days reversed the neurological symptoms and restored glucose oxidation, acetylcholine synthesis and 2-oxoglutarate dehydrogenase activity to normal, transketolase activity remained 30–32% lower than controls. The activities of other TPP-independent enzymes (hexokinase, phosphofructokinase, and glutamate dehydrogenase) were normal in both deficient and reversed animals.Thus, changes in the neurological signs during pyrithiamin-induced thiamin deficiency and in recovery paralleled the reversible damage to a mitochondrial enzyme and impairment of glucose oxidation and acetylcholine synthesis. A more sustained deficit in the pentose pathway enzyme, transketolase, may relate to the anatomical abnormalities that accompany thiamin deficiency.Dedicated to Henry McIlwain.     

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.     

Chemical and pathological oxidative influences on band 3 protein anion-exchanger.

BACKGROUND/AIMS: The erythrocyte is a cell exposed to a high level of oxygen pressure and to oxidative chemical agents. This stress involves SH-groups oxidation, cell shrinkage by activation of K-Cl co-transport (KCC) and elevation of the band 3 tyrosine phosphorylation level. The aim of our study was to test whether oxidative stress could influence band 3-mediated anion transport in human red blood cells. METHODS: To evaluate this hypothesis, normal and pathological (glucose 6 phosphate dehydrogenase (G6PDH) defficient) erythrocytes were treated with known sulphydryl-blocking or thiol-oxidizing agents, such as N-ethylmaleimide (NEM), azodicarboxylic acid bis[dimethylamide] (diamide), orthovanadate, Mg2+ and tested for sulphate (SO4-) uptake, K+ efflux, G6PDH activity and glutathione (GSH) concentration. RESULTS: In normal red blood cells, the rate constants of SO4- uptake decreased by about 28 % when cells were incubated with NEM, diamide and orthovanadate. In G6PDH-deficient red blood cells, in which oxidative stress occurs naturally, the rate constant of sulphate uptake was decreased by about 40% that of normal red cells. Addition of oxidizing and phosphatase inhibitor agents to pathological erythrocytes further decreased anion transport. In contrast, G6PDH activity was increased under oxidative stress in normal as well as in pathological cells and was lower in the presence of exogenous Mg2+ in parallel to a significant increase in sulphate transport. In both cells, the oxidizing agents increased K+ efflux with depletion of GSH. CONCLUSION: The data are discussed in light of the possible opposite effects exerted by oxidative agents and Mg2+ on KCC and on the protein tyrosine kinase (PTK)-protein tyrosine phosphatase (PTP) equilibrium. The decreased sulphate uptake observed in the experimental and pathological conditions could be due to band 3 SH-groups oxidation or to oxidative stress-induced K-Cl symport-mediated cell shrinkage with concomitant band 3 tyrosine phosphorylation.     

Glucose 1,6-bisphosphate-overloaded erythrocytes: a strategy to investigate the metabolic role of the bisphosphate in red blood cells.

Human erythrocytes overloaded with glucose 1,6-bisphosphate were prepared in order to establish the metabolic significance of this phosphorylated sugar in the intact red cell. The intracellular glucose 1,6-bisphosphate concentration was increased six- and twofold over the normal level by encapsulating (i) the commercially available compound and (ii) the glucose 1,6-bisphosphate synthase obtained from rabbit skeletal muscle, respectively. In both experimental conditions, a reduction of glucose utilization by the loaded cells was observed after reequilibration to the steady state. At the steady state, the concentrations of the glycolytic intermediates and of the adenine nucleotides appeared substantially unmodified when compared with those of controls, with the exception of a 50% reduction of glucose and fructose 6-phosphate measured in erythrocytes encapsulated with exogenous glucose 1,6-bisphosphate. Under the considered experimental conditions, the elevated intracellular glucose 1,6-bisphosphate appears to display an inhibitory effect on hexokinase that overcomes the possible activation of phosphofructokinase or pyruvate kinase.