Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

Prostaglandins do not affect ion concentrations in human erythrocytes.

In an attempt to further our understanding of the mode of action of prostaglandins (PGs), experiments were performed in which sodium and potassium ion concentrations were measured in human erythrocytes exposed to various concentrations of PGs. No consistent effect was observed and it is concluded that such changes are not likely to underlie excitability alterations induced by PGs.     

Effect of ion adsorption on the electrokinetic properties of erythrocytes

The microelectrophoresis technique was used to determine the dependence of human erythrocyte surface potential on the concentration of various cations and anions. The interpretation of the results is based on the Gouy--Chapman--Stern theory. Values of pK, characterizing the binding of ions to the external surface of erythrocytes, as well as numbers of binding sites per unit area were determined. The affinities of ions for the red cell membrane were shown to decrease in the sequence: H+ greater than Ca2+ greater than Sr2+ greater than Mg2+ greater than Ba2+ greater than Li+ greater than Na+ congruent to congruent to K+ congruent to NH4+ and trinitrophenol greater than IO4- greater than CIO4- greater than salicylate congruent to I- greater than greater than SCN- greater than H2PO4- greater than Br- greater than Cl- greater than HPO4(2-). Changes in the ionic strength of the medium resulted in changes in numbers of exposed ion-binding sites. This phenomenon is interpreted in terms of ionic strength-dependent structural transformations of the cell surface coat.     

The effect of lysosomotropic detergents on the permeability properties of the lysosome membrane

Compounds such as N-dodecylimidazole and N-dodecylmorpholine kill cells in culture. Their cytotoxicity has been attributed to accumulation in lysosomes where protonation confers detergent properties resulting in membrane destabilization. This hypothesis has been tested by examining the ability of N-dodecylimidazole and N-dodecylmorpholine to decrease the latency of alpha-glucosidase in isolated rat liver lysosomes. No effect was observed. Nor was N-dodecylimidazole apparently able to increase the permeability of isolated rat liver lysosomes to L-alanine, as no diminution of the disruptive effect of L-alanine methyl ester was seen. N-Dodecylimidazole (10-20 micrograms per ml) caused lactate dehydrogenase release from cystinotic fibroblasts, but marginally toxic concentrations failed to induce cystine release, as might have been expected if lysosome membrane damage had occurred. It is concluded that the cytotoxic effects of lysosomotropic detergents may be mediated by a non-lysosomal mechanism.     

Effect of x-ray contrast agents on the deformation properties of human erythrocytes

Using the ectacytometric method the authors observed that the contrast agents: bilimin, triombrast 76%, iodamide-380, bilignost 20%, bilignost 50%, metrizamide decrease red cell deformability. This effect of the contrast agents depends on their osmolality and chemical structure.     

Effect of temperature and pH on magnetic properties of human erythrocytes

Erythrocytes magnetic susceptibility depending on temperature and pH value of buffer were studied by analysing individual erythrocytes moving in a medium in the nonhomogeneous magnetic field near a transversely magnetized thin wire.     

Effect of Triton X-100 on properties of acetylcholinesterase from human erythrocytes

The effect of Triton X-100 on catalytic properties of acetylcholinesterase from human erythrocytes under acetylcholine hydrolysis, on sensitivity of acetylcholinesterase to specific phosphoorganic inhibitors and eserine, and on the mobility and isoenyme spectrum under analytical electrophoresis in polyacrylamide gel is investigated. Triton X-100, independently on its concentration within 0.05-1.0%, slightly changes V and [S]opt values and increases Km value in 2-3 times. The inhibitory effect of Triton X-100 is mainly competitive, 0.5% Triton X-100 decreases bimolecular constant (kII) of the interaction of acetylcholinesterase with phosphoorganic inhibitor and eserine in 2.5-4 times. In the presence of phosphoorganic inhibitor, kII sharply decreased when 0.02% Triton X-100 was added, and then it did not change under the increase of Triton X-100 concentration up to 1.0%. On the basis of these data, an analytical method of estimating Triton X-100 content in protein solution is proposed. The introduction of 0.1% Triton X-100 into polyacrylamide gel results in considerable quantitative redistribution of acetylcholinesterase isoenzyme fractions and in the change of the mobility of one fraction under electrophoresis.     

Effect of hydration on the water content of human erythrocytes.

An ideal, hydrated, nondilute pseudobinary salt-protein-water solution model of the RBC intracellular solution has been developed to describe the osmotic behavior of human erythrocytes during freezing and thawing. Because of the hydration of intracellular solutes (mostly cell proteins), our analytical results predict that at least 16.65% of the isotonic cell water content will be retained within RBCs placed in hypertonic solutions. These findings are consistent not only with the experimental measurements of the amount of isotonic cell water retained within RBCs subjected to nonisotonic extracellular solutions (20-32%) but also with the experimental evidence that all of the water within RBCs is solvent water. By modeling the RBC intracellular solution as a hydrated salt-protein-water solution, no anomalous osmotic behavior is apparent.     

Enfuvirtide effects on human erythrocytes and lymphocytes functional properties.

Enfuvirtide (T-20) is the first inhibitor of human Immunodeficiency Virus type-1 (HIV-1) entrance on a target cell approved for clinical use. Recent studies indicated that its action mechanism involves the interaction with the membrane surface, increasing the concentration in the site of action. In the present study, the in vitro interaction between enfuvirtide and blood cells of healthy human donors, namely erythrocytes and lymphocytes, and the peptide effect on plasma and lymphocyte suspensions supernatant ions were evaluated, in order to better characterize the action of this peptide. Enfuvirtide causes a decrease in the concentration of hemoglobin and in the percentages of methemoglobin and carboxyhemoglobin, together with increased values of P50, pCO2, and [HCO3-], and significant decreases of pO2 and pH, in blood plasma. The supernatants of lymphocyte suspensions derived from blood incubated with enfuvirtide presented a decrease in pH and [HCO3-]. Fluorescence anisotropy measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), used to assess erythrocyte and lymphocyte membrane fluidity, did not yield enfuvirtide-induced changes (an effect could be expected due to peptide partition to lipid bilayers). Erythrocytes incubated with high enfuvirtide concentrations showed a significant decrease in osmotic fragility. As for erythrocyte deformability, enfuvirtide leads to increased elongation indexes for low shear stress values, whereas for high shear stress values it has the opposite effect. Despite the observed statistically significant variations in several parameters, these enfuvirtide-induced changes are not expected to lead to any detectable biomedical outcome for enfuvirtide-treated patients.     

Dependence of calcium permeability of sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The ability of sarcoplasmic reticulum vesicles to retain calcium following ATP-supported calcium uptake in the presence of the calcium-precipitating anions oxalate and phosphate depends on Cao (calcium ion concentration outside the vesicles) and Cai (calcium ion concentration within the vesicles). Calcium efflux rates at any level of Cai are accelerated when Cao is increased. Higher Cao at the time that calcium uptake reactions reach steady state is associated with a spontaneous calcium release that reflects this effect of increased Cao. Increasing Cai at any level of Cao causes little or no acceleration of calcium efflux rate so that calcium permeability coefficients, estimated by dividing calcium efflux rates by Cai, the "driving force", are inversely proportional to Cai. Calcium permability coefficients thus correlate, as a first approximation, with the ratio Cai/Cao, decreasing 1000-fold as this ratio increases over a 3000-fold range (Cao = 0.1 to 3.3 muM, Cai =4 to 750 muM). Oscillations in both the calcium content of the vesicles and Cao are seen as calcium uptake reactions approach steady state, suggesting that calcium permeability undergoes time-dependent variations. Sudden reduction of Cao to levels that markedly inhibit calcium influx via the calcium pump unmasks a calcium efflux that decreases slowly over 60 to 90 s.The maximal calcium permeability observed in the present study would allow the calcium efflux rate from the sarcoplasmic reticulum at a Cai of 100 muM to be approximately 10(-10) mol/cm2/s, which is about 1 order of magnitude less than that estimated for the sarcoplasmic reticulum of activated skeletal muscle in vivo. The release of most of the stored calcium in some experiments indicates that the observed permeability changes can occur over a large portion of the surface of the sarcoplasmic reticulum.     

Phosphorescent analysis of the action of detergents on the internal dynamics of membrane proteins of human erythrocytes

The slow (millisecond) internal dynamics of proteins isolated from human erythrocyte membranes under the action of ionic and nonionic detergents: sodium dodecyl sulfate (0.1-6 mM), sodium deoxycholate (0.16-6 MM), N-Lauroylsarcosine Na+-salt (Sarkosyl) (0.17-6 mM), digitonin (0.025-6 MM), and Tween-20 (0.01-6 mM) has been studied by the method of room-temperature tryptophan phosphorescence. It has been established that the destruction of protein ensembles, the disturbance of protein-lipid interactions, and the unfolding of proteins in membrane result in a considerable increase of slow intramolecular dynamics of proteins. The effects of detergents on the structural and dynamical state of membrane proteins differ depending on their chemical features. On the bases of the results obtained, it has been concluded that the low internal dynamics of membrane proteins in situ, compared with most soluble proteins, is due to the presence of protein ensembles in membrane, the isolation of macromolecules from the aqueous surroundings by the lipid bilayer, and a high content of alpha-helices and beta-sheets in macromolecules.     

Effect of detergents on antibody-antigen interaction.

The effect of the different detergent mixtures on immunodiffusion and immunoprecipitation was studied. The anionic detergent sodium dodecyl sulfate at concentrations above 0.2% ($\text{w}\text{v}$) inhibits the reaction between antigen and antibody by more than 90%. Nonionic detergents at a concentration of 1% ($\text{w}\text{v}$) have little or no detectable effect. In contrast, when we used mixtures of various concentrations of ionic and nonionic detergents the inhibitory effect of the ionic detergent decreased.