Chemically induced fusion of fresh human erythrocytes.

The fusion of fresh human erythrocytes was shown to be induced by calcium and phosphate ions. Prior treatment of erythrocytes with phosphate ion was a pre-requisite for the calcium-induced fusion. ATP levels in cells incubated with phosphate and calcium decreased 46 fold while cell-associated calcium increased 70 fold during 1 hour of incubation at 37°C as compared to cells which were incubated with calcium in saline. Our results suggest that a phosphate complex formed bridges between adjacent erythrocytes causing agglutination followed by aggregation of membrane proteins leading to protein-free areas of lipids. Where these protein-free areas are in close contact fusion may occur.     

The effect of temperature of the rate of photosynthetic electron transfer in chloroplasts of chilling-sensitive and chilling-resistant plants

1. Photochemical activities as a function of temperature have been compared in chloroplasts isolated from chilling-sensitive (below approximately 12 °C) and chilling-resistant plants.2. An Arrhenius plot of the photoreduction of NADP⁺ from water by chloroplasts isolated from tomato (Lycopersicon esculentum var. Gross Lisse), a chilling-sensitive plant, shows a change in slope at about 12 °C. Between 25 and 14 °C the activation energy for this reaction is 8.3 kcal·mole^?1. Between 11 and 3 °C the activation energy increases to 22 kcal·mole^?1. Photoreduction of NADP⁺ by chloroplasts from another chilling-sensitive plant, bean (Phaseolus vulgaris var. brown beauty), shows an increase in activation energy from 5.9 to 17.5 kcal·mole^?1 below about 12 °C.3. The photoreduction of NADP⁺ by chloroplasts isolated from two chilling-resistant plants, lettuce (Lactuca sativa var. winter lake) and pea (Pisum sativum var. greenfeast), shows constant activation energies of 5.4 and 8.0 kcal·mole^?1, respectively, over the temperature range 3–25 °C.4. The effect of temperature on photosynthetic electron transfer in the chloroplasts of chilling-sensitive plants is localized in Photosystem I region of photosynthesis. Both the photoreduction of NADP⁺ from reduced 2,6-dichlorophenol-indophenol and the ferredoxin-NADP⁺ reductase (EC 1.6.99.4) activity of choroplasts of chilling-sensitive plants show increases in activation energies at approximately 12 °C whereas Photosystem II activity of chloroplasts of chilling-sensitive plants shows a constant activation energy over the temperature range 3–25 °C. The photoreduction of Diquat (1,1′-ethylene-2,2′-dipyridylium dibromide) from water by bean chloroplasts, however, does not show a change in activation energy over the same temperature range. The activation energies of each of these reactions in chilling-resistant plants is constant between 3 and 25 °C.5. The effect of temperature on the activation energy of these reactions in chloroplasts from chilling-sensitive plants is reversible.6. In chilling-sensitive plants, the increased activation energies below approximately 12 °C, with consequent decreased rates of reaction for the photoreduction of NADP⁺, would result in impaired photosynthetic activity at chilling temperatures. This could explain the changes in chloroplast structure and function when chilling-sensitive plants are exposed to chilling temperatures.     

Interaction of phloretin with the anion transport protein of the red blood cell membrane

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding and kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25°C, determined from equilibrium binding competition, is $\text{1.8 ± 0.1}\text{μM}$. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin inhibition constant of $\text{1.6 ± 0.4}\text{μM}$. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 μM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.     

Erythrocytes <Emphasis Type="SmallCaps">l</Emphasis>-Arginine y+ Transporter Inhibition by N-Ethylmaleimide in Ice-bath

Erythrocytes l-arginine uptake is conveyed by y+ and y+L membrane transport systems. Pre-incubation with N-ethylmaleimide for 10 min at 37°C inhibits the y+ system. The aim of this study was to determine the ideal pre-incubation temperature in evaluating y+ and y+L systems. Cells were pre-incubated with or without N-ethylmaleimide for 10 min at 4°C and 37°C. l-Arginine uptake was quantified by radioisotope and standard erythrocytes membrane flux methodology. Results demonstrate that erythrocytes l-arginine content is depleted by pre-incubation at 37°C for 10 min, thus changing the V _max measurement. The inhibitory effect of N-ethylmaleimide pre-incubation was temperature independent and already complete after 1 min of incubation. No significant difference in kinetic parameters was detected between cells pre-incubated at 37°C or 4°C, under zero-trans conditions. In conclusion, we suggest that measurement of erythrocytes l-arginine uptake by y+ and y+L systems could be carried out without N-ethylmaleimide pre-incubation at 37°C.     

Electron spin resonance studies on the inorganic-anion-transport system of the human red blood cell Binding of a disulfonatostilbene spin label (NDS-tempo) and inhibition of anion transport

The disulfonatostilbene spin label, NDS-TEMPO, was synthesized (purity over 96%) and the binding of the spin label to human red-cell ghosts was studied. NDS-TEMPO is readily adsorbed to the membrane surface. Both pretreatment of the ghosts with FDNB and DIDS and the presence of DNDS completely prevent the binding of NDS-TEMPO to red-cell ghosts. Chloride and sulfate competitively inhibit the binding of NDS-TEMPO. Conversely, NDS-TEMPO is a strong, competitive inhibitor of chloride and of sulfate transport. The dissociation constants of NDS-TEMPO from the ESR studies were in the range 1.0–2.0 μM (pH 7.6, 20°C). The inhibition constants of NDS-TEMPO as obtained from the flux experiments were in the range 0.5–2.5 μM (pH 7.3, 25°C). The close accordance of the NDS-TEMPO dissociation constants from the ESR studies with the NDS-TEMPO inhibition constants from the flux measurements indicate a specific labeling of the inorganic-anion-transport system.     

Identification of thymus-dependent areas in frozen sections of guinea pig lymphatic tissue by adherence of rabbit erythrocytes

Untreated rabbit erythrocytes adhere to thymus-dependent areas of guinea pig lymphatic tissues as shown with frozen sections. The adherence reaction is temperature dependent. Optimal results were obtained by incubation of the tissue section with the erythrocytes at temperatures between 0 ° and 4 °C. At 37 °C no adherence of erythrocytes was observed. Out of other erythrocytes tested (human, sheep, chicken, rat, mouse) only rat and mouse cells showed weak adherence to guinea pig thymus sections.     

Uptake of hexavalent chromium by bovine erythrocytes and its interaction with cytoplasmic components; The role of glutathione

Hexavalent chromium (Cr(VI)) anion gradually penetrated into bovine erythrocytes and bound with cytoplasmic components. Its penetration was strongly inhibited by the NH₂-reactive agent, 4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonic acid (SITS) and the SH-reactive agent, N-ethylmaleimide (NEM). Gel filtration showed that the intracellular component that bound to chromium was hemoglobin.

The binding affinity of Cr(VI) to hemoglobin in the absence of glutathione in vitro was found to be much less than in intact erythrocytes. However, in the presence of glutathione, the binding affinity of Cr(VI) to hemoglobin became much higher. This indicates that reduction of hemoglobin or Cr(VI) by glutathione is involved in the binding.

Cr(VI) interacted only weakly with the membrane and did not cause hemolysis of bovine erythrocytes, unlike heavy metals such as Hg²⁺.     

Impact of thermohaemolysis-associated membrane alteration on the passive ion permeability and life-span of erythrocytes

Suspending erythrocytes in medium containing sucrose prevented heat-induced lysis at its early stage. This allowed determination of the thermohaemolysis-related ion permeability by measuring the initial rate of the stipulated shrinkage of erythrocytes. Thus, correspondingly, the coefficient P of the ion permeability was calculated for heated human erythrocytes using ouabain-pretreated cells in 37–45°C range and intact cells in 50–58°C range. The values obtained for P obeyed a straight line Arrhenius plot over the entire 37–58°C range suggesting that the ion permeability was activated by a single mechanism earlier identified as relevant to thermohaemolysis. At the 37–58°C range, the activation energy of the P was 250±15 kJ/mol which was markedly different from the value of 56 kJ/mol known for the 10–37°C range. For erythrocytes from five mammals, similar temperature dependencies of the P were obtained over 45–60°C range. For erythrocytes from all species, excluding horse, the P, extrapolated at 37°C, had a value comparable with the known coefficient of the passive, ouabain-insensitive cation permeability at 37°C. For ouabain-treated human erythrocytes at 37°C, the period of thermohaemolysis-related shrinkage in sucrose containing media was found to be about six times shorter than the life-span of intact cells which substantiated the role of the active transport in balancing the thermohaemolysis-related diffusion of ions at 37°C. Consequently, the thermal resistance of erythrocytes, which was earlier related to their sphingomyelin content, was now found also to be in good correlation with their life-span in the circulation of 11 mammals.     

Identification of the Cl− transport site of human red blood cells by a kinetic analysis of the inhibitory effects of a chemical probe

H₂DIDS, the dihydro analog of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) can interact covalently with membrane sites, resulting in an irreversible inhibition of anion exchange. At low temperatures (0°C) and for relatively short times, however, its interaction is largely reversible, so that a kinetic analysis of the nature of its inhibitory effect on Cl^? self exchange can be performed. The effects of variations in the chloride concentration on the inhibitory potency of H₂DIDS are consistent with the concept that Cl^? and H₂DIDS compete for the transport site of the anion exchange system. The value of K_i for H₂DIDS is 0.046 μM, indicating that H₂DIDS has a higher affinity for the transport system than any other inhibitor so far examined. If, as seems probable, the covalent labelling of H₂DIDS occurs at the same site as the reversible binding, H₂DIDS can be used as a covalent label for the transport site. The specific localization of H₂DIDS in the band-3 protein thus indicates that this protein participates directly in anion exchange.     

Temperature dependence of the photosynthetic activities in the thylakoid membranes from the blue-green alga Anacystis nidulans

The photosynthetic electron transport and phosphorylation reactions were measured in the room temperature region in the thylakoid membranes prepared from the blue-green alga, Anacystis nidulans. The Arrhenius plot of the Hill reaction with 2,6-dichlorophenolindophenol showed a distinct break of straight lines at 21°C in the membranes from cells grown at 38°C, and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the Hill reaction with ferricyanide showed a break at 13°C in the membranes from cells grown at 38°C, and at 7°C in those from cells grown at 28°C. On the other hand, the Arrhenius plot of the System I reaction with methylviologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system was composed of a straight line in the membranes from cells grown at 28°C as well as at 38°C. The Arrhenius plot of the System II reaction measured by the ferricyanide reduction mediated by silicotungstate in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea also showed a break at 11°C in the membranes from cells grown at 38°C.The Arrhenius plot of the phosphorylation mediated by N-methylphenazonium methylsulfate showed a break at 21°C in the membranes from cells grown at 38°C and at 12°C in those from cells grown at 28°C. The Arrhenius plot of the phosphorylation mediated by the System I reaction showed a break at 24°C in the membranes from cells grown at 38°C.The characteristic features in the Arrhenius plots of the photosynthetic electron transport and phosphorylation reactions are discussed in terms of the transition of physical phase of the thylakoid membrane lipids.     

An assay of malaria parasite invasion into human erythrocytes: The effects of chemical and enzymatic modification of erythrocyte membrane components

Invasion of erythrocytes by malaria parasites is known to be blocked by proteolytic digestion of merozoite receptors allegedly present in red cell membranes. This information was used in the present work to develop a simple and convenient assay for parasite invasion into red blood cells and for evaluating the role played by red cell membrane components in this process. Synchronized in vitro cultures of Plasmodium falciparum containing only ring stages were subjected to either trypsin or pronase digestion, a treatment that neither affected ring development into schizonts nor mature merozoite release. Cells from this culture were not invaded by the released merozoites. However, upon addition of untreated human red blood cells, marked invasion was observed, either microscopically or as [³H]isoleucine incorporation. The new assay circumvents the need for separating schizonts from uninfected cells and provides a convenient means for assessing how chemical and biochemical manipulation of red blood cells affects their invasiveness by parasites. Using this assay, we verified that sheep and rabbit erythrocytes were resistant to invasion, as were human erythrocytes which had been treated with trypsin, pronase or neuraminidase. Chymotrypsin digestion of human erythrocytes was without effect on invasion. Human erythrocytes which were chemically modified with the impermeant amino reactive reagent H₂DIDS, or with the crosslinker of spectrin, TCEA, were found to resist invasion. The results underscore the involvement of surface membrane components as well as of elements of the cytoskeleton in the process of parasite invasion into erythrocytes.     

Errata

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (E_a = 1–4 kcal · mol^?1). These results are reconcilable with the formation of aqeous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poster, B., Lütkemeier, P., and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196–210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

Effect of extracellular Ca2+, K+ and OH− on erythrocyte membrane potential as monitored by the fluorescent probe 3,3′-dipropylthiodicarbocyanine

Changes in fluorescence intensity of thiodicarbocyanine, DiS-C₃(5), were correlated with direct microelectrode potential measurements in red blood cells from Amphiuma means and applied qualitatively to evaluate the effects of extracellular Ca²⁺, K⁺ and pH on the membrane potential of human red cells. Increasing extracellular [Ca²⁺] from 1.8 to 15 mM causes a K⁺-dependent hyperpolarization and decrease in fluorescence intensity in Amphiuma red cells. Both the hyperpolarization and fluorescence change disappear when the temperature is raised from 17 to 37°C. No change in fluorescence intensity is observed in human red cells with comparable increase in extracellular Ca²⁺ in the temperature range 5–37°C. Increasing the extracellular pH, however, causes human red cells to respond to an increase in extracellular Ca²⁺ with a significant but temporary loss in fluorescence intensity. This effect is blocked by EGTA, quinine or by increasing extracellular [K⁺], indicating that at elevated extracellular pH, human erythrocytes respond to an increase in extracellular Ca²⁺ with an opening of K⁺ channels and associated hyperpolarization of the plasma membrane.     

The effects of anion transport inhibitors on structural transitions in erythrocyte membranes

Red blood cell membranes have been labeled with several covalent and non-covalent inhibitors of anion transport and their heat capacity profiles determined as a function of temperature. Covalent inhibitors include the amino reactive agents 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, pyridoxal phosphate and 1-fluoro-2,4-dinitro benzene. The non-covalent inhibitors include several well known local anesthetics. The study was undertaken in order to identify regions of the membrane involved in anion transport. Covalent modification in all cases resulted in a large upward shift of the C transition, which is believed to involved a localized phospholipid region. Evidence is presented which indicates that Band III protein and this phospholipid region are in close physical proximity on the membrane. Addition of non-covalent inhibitors affects the membrane in either or both of two ways. In some cases, a lowering and broadening of the C transition occurs; in other the B₁ and B₂ transitions are altered. These latter transitions are believed to involve both phospholipid and protein, including Band III. These results may indicate that the non-covalent inhibitors produce their inhibitory effect on anion transport at least in part by interacting with membrane phospholipid.     

Fluorescence labeling of the human erythrocyte anion transport system. Subunit structure studied with energy transfer

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419–2427). The vesicles have a functional anion transport system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescence titrations show a maximum labeling stoichiometry of 1.3 ± 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu²⁺ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion     

Increased susceptibility of stored erythrocytes to anti-band 3 IgG autoantibody binding

When human blood was stored in a citrate-phosphate-dextrose (CPD) solution at 4°C, the susceptibility of the erythrocytes to binding of autologous IgG increased. The autologous IgG binding was partially inhibited by purified Band 3 glycoprotein and its oligosaccharides. The susceptibility of the erythrocytes to binding of ¹²⁵I-labeled anti-band 3 IgG autoantibody similarly increased. The results indicate that the anti-band 3 binding sites composed of Band 3 oligosaccharides were generated on the cell surface. The rate of the increase in the susceptibility of the stored cells to the antibody binding was lowered when blood was stored in a CPD solution containing L-ascorbic acid or erythorbic acid, suggesting involvement of an oxidative mechanism in the generation of the binding sites. The cytoplasmic glutathione level of erythrocytes gradually decreased during the blood storage. Storing blood in a CPD solution containing glutathione monoethylester or glutathione monoisopropylester resulted in partial prevention of the decrease in cytoplasmic glutathione level and of the increase in the IgG-binding ability of the cells. Similar preventive effect of glutathione monoethylester was observed in the binding of ¹²⁵I-labeled anti-band 3 autoantibody to the stored erythrocytes. Thus, the increase in the susceptibility of the stored erythrocytes to anti-band 3 binding may be caused, at least partially, by an oxidative stress resulting in a decreased cytoplasmic glutathione level.     

Heat shock effect on glutathione and superoxide dismutase in Tetrahymena pyriformis

Intracellular levels of total glutathione and cytosolic superoxide dismutase activity were assayed in cells from Tetrahymena pyriformis either exposed to sub-lethal (34°C) or to lethal heat shock (39°C). The results showed that glutathione levels decrease to 60% of normal values after a sub-lethal heat shock for 1 hour. This change is part of the heat shock response, since the effect is reversed as soon as cells are brought to their normal growing temperature. Using actinomycin D, which blocks the synthesis of high molecular weight hsp (Galego and Rodrigues-Pousada, 1985), prior to thermal stress, the fall in total glutathione is not observed, suggesting a partial correlation with the synthesis of these stress proteins. Using cells pre-exposed to a sub-lethal heat shock, a subsequent short severe heat shock does not lead to a significant decrease of the glutathione content. Superoxide dismutase (SOD) activity is not significantly induced after either a short period at 34°C or a prolonged treatment at the same temperature.     

Anion transport in relation to proteolytic dissection of band 3 protein

Sulfate efflux was measured in inside-out vesicles obtained from human red cells. Inhibition was observed in vesicles derived from cells pretreated with DIDS (4,4′-diisothiocyano-2,2′-stilbene disulfonate) or after addition of dipyridamole to the vesicles, both agents being specific and potent inhibitors of anion transport in cells. Trypsinization of the cytoplasmic side of the membrane in order to release a 40 000 dalton fragment from band 3 (the purported anion transport protein) had no effect on sulfate efflux. Further degradation of band 3 to a 17 000 dalton segment, by trypsinization of inside-out vesicles derived from cells that had been pretreated with chymotrypsin, also showed little reduction in transport activity. Furthermore, such vesicles derived from DIDS pretreated cells were inhibited by over 90%. In DIDS-treated cells, the agent is highly localized in band 3. In trypsinized inside-out vesicles, it is largely found in a 55 000 fragment and in trypsinized vesicles derived from cells pretreated with chymotrypsin it is largely located in the 17 000 fragment. The data suggest that both the anion transport and inhibitor binding sites are located in a 17 000 transmembrane segment of band 3.     

Changes in erythrocyte membranes after fractioned hyperthermia

The structural changes in erythrocytes membranes were examined before and after the second heat shock of erythrocytes. Electrophoretic separation of protein erythrocyte membranes for cells incubated at 48.5°C was different from control i.e. from erythrocytes incubated at 37°C. No quantitative or qualitative changes were spotted in comparison with protein membranes isolated from the erythrocytes following single or double heat shock. Fluidity of erythrocytes membranes was determined by using spin labels, 5-doxylstearic acid and 16-doxylstearic acid. The membranes were more rigid in their hydrophobic regions after incubation of cells at 44°C. It can be suggested that erythrocyte membranes play some role in thermotolerance and heat damage of enuclate cells.