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.     

Purification and physical properties of hexokinase from human erythrocytes

A bulk purification is described for hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from human erythrocytes. Following a 110,000-fold purification from 40 litres of blood, 5 mg protein with a specific activity of 22 units/mg were obtained. On application of various separation techniques, the enzyme activity co-migrated with the main protein component. The physical properties, such as the relative molecular mass of 108,000 and sedimentation coefficient of 5.5 S, are similar to those of the enzyme from human heart. In particular, there is a correspondence in the conformational response to glucose 6-phosphate as shown by an association of the enzyme promoted by this metabolite.     

Glucokinase and hexokinase in pig erythrocytes.

We have shown different enzymatic activities responsible for the phosphorylation of glucose in pig erythrocytes. These activities were observed after partial purification from hemolyzed red cells. One of the enzymes involved is the hexokinase which is present in all tissues; the other is similar to hepatic glucokinase. We have determined the kinetic properties of these activities in hemolysates and in partially purified preparations. Their electrophoretic-migration characteristics were studied too.     

Dielectrophoretic study of human erythrocytes

This paper is concerned with the dielectrophoretic study of human erythrocytes under cylindrical field geometry. The influence of physical variables such as the frequency and voltage of the applied electric field, conductivity of the medium in which the cells are suspended, cell concentration and exposure time of the cell to the non-uniform electric field on dielectrophoretic collection rate (DCR) is determined in a systematic manner. It is interesting to note from the DCR spectrum of human erythrocytes that the DCR is minimum at one frequency, maximum at another and there is practically no yield over a certain frequency range. This may be attributed to the variation of complex dielectric constant of the particle and medium over that frequency range. From the DCR spectrum of different groups, it is clear that DCR behaviour is different in the frequency range from 0.3–1.5 MHz, under similar conditions of temperature, conductivity and concentration of erythrocyte suspension and strength of applied AC field. The response of DCR with voltage of the applied field, concentration of cell suspension and square root of elapsed time of the cells confirms the theory of dielectrophoresis.     

Micromethods for the spectrophotometric determination of bacterial protease activities

Microassays for the spectrophotometric determination of bacterial proteases were developed using congo red elastin, a substrate specific for elastolytic activity, and hide powder azure, a substrate sensitive to more general proteolytic activity. The small reaction volume (0.1 ml) allows incubation, filtration and quantitation to be carried out in 96 well microassay plates. Using a simple spin filtration device constructed from microassay plates a large number (768) of microassays can be filtered simultaneously. The microassays are particularly useful for screening large numbers of bacterial colonies for proteolytic mutants since they allow the rapid and efficient handling of multiple samples. These assays also permit the qualitative estimation of enzyme levels.     

Inherited persistence of immature type pyruvate kinase and hexokinase isozymes in dog erythrocytes

1. Red cell pyruvate kinase (EC 2.7.1.40) and hexokinase (EC 2.7.1.1) in high and low potassium (K) dogs were shown to exist as multiple forms which were separable by electrophoresis and ion-exchange chromatography. The R2-type pyruvate kinase, which was determined to be a young type enzyme in canine red cells, was shown to be the predominant form of pyruvate kinase in high K cells. 2. The M2-type pyruvate kinase, a prototype isozyme in erythroid cells, existed in high K dog erythrocytes as well as in high K and low K dog reticulocytes. 3. Isozyme analysis of high K red cell hexokinase also showed a profile similar to that obtained for low K reticulocytes. 4. These results seem to reflect the immaturity of high K erythrocytes, which suggest that an abnormal cell differentiation or maturation may occur at an early stage of erythroid cell proliferation in high K dogs.     

A study on the reaction of human erythrocytes with hydrogen peroxide

Membrane fluidity of human erythrocytes treated with H2O2 (1--20 mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2- or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation. The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (tau c) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes. The ESR spectrum of H2O2-treated erythrocytes at -196 degrees C is characterized by signals of nonheme ferric iron type (g equal to 4.3), low spin ferric iron, and free radical type at g equal to 2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g equal to 2.00 or 4.3. These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.     

A study of porous structure of cellular membranes in human erythrocytes

Properties of cell membrane of human erythrocytes are studied using the mechanistic formalism of membrane transport developed earlier. We estimate that an erythrocyte with a membrane surface of 176 x 10(6)nm2 has about 1900 water-permeable pores with cross-section areas ranging from 0.07 to 0.2 nm2.     

Enzymatic properties of overexpressed human hexokinase fragments

Full-length hexokinase (HK; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), a truncate form of the enzyme lacking the first 11 amino acids (HK-11aa) and the 50 kDa C-terminal half (mini-HK) containing the catalytic domain, were overexpressed and purified to homogeneity to investigate the influence of the N-terminal region of human hexokinase type I (HK) on its regulatory properties. All forms of the enzyme are catalytically active with the HK-11aa being the most active. All the forms of HK showed the same affinity for glucose and MgATP and were also inhibited by glucose 6-phosphate (Glc 6-P) competitively vs. MgATP with similar Kis (28.5-37 M). Glucose 1,6-bisphosphate (Glc 1,6-P2) was also a strong inhibitor of all HKs without significant differences among the different truncate forms of the enzyme (Kis 49.5-59 M). At low concentrations (0-3 mM), Pi was able to reverse the sugar phosphate inhibition of the full-length HK and HK-11aa but not of the mini-HK. In contrast, at high concentrations Pi was an inhibitor of all the hexokinases investigated. These findings confirm that Pi has a low affinity binding site on the C-terminal of HK while counteracts glucose 6-phosphate inhibition by binding to or requiring the N-terminal half of the enzyme. The first 11 N-terminal amino acids influence the specific activity of HK but are unable to affect the kinetic properties investigated.     

Isolation of Plasmodium berghei by hemolysin lysis of infected erythrocytes and evidence for a parasite hexokinase.

A rapid and simple procedure has been developed for the isolation of Plasmodium berghei parasites from infected-mouse erythrocytes employing the heat stable hemolysin produced by Pseudomonas aeruginosa. Using parasites isolated by this method, the presence of a parasite specific hexokinase has been demonstrated, providing an explanation for the increased glucose consumption observed with infected cells. Enzyme assays and serology were employed in determining the purity and yield of purified parasites. The enzyme assays showed that about 25% of the parasites in infected RBCs were recovered in the purified state. The purified parasites were not agglutinated by rabbit-anti-mouse RBC serum which indicated the purified parasites were not contaminated by RBC components.     

The use of fluorescence probes in the study of the beta-adrenoreceptor function of human erythrocytes

Possibility of making use of fluorescent probe 4-(p-dimethyl aminostyril)-1-methyl pyridinium p-toluene-sulphonate (DSM) for observing the interaction of beta-adrenoactive substances (adrenaline and propranolole) and specific receptors of human blood erythrocytes has been studied. Two types of DSM bindings with erythrocyte membranes have been revealed--specific and non-specific. It has been found that DSM reacts selectively to preliminary influence of beta-adrenoactive substances: antagonist propranolole diminishes both specific and nonspecific bindings and agonist adrenaline increases specific binding with the membrane erythrocytes. The conclusion is made as to possible use of DSM probe for investigating beta-adrenoreceptive function of the cell membranes.