High-pressure-induced hemolysis in papain-digested human erythrocytes is suppressed by cross-linking of band 3 via anti-band 3 antibodies

Upon exposure of human erythrocytes to a high pressure of 200 mPa, both hemolysis and vesiculation occur. The hemolysis of erythrocytes at 200 mPa was enhanced by removal of sialic acids from the membrane surface with papain. However, such enhancement was suppressed by cross-linking of band 3 via an anti-band 3 antibody (AB3A), which recognizes the exofacial domain of band 3, or by clustering of band 3 via Zn2+. On the other hand, the size of high-pressure-induced vesicles increased from 423 to 525 nm in diameter upon exposure to papain of erythrocytes, but decreased to 444 nm with following treatment with AB3A. In these vesicles, the content of spectrin relative to band 3 was almost the same. Furthermore, the band 3-cytoskeleton interactions in erythrocyte membranes remained unaltered upon treatment with papain and AB3A. Flow cytometric analysis demonstrated that papain-pretreated erythrocytes mainly produce open ghosts at 200 mPa and that the production of such open ghosts is suppressed by AB3A. Thus, upon removal of negative charges from the membrane surface, open ghosts are readily produced due to the release of larger vesicles under pressure. Upon cross-linking of band 3 via AB3A, however, the release of smaller vesicles at 200 mPa is facilitated so that high-pressure-induced hemolysis is suppressed.     

Effect of nickel,copper, and zinc on emulsifier production and saturation of cellular fatty acids in the filamentous fungus Curvularia lunata

In the first step of this investigation the toxicity of Ni²⁺, Cu²⁺, and Zn²⁺ ions to the emulsifier producing strain of Curvularia lunata was assessed. Among all the heavy metals studied, Ni²⁺ ions were found to be the most toxic to C. lunata, whereas Zn²⁺ ions exhibited the lowest toxicity. Moreover, only Ni²⁺, when used at sublethal concentration (5 mM) caused lysis of some hyphal tip cells after a short-term exposure (5 h). In the next step, emulsifier production, accumulation of heavy metals by mycelia and emulsifier as well as saturation of cellular fatty acids were examined in 48-h-old cultures where fungal growth intensity was not inhibited by heavy metals (in the presence of Ni²⁺, Cu²⁺, and Zn²⁺ ions at the initial concentration of 1, 5, and 15 mM, respectively) and in cultures where approximately 50% biomass inhibition occurred (in the presence of Ni²⁺, Cu²⁺, and Zn²⁺ ions at the initial concentrations of 3, 10, and 17.5 mM, respectively). Among all the heavy metals studied only Ni²⁺ ions did not induce emulsifier production. As compared with the control, only biomass treated with Ni²⁺ ions displayed an increase in total lipid saturation. This effect resulted mainly from the decrease in linoleic acid (18:2) content correlated with the increase in the amount of stearic acid (18:0). The possible mechanisms by which Ni²⁺ ions could alter the fatty acid profile of C. lunata and the protective role of the emulsifier were also discussed.     

Modulation of linoleic acid-binding properties of human serum albumin by divalent metal cations

Human serum albumin (HSA) is an abundant multiligand carrier protein, linked to progression of Alzheimer’s disease (AD). Blood HSA serves as a depot of amyloid β (Aβ) peptide. Aβ peptide-buffering properties of HSA depend on interaction with its ligands. Some of the ligands, namely, linoleic acid (LA), zinc and copper ions are involved into AD progression. To clarify the interplay between LA and metal ion binding to HSA, the dependence of LA binding to HSA on Zn²⁺, Cu²⁺, Mg²⁺ and Ca²⁺ levels and structural consequences of these interactions have been explored. Seven LA molecules are bound per HSA molecule in the absence of the metal ions. Zn²⁺ binding to HSA causes a loss of one bound LA molecule, while the other metals studied exert an opposite effect (1–2 extra LA molecules are bound). In most cases, the observed effects are not related to the metal-induced changes in HSA quaternary structure. However, the Zn²⁺-induced decline in LA capacity of HSA could be due to accumulation of multimeric HSA forms. Opposite to Ca²⁺/Mg²⁺-binding, Zn²⁺ or Cu²⁺ association with HSA induces marked changes in its hydrophobic surface. Overall, the divalent metal ions modulate LA capacity and affinity of HSA to a different extent. LA- and Ca²⁺-binding to HSA synergistically support each other. Zn²⁺ and Cu²⁺ induce more pronounced changes in hydrophobic surface and quaternary structure of HSA and its LA capacity. A misbalanced metabolism of these ions in AD could modify interactions of HSA with LA, other fatty acids and hydrophobic substances, associated with AD.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Differential resistance to calcium-induced bilirubin-dependent hemolysis in mammalian erythrocytes

Washed erythrocytes from human, buffalo, sheep and goat preincubated with different concentrations of calcium chloride (16.7–1830 μM) showed significantly different rates of hemolysis (up to 62%) after addition of bilirubin (72 μM). Goat erythrocytes displayed marked resistance to hemolysis with only 11% hemolysis observed at the highest calcium concentration. Similar trend in hemolysis was also observed when the concentration of CaCl₂ was fixed (330 μM) and bilirubin concentration varied (0–72 μM). (Ca²⁺–Mg²⁺)-ATPase levels were found significantly lower in goat and sheep erythrocyte membranes compared to human and buffalo erythrocyte membranes. This was correlated well with the observed hemolysis in various mammalian erythrocytes.     

Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

Fusion of human erythrocytes induced by uranyl acetate and rare earth metals

Incubation of human erythrocytes with either uranyl ions (UO₂²⁺) or rare earth metals (La³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Tb³⁺, Dy³⁺ and Yb³⁺) at 37°C for 30–45 min resulted in the fusion of erythrocytes. Redistribution of membrane-associated particles was observed using colloidal-iron charge labelling and freeze-fracture electron microscopy. The fusion of erythrocytes induced by these agents, unlike Ca²⁺, did not exhibit the absolute requirement for phosphate. Moreover, agglutination and fusion by these agents was observed in neuraminidase-treated erythrocytes in contrast to Ca²⁺- and phosphate-induced fusion. Inhibitors of intrinsic transglutaminase activity partially inhibited (35–45%) the fusion induced by UO₂²⁺ suggesting that cross-linking of membrane proteins results in protein-free areas of lipid where fusion may be initiated.     

Purification and characterization of the inactive Ca2+, Mg2+-activated adenosine triphosphatase of the unc A- mutant Escherichia coli AN120.

Resealing of erythrocyte ghosts in the presence of 4.5 mm Ca²⁺ induces the segregation of small membrane vesicles with a very high phospholipid:protein ratio and a high lysolecithin content. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the vesicles consist mainly of the high molecular spectrin peptides, the Ca²⁺-induced increase of band IIa (M_r 198,000) which is not extractable at low ionic strength, and a weak peptide band in the 72,000 M_r region. Ca²⁺ ghosts and vesicles show significant differences with regard to the specific activities of several membrane-associated enzymes. The segregated vesicles dispose of an efficient outwarddirected Ca²⁺-transport system.     

The role of intracellular zinc in H2O2-induced oxidative stress in human erythrocytes

In vitro exposure of human erythrocytes to H₂O₂ at concentrations of 30–1000 μM resulted in a dose-dependent increase of the intracellular levels of Zn²⁺ and inhibition of the cytosolic esterase activity, which is a major marker of erythrocyte viability. The observed effect depended on the concentration of H₂O₂ and the duration of exposure of the cells to this compound. An inverse relationship between the changes in the intracellular level of labile zinc ions and esterase activity in the cells exposed to hydrogen peroxide was detected; this was indicative of the role of Zn²⁺ in the programmed death of red blood cells. The combined action of hydrogen peroxide and N',N'-tetrakis-(2-pyridyl-methyl)-ethylenediamine, an intracellular zinc ion chelator, has been found to eliminate the cytotoxic effect of H₂O₂, whereas the addition of Zn²⁺ to the erythrocyte incubation medium enhanced the effects of hydrogen peroxide. The reduction of the concentration of non-protein thiol groups due to a decrease of the level of reduced glutathione was shown to contribute to the release of Zn²⁺ from the intracellular binding sites during oxidative stress induced by H₂O₂ in human erythrocytes.

     

Effect of metabolic state on phytohemagglutinin-P agglutination of normal human erythrocytes

A reproducible quantitative assay for the lectin-mediated agglutination of human erythrocytes, depending on different rates of settling of agglutinated and non-agglutinated erythrocytes, was developed. This assay was used to study the aggregation of human erythrocytes by phytohemagglutinin-P. The aggregation of human erythrocytes by phytohemagglutinin-P was found to depend upon the metabolic state of the cells. Metabolically depleted erythrocytes agglutinated much less readily than did similar cells supplied with adenosine. this was not due to swelling and rigidity of the cells, since erythrocytes in hypotonic solution did not exhibit significantly altered phytohemagglutinin-P agglutination.Metabolically depleted erythrocytes, or erythrocytes from blood stored 8 weeks, lysed and resealed in the presence of ATP, were agglutinated by phytohemagglutinin-P to a much greater extent than control samples without ATP. The presence of Mg²⁺, either alone or with ATP, had little effect on the agglutinability of the resealed membranes. Low concentrations of Ca²⁺ (0.2 mM) had little effect on agglutinability, although high Ca²⁺ (5 mM) inhibited agglutinability of the resealed membranes somewhat.Both metabolically depleted erythrocytes and depleted erythrocytes, previously treated with adenosine, when treated with trypsin released similar amounts of sialic acid. The agglutinability of the trypsinized adenosine-supplemented cells increased more readily than did that of trypsinized metabolically depleted cells.The agglutination of erythrocytes was not affected by cytochalasin B (40 μg/ml). Vinblastine (0.2 mM) caused depleted erythrocytes to agglutinate similarly to adenosine-supplemented erythrocytes, but had no effect on the agglutination of adenosine-supplemented erythrocytes.It is concluded that ATP in the human erythrocyte probably participates in the modulation of phytohemagglutinin-P agglutinability. This is not a consequence of the more rigid membrane known to accompany ATP depletion in the erythrocyte, or of the effect of ATP levels on Ca²⁺ or Mg²⁺ content. It appears likely that ATP modulates human erythrocyte phytohemagglutinin-P agglutinability through interaction, direct or indirect, with a membrane-associated component, which might also be sensitive to vinblastine.     

An immunochemical approach for the analysis of membrane protein alterations in Ca2+-loaded human erythrocytes

An increase in the intracellular concentration of Ca²⁺ in human erythrocytes results in the formation of γ-glutamyl-?-lysine cross-linked membrane protein polymers. Following solubilization of the membranes with SDS, these polymers can be isolated on a Lubrol-containing sucrose gradient. Immunoelectrophoresis of the polymeric material with a polyspecific rabbit antibody against human ghosts gave rise to a single, but heterogeneous, precipitate. The polymer was amphiphilic and, on addition to Triton-solubilized erythrocyte membrane proteins, it coprecipitated with spectrin. When the antighost antibody was absorbed with the polymer prior to cross immunoelectrophoresis of normal erythrocyte membrane proteins, the precipitates of glycophorin, acetylcholinesterase, and hemoglobin were normal, whereas the anti-body liters against band 3 protein, spectrin, and ankyrin became reduced. Furthermore, a rabbit antibody raised against the isolated human polymer reacted selectively with the same three membrane proteins. No reactions occurred with lysate proteins.     

Heavy metal ions are potent inhibitors of protein folding

Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd²⁺, Hg²⁺ and Pb²⁺ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC₅₀ in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far.     

Calcium effects on human erythrocyte membrane proteins

The effects of Ca²⁺ on human erythrocyte membrane proteins were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Ca²⁺ had several effects on normal human erythrocyte membrane proteins. It affected the binding of cytoplasmic proteins to the membrane, produced a non-reversible aggregation of several membrane proteins and activated apparent proteolysis of membrane proteins. The Ca²⁺ effect could be obtained with isolated, washed membranes when the erythrocyte cytoplasm was added. These studies indicate that the Ca²⁺-induced membrane proteolysis and aggregation effects are not due simply to its presence at the time of hemolysis as previously suggested (Carraway, K.L., Triplett, R.B. and Anderson, D.R. (1975) Biochim. Biophys. Acta 379, 571–581), but are the result of more complex interactions between the erythrocyte membrane and cytoplasmic factors.     

Formation of γ-glutamyl-ϵ-lysine bridges between membrane proteins by a Ca2+-regulated enzyme in intact erythrocytes

A rise in the intracellular concentration of Ca²⁺-ions in human erythrocytes causes the formation of high-molecular-weight membrane protein polymers, cross-linked by γ-glutamyl-?-lysine side chain bridges. Cross-linking involves proteins at the cytoplasmic side of the membrane (band 4.1, spectrin, and band 3 materials) and the reaction is catalyzed by the intrinsic transglutaminase. This enzyme is regulated by Ca²⁺-ions and it exists in a latent form in normal cells. The protein polymer, isolated from the membranes of Ca²⁺-loaded intact human red cells, is heterogeneous in size and may contain as many as 6 moles of γ-glutamyl-?-lysine cross-links per 100,000 gm of protein. Synthetic compounds, which either compete against the ?-lysine cross-linking functionalities of the protein substrates (eg, histamine, aminoacetonitrile, cystamine) or directly inactivate the transamidase (eg, cystamine), inhibit the membrane polymerization reaction in intact human erythrocytes. They also interfere with the Ca²⁺-induced irreversible shape change from discocyte to echinocyte and inhibit the irreversible loss of membrane deformability. Thus, the transamidase-catalyzed production of γ-glutamyl-?-lysine cross-links in the membrane may be a common denominator in these cellular manifestations.     

Alkaline phosphatase conductometric biosensor for heavy-metal ions determination

Alkaline phosphatase conductometric biosensors consisting of interdigitated gold electrodes and enzyme membranes have been used for assessment of heavy-metal ions in water. These analytes act as enzyme inhibitors. Enzyme residual activity has been measured in Tris-nitrate buffer without metal preincubation in the presence of Mg²⁺ ions as activator. The results indicate that the toxicity of the various metals tested toward immobilized phosphatase is ranged as follows: Cd²⁺ > Co²⁺ > Zn²⁺ > Ni²⁺ > Pb²⁺. Detection limits were about 0.5 ppm for Cd²⁺, 2 ppm for both Zn²⁺ and Co²⁺, 5 ppm for Ni²⁺ and 40 ppm for lead ions. In addition, the responses during 10 h were stable (RSD 4%) and a drift of about 7% per day was observed. The storage stability in buffer solution at 4 °C remained stable for more than one month.     

An amperometric biosensor based on horseradish peroxidase immobilized onto maize tassel-multi-walled carbon nanotubes modified glassy carbon electrode for determination of heavy metal ions in aqueous solution

A biosensor for trace metal ions based on horseradish peroxidase (HRP) immobilized on maize tassel-multiwalled carbon nanotube (MT-MWCNT) through electrostatic interactions is described herein. The biosensor was characterized using Fourier transform infrared (FTIR), UV–vis spectrometry, voltammetric and amperometric methods. The FTIR and UV–vis results inferred that HRP was not denatured during its immobilization on MT-MWCNT composite. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H₂O₂, before and after incubation in trace metal standard solutions. Under optimum conditions, the inhibition rates of trace metals were proportional to their concentrations in the range of 0.092–0.55 mg L⁻¹, 0.068–2 mg L⁻¹ for Pb²⁺ and Cu²⁺ respectively. The limits of detection were 2.5 μg L⁻¹ for Pb²⁺ and 4.2 μg L⁻¹ for Cu²⁺. Representative Dixon and Cornish-Bowden plots were used to deduce the mode of inhibition induced by the trace metal ions. The inhibition was reversible and mixed for both metal ions. Furthermore, the biosensor showed good stability, selectivity, repeatability and reproducibility.     

Further Studies on Zn2+-Mediated Domain–Domain Communication in Human Erythrocyte Band 3

Human erythrocyte band 3 is purified and reconstituted into vesicles, forming right-side-out proteoliposomes. Zn²⁺entrapped inside the proteoliposomes inhibits the anion transport activity of band 3, and removal of the cytoplasmic domain of band 3 is able to diminish Zn²⁺ inhibition. Thus, the inhibition of activity of band 3 results from the Zn²⁺ induced conformational change of the cytoplasmic domain, which in turn is transmitted to the membrane domain. The results of intrinsic fluorescence and its quenching by HB and the ³⁵Cl NMR study indicate that the cytoplasmic domain is essential for the conformational change induced by Zn²⁺.SH-blocking reagents, CH₃I and GSSG, are used to modify the cytoplasmic domain, where they specifically bind to Cys201 and Cys317. It is observed that the Zn²⁺ induced inhibition of anion transport activity is blocked. This demonstrates that Cys201 and Cys317 are required in Zn²⁺-mediated domain–domain communication.     

Hemolysis and clearance of erythrocytes in Scylla serrata are related to the agglutination by the native sialic acid-specific lectin

The hemolymph of the crab Scylla serrata contains a lectin specific for N-glycolylneuraminic acid. The role of the sialic acid-specific lectin on natural immunity of the crab is studied by using several kinds of mammalian erythrocytes as a pathogen model. A significant correlation is observed between in vivo clearance of exogenous erythrocytes with the extent of erythrocyte agglutination by the lectin. Similarly, another correlation is noticed between the susceptibility of erythrocytes to lectin-dependent hemocytc-mediated hemolysis and the extent of lectin-mediated erythrocyte agglutination. Two hours after administration of the erythrocytes into the hemocoel, induced augmentation of hemagglutinating activity was observed against all erythrocytes, whether agglutinated highly or least by the lectin, suggesting an increase in the circulating lectin. This study documents that “opsonization” of foreign pathogen by the native lectin is an important step in hemocyte recognition, hemolysis and clearance of the pathogen.     

A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon

Zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator’s versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn²⁺, Cu²⁺, and Co²⁺ were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, an optimized method that allows the facile determination of Zn²⁺, Cu²⁺, and Co²⁺ with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn²⁺ and Cu²⁺ within the same sample is described. Using the prototypical Cu²⁺/Zn²⁺-protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantification in metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins.