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.     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H⁺ + K⁺)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg²⁺-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol P_i and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K⁺ (up to 0.69 ± 0.09 nmol P_i incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K⁺-stimulated ATPase and p-nitrophenylphosphatase activities and K⁺-sensitive phosphorylation: Mg²⁺-ATPase (μmol P_i/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K⁺-stimulated); Mg²⁺-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K⁺-stimulated); Mg²⁺-phosphorylation (nmol P_i/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K⁺). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H⁺ + K⁺)-ATPase in a still active structure.     

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 μmol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [¹⁴C]pyruvate to haemoglobin and the rate of efflux of [¹⁴C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent K_m and V_max for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.     

Magnetic resonance studies on interaction of bovine brain hexokinase with manganous ion, glucose, and glucose 6-phosphate

Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, K_D = 25 ± 4 μM and K′_D = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ?_b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ?_b = 3.5 ± 0.4 for tight binding sites and an average ?_b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s^?1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.     

Nδ-acetylornithine and S-methylcysteine in blood plasma

N^δ-Acetylornithine and S-methylcysteine have been identified as minor components of deproteinized blood plasma of human and bovine blood. Human blood plasma contains a variable amount of acetylornithine, averaging 1.1 ± 0.4 μmol/l (range 0.8–0.2 μmol/l). Urine contains a very small amount of acetylornithine, approximately 1 nmol/mg creatinine (1 μmol/day). Human blood plasma contains 3.9 ± 1.9 μmol/l (range 1.4–6.5 μmol/l) of S-methylcysteine. Urine contains approximately 5 nmol/mg creatinine; after acid hydrolysis the amount is increased to 20 nmol/mg creatinine.     

Rabbit heart mitochondrial hexokinase: Solubilization and general properties

In rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent K_m for glucose (0.065 mm), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent K_m for ATPMg than the solubilized enzyme, whereas the apparent K_m for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968)Proc. Natl. Acad. Sci. USA61, 598–605]. Furthermore, low concentrations (0.1 mm) of glucose 6-phosphate (G6P) or ATP^4? specifically solubilize hexokinase. The solubilizing effect of G6P and ATP^4?, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with P_i or Mg²⁺. In addition, enzyme solubilization by G6P can be reversed by Mg²⁺ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J.37, 18–19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one.     

The effect of age on beta-adrenergic receptors and adenylate cyclase activity in rat erythrocytes.

Beta-adrenergic receptors and catecholamine-sensitive adenylate cyclase activity were studied in erythrocytes obtained from rats 6 weeks, 6 months, and 15 months of age. Intact erythrocytes from 6 week old rats contained significantly more beta receptors (411 ± 31 sites/cell) than 6 month (328 ± 21) or 15 month old rats (335 ± 16), as determined by binding of [¹²⁵I] iodohydroxybenzylpindolol. Erythrocytes from 6 week old rats also contained significantly greater isoproterenol-sensitive adenylate cyclase activity (95.0 ± 9.4pmoles/10⁹ cells) than erythrocytes from 6 month (27.9 ± 3.3) or 15 month old rats (23.7 ± 3.6). The erythrocyte population of 6 week old rats was bigger (mean corpuscular volume = 62 ± 2μ^3/cell) than the older rat erythrocytes (47 ± 1μ³ and 48 ± 1μ³). When the data were expressed relative to a unit of cell volume, there was no difference in the density of beta receptors among all three populations but a progressive and significant fall in hormone-sensitive adenylate cyclase activity. In the rat erythrocyte, the age-related loss of adenylate cyclase activity is not accompanied by changes in β-receptor density.     

The effects of synthetic estrogens on the metabolic clearance and production rates of estrone and estradiol

The metabolic clearance rates (MCR) of estrone (1) and estradiol were determined by pulse injections and constant infusions of ³H-estrone and ³H-estradiol in seven women taking mestranol-containing compounds and in seven women taking ethinyl estradiol-containing compounds. These results were compared with the results previously obtained in our laboratory (2, 3, 4, 5) in comparable women not taking these compounds. In the women taking mestranol the mean (± SE) MCR for estradiol, 750 ± 600 1/day/m², was similar to our normal mean value, 790 ± 30 1/day/m². However, the mean MCR for estrone was less 1,010 ± 60 1/day/m² than that in normals 1,230 ± 30 1/day/m². In the women taking ethinyl estradiol the mean MCR for estradiol, 1,070 ± 60 1/day/m² was significantly (P < 0.01) greater than the normal value. The mean MCR for estrone, 1,180 ± 80 1/day/m² was not different from the normal.Using an immunoassay to measure the concentrations of estradlol and. estrone in plasma, the mean level of estradlol in mestranol users was 40 ± 7 Pg/ml and in ethinyl estradlol users 69 ± 4 pg/ml.The mean calculated production rate for estradlol in the mestranol users was 47 ± 8 μg/day and in the ethinyl estradlol users was 120 ± 22 μg/day. The mean calculated, production rates for estrone were 71 ± 12 and 93 ± 12 μg/day in the respective groups.Thus while mestranol appears to have little effect on endogenous estrogen metabolism, the use of ethinyl estradiol appears to increase the MCR of estradlol, but not of estrone. The MCR of estradlol returns to the normal range when ethinyl estradlol is stopped.     

Role of diagnostic factors associated with antioxidative status and expression of matrix metalloproteinases (MMPs) in patients with cancer therapy induced ocular disorders

Background

Cancer patients when treated with different chemotherapeutic drugs often develop mild to severe sight threatening diseases during or after chemotherapy. The mechanism involved in the pathogenesis of ocular toxicities is poorly understood. Oxidative stress, inflammation and MMPs (angiogenic factor) are involved in the progression of chemotherapy related ocular disorders.

In vitro inhibition of human erythrocyte hexokinase by various hyperglycemic drugs

Hemolysis is the red blood cell abnormality most often associated with adverse effect of drug therapy. Drug‐induced or drug‐associated hyperglycemia could decrease the activity of hexokinase. The aim of this study was to investigate the inhibitory effects of some commonly used drugs that have hyperglycemic side effect on the human erythrocyte hexokinase enzyme in vitro. Hexokinase was purified from human erythrocytes using sequential chromatography, with a specific activity of 0.96 ± 0.18 U/g hemoglobin, and assayed in the presence of selected drugs that have hyperglycemic side effect. The IC₅₀ were determined from the regression analysis graph. Correlation analysis showed that there was positive correlation between the hyperglycemic side effect of some of the tested drugs and decrease of hexokinase activity. This suggests that, at least in part, these drugs exert their hyperglycemic effect by inhibiting glucose phosphorylation by the hexokinase, which consequently causes the glucose accumulation.     

Urea production and arginine metabolism are reduced in the growth restricted ovine foetus

Urea production may be impaired in intrauterine growth restriction (IUGR), increasing the risk of toxic hyperammonaemia after birth. Arginine supplementation stimulates urea production, but its effects in IUGR are unknown. We aimed to determine the effects of IUGR and arginine supplementation on urea production and arginine metabolism in the ovine foetus. Pregnant ewes and their foetuses were catheterised at 110 days of gestation and randomly assigned to control or IUGR groups. IUGR was induced by placental embolisation. At days 120 and 126 of gestation, foetal urea production was determined from [¹⁴C]-urea kinetics and arginine metabolism was determined from the appearance of radioactive metabolites from [³H]-arginine, both at baseline and in response to arginine or an isonitrogenous mixed amino acid supplementation. Urea production decreased with gestational age in the embolised animals (13.9 ±  3.1 to 11.2 ±  3.0 μmol/kg per min, P ≤ 0.05) but not in the controls (13.3 ±  3.5 to 14.8 ±  6.0 μmol/kg per min). Arginine supplementation increased urea production in both groups, but only at 126 days of gestation (control: 15.0 ±  8.5 to 17.0 ±  9.4 μmol/kg per min; embolised: 11.7 ±  3.1 to 14.3 ±  3.1 μmol/kg per min, P ≤ 0.05). Embolisation reduced foetal arginine concentrations by 20% ( P ≤ 0.05) while foetal arginine consumption was reduced by 27% ( P ≤ 0.05). The proportions of plasma citrulline and hydroxyproline derived from arginine were reduced in the embolised animals. These data suggest that foetal urea production and arginine metabolism are perturbed in late gestation after placental embolisation.     

Measurement of fluxes through the pentose phosphate pathway in erythrocytes from individuals with sickle cell anemia by carbon-13 nuclear magnetic resonance spectroscopy

Erythrocytes from individuals with sickle cell anemia have previously been shown to have increased levels of intracellular oxidants and increased oxidative damage. Oxidative damage has been implicated in the events leading to the painful crises and hemolytic anemia found in sickle cell anemia. Since the pentose phosphate pathway (PPP) is an important source of reducing capacity in erythrocytes, we have investigated the fluxes through the PPP in normal and sickle cell erythrocytes using [2-¹³C]D-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Our results indicate that sickle cell erythrocytes have a flux through the PPP of 0.13±0.02 μmol/h per ml erythrocytes that is comparable to that in normal erythrocytes, 0.21±0.02 μmol/h per ml erythrocytes. However, when stimulated with methylene blue, sickle cell erythrocytes show a decreased response, 0.59±0.10 μmol/h per ml erythrocytes, compared to normal erythrocytes, 1.64±0.10 μmol/h per ml erythrocytes. When homogeneous populations of sickle cell erythrocytes are isolated by density gradient centrifugation, the rate of flux through the PPP in methylene blue-stimulated sickle cell erythrocytes, 1.16±0.16 μmol/h per ml erythrocytes, approaches that in methylene blue-stimulated normal erythrocytes. In addition, by analyzing the dose response to methylene blue, we have found that the decreased stimulation of the PPP by methylene blue in heterogeneous populations of sickle cell erythrocytes is a failure of methylene blue to simulate the PPP rather than a deficiency in the PPP in sickle cell erythrocytes.     

Desensitization to glucose 6-phosphate of phosphoenolpyruvate carboxylase from maize leaves by pyridoxal 5′-phosphate

Incubation of the nonphosphorylated form of maize-leaf phosphoenolpyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), PEPC, EC 4.1.1.31) with the reagent pyridoxal 5′-phosphate (PLP) resulted in time-dependent, reversible inactivation and desensitization to the activator glucose 6-phosphate (Glc6P) and other related phosphorylated compounds. Both processes are not connected, since (i) when the PLP-modification was carried out in the presence of saturating ligands of the active site, which prevents inactivation, the desensitization to Glc6P is still observed, and (ii) under some experimental conditions the desensitization reaction is 4-times faster than the inactivation. Desensitization to Glc6P is first order with respect to PLP and has a second-order forward rate constant of 4.7±0.3 s⁻¹ M⁻¹ and a first-order reverse rate constant of 0.0046±0.0002 s⁻¹. Correlation studies between the remaining Glc6P sensitivity and mol of PLP residues incorporated per mol of enzyme subunit indicate that one lysyl group for enzyme monomer is involved in the sensitivity of the enzyme to Glc6P. The reactivity of this group is increased by polyethylene glycol and glycerol, while the reactivity of the lysyl group of the active site is not affected by these organic cosolutes. In the presence but not in the absence of the organic cosolutes, Glc6P by itself offers significant protection against desensitization, while increases the extent of inactivation. Free PEP or PEP-Mg have opposite effects, protecting the enzyme against inactivation and increasing the degree of desensitization. They also increases the protection against desensitization afforded by Glc6P. Finally, the PEPC inhibitor malate provides some protection against both inactivation and desensitization. Taken together, these results are consistent with PLP-modification of a highly reactive lysyl group at or near the allosteric Glc6P-site.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca²⁺ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca²⁺-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca²⁺, Sr²⁺, or Ba²⁺, but not by Mg²⁺ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.     

Substrate and metal ion binding to carbamate kinase: NMR and EPR studies

Metal ion and substrate binding to carbamate kinase from Streptococcus faecalis was studied by nuclear magnetic resonance (NMR) and electron paramagnetic resonance using Mn²⁺ as the paramagnetic probe. The enzyme binds Mn²⁺ weakly (K_D = 0.45 ± 0.05 mm) with a stoichiometry of one per two subunits. However, in the presence of nucleotides, tighter binding of Mn²⁺ was observed with K_D = 44 ± 4 μm in the presence of ADP and K_D = 23 ± 4 μm with ATP present. Proton relaxation rate enhancement studies were conducted on water molecules interacting with ternary enzyme-Mn²⁺-nucleotide and binary enzyme-Mn²⁺ complexes. Mn²⁺ bound to carbamate kinase enhances the proton relaxation rate of water giving a binary enhancement value of ?_b = 9.3 ± 0.4. When enzyme-Mn²⁺ was titrated with ADP or ATP, a bell-shaped titration curve was obtained typical of many other enzyme-Mn²⁺-nucleotide ternary complexes. Computer fits to the titration data gave ternary enhancement values of ?_t^ADP = 14 ± 1 and ?_t^ATP = 19 ± 1. The dissociation constants for Mn-ADP and Mn-ATP binding to carbamate kinase were also obtained from these data analyses and are K₁ = 2.5 ± 0.5 μm and K₁ = 50 ± 8 μm, respectively. Therefore, these data demonstrate the formation of a ternary enzyme-metal-nucleotide bridge complex at the nucleotide substrate site of carbamate kinase. Distance measurements were conducted by NMR techniques with ¹³C-enriched carbamate and demonstrate that carbamate is 4–8 Å from enzyme-bound Mn²⁺. Thus carbamate binds near the metal-nucleotide substrate site of carbamate kinase.     

Allophycocyanin B (λmax 671, 618 nm)

A hitherto undescribed red fluorescent phycobiliprotein (maximum emission at ∼ 680 nm), characterized by long wavelength absorption maxima in the visible region at 671 nm (ε=172000 M⁻¹·cm⁻¹ per monomer of mol. wt. 30600) and 618 nm, has been purified to homogeneity from a unicellular cyanobacterium, Synechococcus sp., and from a filamentous cyanobacterium, Anabaena variabilis. The name allophycocyanin B has been proposed for the new protein. A. variabilis allophycocyanin B is characterized by a native molecular weight of 89000 ± 5000 (in 0.05 M phosphate at pH 7.2), an isoelectric point of 5.09, and a subunit molecular weight, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of 15300. The protein contains one phycocyanobilin chromophore per subunit. In common with allophycocyanin from the same organism, allophycocyanin B does not contain either histidine or tryptophan. In other respects, the amino acid compositions of the two proteins are significantly different. Synechococcus sp. (Anacystis nidulans) allophycocyanin B gives two components of 16000 and 17000 mol. wt., of equal staining intensity, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Allophycocyanins B from both organisms cross-react with rabbit antisera directed against either Synechococcus sp. or Anabaena sp. allophycocyanin, but not with antisera against the phycocyanins of the same organisms. It is suggested that allophycocyanin B occupies a position between allophycocyanin and chlorophyll a in the energy transfer path from the accessory pigments to species of chlorophyll a with absorption maxima at λ>670 nm.     

Measurement of blood volume in the elasmobranch fish Scyliorhinus canicula following acute and long‐term salinity transfers

A technique using ⁵¹chromium‐labelled erythrocytes was used to measure blood volume in Scyliorhinus canicula following long‐term and acute salinity transfers. Basal whole‐blood volume was 5·6 ± 0·2 ml 100 g^?1 (mean ±s .e .), this increased (6·3 ± 0·2 ml 100 g^?1) following +14 day acclimation to 80% sea water (SW) and decreased (4·6 ± 0·2 ml 100 g^?1) following acclimation to 120% SW. These changes were shown to be primarily due to changes in plasma volume, with no significant changes in extrapolated red‐cell volume being demonstrated. Blood volume was also measured in the same animals during 10 h acute transfer to 100% SW. Plasma volume in S. canicula during acclimation from 80% SW was significantly reduced (4·5 ± 0·3 ml 100 g^?1) after 6 h of transfer to 100% SW. Blood volume in animals during acclimation from 120% SW was significantly increased (4·8 ± 0·2 ml 100 g^?1) after 4 h of acute transfer. The osmoregulatory implications of these different timeframes during hyposaline and hypersaline transfer are discussed, along with the importance of this in vivo technique as context for in vitro studies with haemo‐dynamic stimuli.     

Toxic copper level increases erythrocyte glycolytic rate,glutathione production and alters electrolyte balance in male Wistar rats

BackgroundCopper is a micronutrient vital to several cellular energy metabolic processes and drives erythropoiesis. However, it disrupts cellular biological activities and causes oxidative damage when in excess of cellular needs. This study investigated the effects of copper toxicity on erythrocyte energy metabolism in male Wistar rats.MethodsTen Wistar rats (150–170 g) were randomly divided into 2 groups: control (given 0.1 ml distilled water) and copper toxic (given 100 mg/kg copper sulphate). Rats were orally treated for 30 days. Blood, collected retro-orbitally after sodium thiopentone anaesthesia (50 mg/kg i.p.) into fluoride oxalate and EDTA bottles, was subjected to blood lactate assay and extraction of red blood cell respectively. Red blood cell nitric oxide (RBC NO), glutathione (RBC GSH), adenosine triphosphate (RBC ATP) levels, RBC hexokinase, glucose-6-phosphate (RBC G6P), glucose-6-phosphate dehydrogenase (RBC G6PDH), and lactate dehydrogenase (RBC LDH) activity was estimated spectrophotometrically. Values (Mean±SEM, n = 5) were compared by Student’s unpaired T-test at p < 0.05.Results and conclusionCopper toxicity significantly increased RBC hexokinase (23.41 ± 2.80 µM), G6P (0.48 ± 0.03 µM), G6PDH (71.03 ± 4.76nmol/min/ml) activities, ATP (624.70 ± 57.36 µmol/gHb) and GSH (3.08 ± 0.37 µM) level compared to control (15.28 ± 1.37 µM, 0.35 ± 0.02 µM, 330.30 ± 49.58 µmol/gHb, 54.41 ± 3.01nmol/min/ml and 2.05 ± 0.14 µM respectively, p < 0.05). Also, RBC LDH activity (145.00 ± 19.88mU/ml), NO (3.45 ± 0.25 µM) and blood lactate (31.64 ± 0.91 mg/dl) level were lowered significantly compared to control (467.90 ± 94.23mU/ml, 4.48 ± 0.18 µM and 36.12 ± 1.06 mg/dl respectively). This study shows that copper toxicity increases erythrocyte glycolytic rate and glutathione production. This increase could be connected to a compensatory mechanism for cellular hypoxia and increased free radical generation.     

The effects of different light intensities on the culture of Gigartina skottsbergii (Rhodophyta,Gigartinales) tetrasporophytes and gametophytes in the Magellan region,Chile

Gigartina skottsbergii is a red seaweed used as raw material for extracting carrageenans, constituting an important economic resource for Chile. In 2009, extraction in the Magellan region reached 15,064 t. The growing demand has adversely affected the sustainability of natural beds, creating an interest in the culture of this resource. In order to provide information relevant to the culture and regeneration of this seaweed, the present study addresses the effects of different light intensities on the growth of G. skottsbergii gametophytes and tetrasporophytes during the early stages of development. Mature reproductive fronds were induced to release spores in the laboratory by a drying process. Gametophytes cultured at different light intensities showed an increase in diameter, which reached 519.13?±?108.95 μm with 4 μmol photons m^?2 s^?1, while tetrasporophytes showed a greater increase in diameter, reaching 714.11?±?116.45 μm with 8 μmol photons m^?2 s^?1. Results indicate that both stages of the reproductive cycle are influenced by different light intensities within a limited range. Therefore, both phases require different and specific ranges of light intensity.     

Plasmodium knowlesi: glycolytic intermediate levels of enzyme activities of infected rhesus monkey erythrocytes

In vitro glycolytic enzyme activities and in vivo glycolytic intermediate concentrations were assayed in Plasmodium knowlesi-infected rhesus monkey erythrocytes and control erythrocytes. The enzyme activities of infected erythrocytes were greater than controls indicating that P. knowlesi had its own glycolytic system and that parasite glycolysis was the source of the increased rate of glucose consumption by infected erythrocytes. The P. knowlesi glycolytic enzymes phosphofructokinase and hexokinase were less sensitive to acid inhibition than uninfected red cells.P. knowlesi-infected monkey erythrocytes and Plasmodium berghei-infected mouse erythrocytes had similar in vivo glycolytic profiles and in vitro enzyme activity increases.