Interaction of molybdenum with human erythrocyte membrane proteins

This study describes the interaction of molybdenum with blood components. Molybdenum-99 was added to blood, and after four washings, 3% of the total radioactivity was found in red cells. More specifically, the radioactivity was determined to be associated with the cell membrane. Molybdenum-99 in the +VI form did not interact with the human erythrocyte membrane; however, Mo(V) forms did interact. Of five different compounds, the highes uptake was observed with a brown Mo(V)-ascorbate complex generated from Mo(VI) and ascorbic acid in the molar ratio 1∶20. A membrane suspension of Mo-ascorbate-treated human erythrocytes was prepared and the solubilized proteins were separated on a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS). Molybdenum-99 binding to spectrin was demonstrated, as well as some minor interactions with membrane hemoglobin and bands 6 and 8.     

Insulin control of a transplasma membrane NADH dehydrogenase in erythrocyte membranes

A study of NADH ferricyanide reductase activity in oriented vesicles or open ghosts of human and porcine erythrocytes shows that the dehydrogenase activity can have three types of orientation in the membrane. There is activity which responds only to acceptors and NADH exclusively on the inside face, or exclusively on the outer surface. There is also activity which requires exposure of both sides of the membrane and thus is transmembranous. The transmembrane activity is inhibited by insulin, whereas the internal and external enzymes do not respond to insulin. The transmembrane dehydrogenase can be a basis for proton transport in the plasma membrane.     

Human erythrocyte galactosyltransferase. Characterization, membrane association and sidedness of active site

Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-α-d-galactopyranosylpeptide galactose transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements, Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate-inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase.     

Spectrin involvement in a 40 degrees C structural transition of the red blood cell membrane

Proteins involved in a structural transition detected in red blood cell membranes at 40 degrees C by spin labeling methods have been investigated. Antibodies specific for spectrin, band 3, and protein 4.1 have been used as specific probes to modify membrane thermotropic properties. Spectrin seems to be involved in a 40 degrees C transition detected in ghosts by both a stearic acid spin label (16-doxyl stearic) and a sulfhydryl-specific maleimide analogue spin label. Circular dichroism and maleimide spin labeling studies of purified spectrin show a slow unfolding of the protein structure starting at 25-30 degrees C and a massive transition with an onset temperature of 48 and 40 degrees C, respectively. This thermotropic behavior of spectrin could be the process that modifies membrane physicochemical properties above 40 degrees C that are detected by the stearic acid spin label. The transition detected by the stearic acid spin label was modified both by antispectrin antibodies and anti-4.1 protein antibodies, but not by antibodies specific for the cytoplasmic domain of band 3. These results suggest an involvement of protein 4.1 in regulating spectrin unfolding at the membrane level. A selective inhibition of the transition detected by the maleimide spin label has been obtained with a monoclonal antispectrin antibody at 1:1 molar ratio. The involvement in this transition of a localized spectrin domain(s) containing few exposed sulfhydryl groups is proposed.     

Modification of the erythrocyte membrane by a non-specific lipid transfer protein

The non-specific phospholipid transfer protein purified from bovine liver has been used to modify the phospholipid content and phospholipid composition of the membrane of intact human erythrocytes. Apart from an exchange of phosphatidylcholine between the red cell and PC-containing vesicles, the protein appeared to facilitate net transfer of phosphatidylcholine from the donor vesicles to the erythrocyte and sphingomyelin transfer in the opposite direction. Phosphatidylcholine transfer was accompanied by an equivalent transfer (on a molar basis) of cholesterol. An increase in phosphatidylcholine content in the erythrocyte membrane from 90 to 282 nmol per 100 μl packed cells was observed. Phospholipase C treatment of modified cells showed that all of the phosphatidylcholine which was transferred to the erythrocyte was incorporated in the lipid bilayer. The nonspecific lipid transfer protein used here appeared to be a suitable tool to modify lipid content and composition of the erythrocyte membrane, and possible applications of this approach are discussed.     

Incorporation of the human erythrocyte sialoglycoprotein into recombined membranes containing cholesterol

Summary Glycophorin, the major sialoglycoprotein from the human erythrocyte membrane, has been isolated and recombined with phosphatidylcholine and cholesterol. Sucrose density gradient analysis of the recombinants shows that it is possible not only to recombine this protein with phospholipid, but also with phospholipid-cholesterol mixtures. Surprisingly, by the same analysis, it was possible to make a recombinant with cholesterol and glycophorin, only, in the absence of added phospholipid. The accessibility of the protein to trypsin was tested in each of these recombinants. In all the recombinants which contained either phospholipid, or phospholipid and cholesterol, the protein was protected from extensive hydrolysis. This is consistent with closed vesicles and incorporation of the protein into the recombinant membrane. Extensive hydrolysis of the protein occurred in the cholesterol-glycophorin recombinant indicating some differences in structure. Freeze-fracture electron microscopy of the phospholipid and the phospholipid-cholesterol recombinants showed mostly unilamellar vesicles, 1000 to 5000 Å in diameter. Intramembranous particles were observed on both fracture faces, and the fracture planes were those expected for phospholipid bilayers. The glycophorin-cholesterol recombinants also showed fracture planes consistent with bilayers, and revealed intramembranous particles. Pieces of membrane-like structures as well as apparent vesicular structures were observed. Finally in the recombinants of glycophorin with phospholipid and cholesterol, cholesterol is shown to reduce the population of the motionally restricted phospholipid headgroup environment, in proportion to the mole percent cholesterol content.     

Effects antifreeze peptides on the thermotropic properties of a model membrane

In this paper, we report on the effect of short segments of type I antifreeze protein (AFP I) on the thermotropic properties of a model membrane. Two different types of dimyristoylphosphatidylcholine model membranes were used, multilamellar vesicles and small unilamellar vesicles. The membrane properties were studied by differential scanning calorimetry (DSC) and fluorescence anisotropy. With the incorporation of AFP I and its short segments, the order of the model membrane increased both in the gel state and in the liquid crystalline state. The interaction of AFPs with the model membrane caused a shift in the phase transition to lower temperatures, which is accompanied by a broadening of the DSC thermogram. This preferential stabilization to a more ordered phase by the AFPs could be due to ordering the hydrophobic membrane core and separation into domains. Overall, this approach of employing short segments of AFP I simplifies the correlation between antifreeze protein characteristics and the effect of these parameters on the interaction mechanism of AFP with cell membranes. The success of this approach can lead to the identification of short peptides with high antifreeze activity.     

Alterations in erythrocyte membrane fluidity in children with trisomy 21: a fluorescence study.

Membrane fluidity of erythrocytes obtained from 15 children with trisomy 21 and 20 healthy controls were studied by measuring steady-state fluorescence anisotropy and fluorescence lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in hemoglobin-free erythrocyte membranes. Our results demonstrate a significant decrease in DPH fluorescence anisotropy and a significant increase in TMA-DPH fluorescence anistropy in erythrocytes from subjects with trisomy 21. No significant differences between the two groups were observed in the fluorescence lifetime of DPH and TMA-DPH. These data suggest an increase in membrane fluidity in the interior part of the membrane and a decrease in fluidity at the lipid-water interface region. This could be in part attributed to an increased oxidative damage in trisomy 21.     

Structural changes in the erythrocyte membrane of the trout Salmo irideus at seasonal acclimatization

Differences of thermostability were studied in red blood cells of the trout Salmo irideus differing in sex and age, as well as structural-dynamic characteristics of erythrocyte membrane proteins at seasonal acclimatization in the interval of reservoir water temperature of 0–19°C. An increase of resistance of erythrocytes to temperature lysis with elevation of the environmental temperature was revealed to be accompanied by a rise of the proteins segmental mobility and a decrease of intermolecular interactions in spectrin-actin cytoskeleton from the data of the ESR spin labeling method. Regulation of erythrocyte stability during acclimation was concluded to occur both changes of the fatty acids chain package at the variations of lipid composition and by changes of the cytoskeleton structural lability. Thereby this provides an increase of the bilayer firmness, on the one hand, while, on the other hand, a rise of elasticity and expansibility of the membrane on the whole, which increases resistance of cells to colloidal-osmotic hemolysis. Changes of concentration of oxygen dissolved in water, which are caused by temperature fluctuations, do not deem to be of crucial importance for structural stability of erythrocytes, as it can be compensated by another mechanism, specifically by changes of affinity of hemoglobin to oxygen.     

Role of membrane thermotropic properties on hypotonic hemolysis and hypertonic cryohemolysis of human red blood cells

The hypothesis of a correlation between the effects of temperature on red blood cells hypotonic hemolysis and hypertonic cryohemolysis and two thermotropic structural transitions evidenced by EPR studies has been tested. Hypertonic cryohemolysis of red blood cells shows critical temperatures at 7 degrees C and 19 degrees C. In hypotonic solution, the osmotic resistance increases near 10 degrees C and levels off above 20 degrees C. EPR studies of red blood cell membrane of a 16-dinyloxyl stearic acid spin label show, in the 0-50 degrees C range, the presence of three thermotropic transitions at 8, 20, and 40 degrees C. Treatments of red blood cells with acidic or alkaline pH, glutaraldehyde, and chlorpromazine abolish hypertonic cryohemolysis and reduce the effect of temperature on hypotonic hemolysis. 16-Dinyloxyl stearic acid spectra of red blood cells treated with glutaraldehyde and chlorpromazine show the disappearance of the 8 degrees C transition. Both the 8 degrees C and the 20 degrees C transitions were abolished by acidic pH treatment. The correlation between the temperature dependence of red blood cell lysis and thermotropic breaks might be indicative of the presence of structural transitions producing areas of mismatching between differently ordered membrane components where the osmotic resistance is decreased.     

Role of the bilayer in the shape of the isolated erythrocyte membrane

Summary The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5mm NaP_i (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1mm 2,4-dinitrophenol or 0.1mm lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine.In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.     

大强度训练及恢复后大鼠红细胞变形能力和红细胞膜蛋白的变化

目的 :探讨运动对红细胞变形性和红细胞膜蛋白的影响及其相互关系。方法 :设计不同强度的训练方案 ,用激光衍射法测定红细胞变形能力 ,用SDS PAGE方法测定一定体积大鼠红细胞膜中的重要蛋白带 3蛋白 (band 3)和肌动蛋白 (actin)的含量 ,研究运动即刻和恢复后红细胞变形性及膜蛋白的变化。结果 :长期的运动训练会促进大鼠红细胞变形能力的改善和红细胞膜band 3蛋白和actin的良好发展 ,一次大强度训练会引起红细胞膜band 3蛋白和actin含量的减少 ,大鼠红细胞变形能力降低 ,一周和二周的大强度训练会提高恢复期大鼠红细胞的变形能力和红细胞膜band 3蛋白和actin含量。结论 :运动训练造成的红细胞膜蛋白含量的变化 ,导致了红细胞膜结构的改变 ,从而影响红细胞变形能力 ,可能是训练对红细胞变形能力的作用机制之一。     

The erythrocyte membrane in essential hypertension: Modified temperature-dependence of Li+ efflux

The rate of ouabain-resistant Li⁺-efflux was studied in erythrocytes of normal controls and of patients with essential hypertension. Despite variability in rate, erythrocytes from normotensive persons revealed a uniform pattern of temperature dependence of the efflux, with two slopes (K_a = 9.4 and 19.1 kcal/mol, respectively) and a transition at about 25°C. Erythrocytes from the patients showed both a higher rate of Li⁺ efflux and significant changes in the temperature repsonse, with essentially a single slope (K_a = 14 kcal/mol). The data indicate localized changes in the membrane organization of hypertensive erythrocytes, involving lipid-protein interaction.     

Transport of benzenesulfonic acid derivatives through the rat erythrocyte membrane

Summary Transport of benzenesulfonic acid derivatives through the rat erythrocyte membrane was studied. The transport properties, such as pH-dependence and effects of reagents reacting with amino-groups, were similar to those of anions like Cl^– through the human erythrocyte membrane. The rate of transport of anions through rat erythrocyte membranes is higher than through those of other mammals, such as guinea pig and bovine erythrocyte membranes. This relatively high rate of transport makes the rat erythrocyte membrane suitable for use in comparative studies on the transports of slowly penetrating substances, such as organic anions. The transport velocities of benzenesulfonic acid derivatives were compared with their physico-chemical properties. It was shown that the hydrophobicity has no effect on the transport, but the electronic property has a significant effect: the transport rate is mainly dependent on thee ^– donor capacities. This feature is the inverse to the well-known inhibitory effect of these derivatives on other anion transport: the inhibition is mainly dependent on thee ^– acceptor capacities. It is suggested that the transport is regulated by the binding capacity of anions to the transport site.     

Lipid unsaturation determines the interaction of AFP type I with model membranes during thermotropic phase transitions

We have previously shown that antifreeze protein (AFP) type I from winter flounder interacts with the acyl chains of lipids in model membranes containing a mixture of dimyristoylphosphatidylcholine (DMPC) and the plant thylakoid lipid digalactosyldiacylglycerol (DGDG), most likely through hydrophobic interactions. By contrast, in studies with pure phospholipid membranes, no such interaction was seen. DGDG is a highly unsaturated lipid, which renders these studies quite different from the previous studies of AFP-membrane interaction where the lipids were saturated or trans-unsaturated. Therefore, it seemed possible that either the digalactose headgroups or the unsaturated DGDG acyl chains, or both, may be important for interactions of membranes with AFP type I. To distinguish between these possibilities, we catalytically hydrogenated the DGDG to obtain a galactolipid with completely saturated fatty acyl chains. The results with the hydrogenated DGDG were strikingly different from those obtained previously with the unsaturated DGDG; the clear binding of AFPs to the bilayer appeared to be lost. Nevertheless, the temperature-dependent folding of AFP type I was inhibited in the presence of liposomes containing either the unsaturated or the hydrogenated DGDG. The results indicate that the liposomes and protein still interact, even following hydrogenation of the acyl chains, perhaps at the membrane-solution interface.     

Cigarette smokers develop altered erythrocyte membrane composition: an investigation unmasking the role of membrane bound integral protein GLUT 1

Erythrocytes in cigarette smokers are prone to oxidative damage. Here, we sought to elucidate the facts behind modifications and possible defense system developed in erythrocyte of cigarette smokers. We observed significant increase in stomatocytes and spherocytes, and osmotic fragility of erythrocyte, along with reduced level of protein thiol and increased fluorescence anisotropy in isolated membrane. Denaturing gel electrophoresis indicated alterations in band 3, band 4.2 and band 4.5. Among those, Glut 1 (i.e. band 4.5), which transports glucose (insulin independent) and dehydroascorbate (DHA), was selectively chosen for its long history in reducing reactive oxygen species (ROS). The increased Glut 1 level in smokers was confirmed by immunoblotting and immunocytochemistry. Furthermore, smokers showed significantly higher glucose uptake in whole blood. The intracellular (Ic) ROS (as indicated by 2′,7′-dichlorofluorescin) was significantly higher in smokers as evidenced by flow cytometric assay. Glucose and DHA alone or together significantly reduced IcROS at higher rate in smokers. However, in presence of Glut 1 specific blocker, phloretin, neither glucose nor DHA could reduce IcROS in both non-smokers and smokers. This confirms that Glut 1 by transporting glucose or DHA attenuates IcROS. Therefore, we conclude that erythrocytes, although altered morphologically, also develop a defense system by upregulating Glut 1 to combat with enhanced Ic oxidative insult in cigarette smokers.     

Lead effect on the oxidation resistance of erythrocyte membrane in rat triton-induced hyperlipidemia

The anemia observed in severe chronic lead poisoning is in part attributable to alterations in the erythrocyte physicochemical properties. Since they are partly related to the membrane lipid composition, the aim of the present study was to determine the effects of a triton-induced hyperlipidemia on the resistance to oxidation of erythrocyte membranes in lead-treated Wistar rats. Our results showed that triton administration to lead-treated rats induced an increase in erythrocyte choline phospholipid levels together with a significant decrease in the erythrocyte membrane lipid resistance to oxidation. These results led us to suggest that anemia in lead poisoning is linked to interactions between lead present in the membrane and plasma phospholipids. Their increase in rat hyperlipidemia induced by triton resulted in a decrease in the membrane resistance to oxidation and finally in an erythrocyte fragility leading to their destruction.     

Transport of hydrophobic ions in erythrocyte membrane: I. Zero membrane potential properties

Summary The permeability and partition coefficients of tetraphenylarsonium (TPA) and several other organic cations were studied in the human erythrocyte using an ion-selective electrode. The permeability constant for the different cations could be explained quite well by differences in oil/water partition coefficients. No evidence for facilitated transport could be found. Binding of the organic ions occurred to both the cell membrane and to intracellular contents. Partitioning to the membrane remained relatively constant despite variation from ion intracellular binding with blood samples from different donors. TPA flux is stimulated by substoichiometric amounts of tetraphenylboron and other organic anions, suggesting an ion-pairing mechanism.     

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.     

Effects of nonionic amphiphiles at sublytic concentrations on the erythrocyte membrane

The interactions of octaethyleneglycol alkylethers (C10-C16), pentaethyleneglycol dodecylether, and dodecyl D-maltoside with the human erythrocyte membrane were studied. All the amphiphiles protected erythrocytes against hypotonic haemolysis. At concentrations where the amphiphiles protected erythrocytes against hypotonic haemolysis they reduced phosphate efflux. The potency of the amphiphiles, at equiprotecting concentrations, was correlated negatively to the length of the alkyl chain. Potassium fluxes were increased by all the amphiphiles at protective concentrations. The relative potency of the amphiphiles varied but it was not simply related to the length of the alkyl chain. The only amphiphile affecting active potassium influx was octaethyleneglycol decylether which induced a slight decrease. It is concluded that the increase in passive cation fluxes caused by the amphiphiles is due to an increased permeability of the lipid bilayer induced through a nonspecific interaction of the amphiphiles with the bilayer. The effect of the amphiphiles on ion transport mediated by membrane proteins is proposed to be due to an alteration of the state of the transporting protein.