Reaction of Se with SH groups in spectrin is involved in the stabilization of erythrocyte membrane skeleton

Na2SeO3 supplementation in the dialysis medium could obviously prevent the dissociation of spectrin from the erythrocyte membranes. Such Se effect could be eliminated by pretreatment of erythrocyte membranes with a SH-blocking reagent, iodoacetamide(IAA) or addition of a SH-compound, dithio-threitol. The fluorescence intensity of erythrocyte membranes labelled with the fluorescent probe N-(3-pyrenyl)-maleimide decreased with increasing Na2SeO3 concentration used for pretreatment of ghosts. 31P-NMR spectra of erythrocyte membrane dialyzed in the presence or absence of Na2SeO3 concentration showed a difference in chemical shift anisotropy (delta sigma) between these two samples. These data suggest that the stabilization effect is based on changes in lipid-protein interaction and conformation of membrane skeletal components induced by reaction of their SH groups with Na2SeO3.     

Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane.

The effect of ionic strength on the proteolysis by trypsin of the major membrane-penetrating protein (polypeptide 3) in the erythrocyte membrane was studied. Both the intracellular and extracellular regions of the protein are susceptible to trypsin proteolysis under hypo-osmotic conditions, whereas under iso-osmotic conditions the extracellular region of the protein is resistant to trypsin, and the intracellular region yields only two cleavage products with trypsin. Studies of the fragments obtained from polypeptide 3 by trypsin digestion under iso-osmotic conditions of 'ghosts' radioiodinated with lactoperoxidase confirmed our earlier conclusions that the polypeptide chain of polypeptide 3 traverses the membrane twice. Ionic-strength-dependent changes were also observed in the incorporation of iodine by lactoperoxidase into the individual extracellular tyrosine sites of the protein. These results show that polypeptide 3 undergoes ionic-strength-dependent changes in structure.     

The diversity of sulfhydryl groups in the human erythrocyte membrane

Human bank blood erythrocytes were exposed to the mercurials p-chloromercuribenzoate (PCMB), chlormerodrin (CM), p-chloromercuribenzenesulfonate (PCMBS), and 1-bromomercuri-2-hydroxypropane (BMHP) for different time intervals, at different concentrations and in combination with n-ethylmaleimide (NEM) added before, and 2-mercaptoethylguanidine (MEG) and reduced glutathione (GSH) added after the mercurial. Binding patterns of the mercurials to the cells and effects on permeability of the cells were measured. The results indicate that the erythrocyte membrane contains multiple classes of sulfhydryl groups, alteration of which has a variety of effects on cell permeability. PCMB, chlormerodrin and PCMBS react with at least three classes of sulfhydryls, two of which are associated with the sodium-potassium barrier and, when altered, result in potassium loss, sodium accumulation and hemolysis. BMHP reacts with at least two classes of sulfhydryls, one of which is associated with permeability, and, when altered, results in hemolysis in isotonic solutions of choline chloride or lactose. The results provide additional insight into the structure and function of the erythrocyte membrane.     

Platelet and erythrocyte membrane changes in Alzheimer's disease

Previous reports have suggested that the physical properties of cell membranes and calcium homeostasis in both the central and peripheral nervous system are changed in Alzheimer's disease (AD). This study has examined the biophysical properties of erythrocyte and platelet membranes by measuring the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and possible related changes in lipid peroxidation. In addition, we have studied calcium homeostasis by measuring thrombin-stimulated changes in intraplatelet free calcium and Ca2(+)-ATPase activity in AD and healthy age and sex-matched controls. Our results show that there was no significant difference in the fluorescence anisotropy of DPH in erythrocyte membranes isolated from the three groups. There was also no significant difference in lipid peroxidation levels in erythrocytes and plasma of AD patients compared to controls. However, there was a significant reduction in the fluorescence anisotropy of DPH in platelet membranes from AD patients, compared with healthy controls. Recent evident suggests that the increase in platelet membrane fluidity results from alterations in internal membranes. We measured the specific activities of enzyme markers associated with intracellular and plasma membranes in platelets from AD patients and healthy controls. There was a significant reduction in the specific activity of antimycin A-insensitive NADH-cytochrome-c reductase (a specific marker for smooth endoplasmic reticulum (SER)), in AD patients compared to controls, but no change in the specific activity of bis(p-nitrophenyl)phosphate phosphodiesterase (a specific marker for plasma membrane). We have also shown that SER mediated [Ca2+] homeostasis is possibly impaired in AD platelets, i.e., the percentage of thrombin-stimulated increase in intraplatelet [Ca2+] above basal levels was significantly higher in AD compared to matched controls and there were significant reductions in the specific activities of Ca2+/Mg2(+)-ATPase and Ca2(+)-ATPase (but not Mg2(+)-ATPase) in AD platelets. Finally electron microscopic analysis of platelets showed that there was a significant increase in the incidence of abnormal membranes in AD patients compared to controls. The ultrastructural abnormalities seem to consist of proliferation of a system of trabeculated cisternae bounded by SER. These results suggest that both SER structure and function might be defected in AD platelets, which could explain the fluidity changes observed here.     

Radiation-induced changes in the structure of erythrocyte membrane proteins

Structural changes in proteins of erythrocyte membranes induced by gamma-radiation at doses of 10-10(3) Gy were studied using the method of tryptophan fluorescence quenching by acrylamide. It was found that the exposure to ionizing radiation leads to a decrease in intramolecular dynamics of membrane proteins.     

Relationship between membrane ATPase and shape changes in the dog erythrocyte

The effects of heating blood to 57 degrees C on intraerythrocytic calcium, membrane ATPase activity and cell shape have been studied in canine blood. Intraerythrocytic calcium was determined by use of arsenazo III, membrane ATPase activity was determined by inorganic phosphorous formation and erythrocyte shape was determined by scanning electron microscopy. The results of this study showed that this degree of thermal trauma would cause a 27% increase in intraerythrocytic calcium and a 38% decrease in ATPase activity. During these changes in calcium and ATPase activity the erythrocyte changed form from biconcave to spherical. Addition of catalase (3,200 U/ml) to the blood prior to heating prevented the changes observed in intraerythrocytic calcium, membrane ATPase activity and shape. The addition of the free-radical generating combination of hypoxanthine-xanthine oxidase to blood produced a 20% decrease in membrane ATPase activity and a change in erythrocyte shape, but did not alter intraerythrocytic calcium. These results suggest that free-radicals are responsible for the changes in membrane ATPase activity. The observation that shape change occurs when ATPase activity has been decreased, but calcium has not been increased, suggests that membrane ATPase activity levels are more important in producing changes in erythrocyte shape than are intraerythrocytic calcium levels.     

Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane–cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane–cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.     

Presence of horse blood group antigens in the major glycoprotein fraction of the erythrocyte membrane

The aqueous phase of the chloroform/methanol extract of the horse erythrocyte membrane contained the blood group activities Ad, Dc or Dd. The factors Ad and Dc could be separated by gel filtration.     

Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. I. Impairment of resealing and formation of aqueous pores in the ghost membrane after modification of SH groups.

Resealed human erythrocyte ghosts prepared by a two-step procedure were shown to have small residual barrier defects with the properties of aqueous pores, such as size discrimination of hydrophilic nonelectrolytes (erythritol to sucrose), indicative of an apparent pore radius of about 0.7 nm, and a low activation energy (about 12-20 kJ/mol (mannitol, sucrose)) of the leak fluxes. As in other cases (Deuticke et al. (1991) Biochim. Biophys. Acta 1067, 111-122) these leak fluxes can be inhibited by phloretin. Treatment of such resealed ghosts with the mild SH oxidizing agent, diamide, induces additional membrane leaks to the same extent and with the same properties as in native erythrocytes (Deuticke et al. (1983) Biochim. Biophys. Acta 731, 196-210), including reversibility of the leak by SH reducing agents, inhibition by phloretin and stimulation by alkanols. In contrast, resealed ghosts prepared either from diamide-treated erythrocytes or by adding diamide to the 'open' membranes prior to reconstitution of high ionic strength and raising the temperature, exhibit a state of greater leakiness. This leakiness is somewhat different in its origin from the former class of leaks, since it can also be produced by N-ethylmaleimide, which is essentially ineffective when added to the membrane in its 'tight' state. The leaks induced in the 'open' state of the membrane, which can be regarded as a consequence of an impaired resealing, are nevertheless reversible by reducing agents added after resealing and are comparable in many, but not all their characteristics to leaks induced in the 'tight' state of the membrane. Resealing in the presence of the isothiocyanostilbenes DIDS or SITS mimicks the leak forming effect of diamide by modifying a small population of SH groups, while amino groups seem not to be involved. The findings indicate and substantiate an important role of the redox state of membrane skeletal protein sulfhydryls in the maintenance and the re-establishment of the barrier function of the erythrocyte membrane.     

Diversity of the human erythrocyte membrane sialic acids in relation with blood groups

The aim of the present study was to detect defective structural properties in bilayers of mitochondrial phospholipids after oxidative stress of isolated mitochondria in vitro, reportedly during respiration state IV. The structural behaviour of extracted phospholipids was studied by electron paramagnetic resonance (EPR) spectrometry in oriented phospholipid bilayers spin-labelled with 5-doxyl-lecithin, by detecting of the degree of EPR spectral anisotropy loss, indicative of the phospholipid bilayer packing order. Bilayers of phospholipids from untreated mitochondria showed the highest spectral anisotropy, hence highly ordered structure, while chemically oxidised phospholipid yielded almost completely disordered supported phospholipid bilayers. Samples from mitochondria after respiration state IV showed bilayer disorder increasing with oxidation time, while inclusion of the antioxidant resveratrol in the respiration medium almost completely prevented bilayer disordering. On the other hand, β-n-doxylstearoyl-lecithin spin-labelled mitochondria showed unchanged order parameter S at C positions 5, 12 and 16 after respiration state IV, confirming the insensitivity of this parameter to phospholipid oxidative stress. It is concluded that reactive oxygen species attack to the membrane affects lipid packing order more than fluidity, and that EPR anisotropy loss reveals oxidative damage to the bilayer better than the order parameter.     

Active transport and enzymes of the erythrocyte membrane under protein deprivation

1. Starvation for 3 days causes membrane damage of the rat erythrocyte manifested by several alterations. The adenosine-triphosphatase activity is decreased but that of acetylcholinesterase is not affected. 2. The ouabain-sensitive adenosine-triphosphatase activity increases at the expense of the non-sensitive enzyme moiety. 3. The Rb(+) uptake is not altered but the galactose transport is accelerated by the stated experimental conditions. 4. The modifications induced by starvation do not recover on re-feeding.