Effect of reduced and oxidized glutathione on physico-chemical properties of erythrocyte membranes]

The parameters describing the structural and functional state of membranes depending on the level of reduced glutathione in erythrocytes were studied. It was shown, that the decrease in the concentration of reduced intracellular glutathione in erythrocytes upon metabolic depletion (prolonged incubation of cells at 37 degrees C in the absence of glucose) or a rapid irreversible depletion of glutathione with 1-chloro-2,4-dinitrobenzene enhances lipid peroxidation processes in membranes, inhibits the membrane-bound NAD.H methemoglobin reductase activity and decreases the intensity of 1,6-diphenyl-1,3,5-hexatrien fluorescence. The data obtained suggest that the depletion of reduced intracellular glutathione causes changes in the physicochemical state of the erythrocyte membrane: the accumulation of lipid peroxidation products, changes in the physical state of lipid bilayer and the inhibition of membrane-bound NAD.H-methemoglobin reductase activity.     

Changes in the lipid physical state in human erythrocyte membranes subjected lead exposure in vitro

The effect of lead acetate on the physical state of membrane lipids in human erythrocytes in vitro was studied using the lipophilic fluorescence probe 1,6-diphenyl-1,3,5-hexatriene and spin probes 16-doxyl-stearate and iminoxyl palmitic acid. It was shown that 2-10 microM lead acetate causes an increase in both intensity and polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, indicating changes in the microviscosity of the lipid bilayer of erythrocyte membranes. Judging from the parameters of EPR spectra of 16-doxyl stearate and iminoxyl palmitic acid incorporated into erythrocyte membranes, 2-10 microM lead acetate increases the heterogeneity of the lipid bilayer in surface and deep hydrophobic layers of the erythrocyte membrane.     

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.     

Excimer-forming lipids in membrane research

Pyrenedecanoic acid and pyrene lecithin are optical probes well suited to investigate lipid bilayer membranes. The method is based on the determination of the formation of excited dimers or excimers. The rate of excimer formation yields information on the dynamic molecular properties of artificial as well as of natural membranes. This article will review applications of the excimer-forming probes.Pyrene lipid probes are used to determine the coefficient of the lateral diffusion in fluid lipid membranes. Results in artificial membranes are comparable to the values obtained in erythrocyte membranes.Moreover, the excimer formation rate is a very sensitive measure of changes in membrane fluidity. Membrane fluidity is an important regulator of membrane functional proteins. For example, there is a correlation between membrane fluidity and enzyme activities of the adenylate cyclase system.The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transversal mobility, that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. Again, artificial as well as natural membranes can be investigated by this technique.Another important area of investigation in membrane research is the interaction between lipids and proteins. Lipids, in the presence of a protein, show a different dynamic behavior from free lipids. Because of changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, our methods could also be applied to the examination of phase separation phenomena and to lipid-protein interactions.     

The in vitro influence of the external electrostatic field on the physical parameters of erythrocyte membranes

The in vitro influence of external electrostatic fields with 200 kV/m tension on the biophysical parameters of the erythrocyte membranes and their ghosts of white outbred rats was studied. The investigation on the parameters of erythrocyte membranes and their ghosts, particularly, their microviscosity, the amount and degree of membrane proteins submersion in lipids, polarity in depth of the membrane bilayer and its viscosity was carried out by the spectrofluorimeteric method using pyrene as a hydrophobic fluorescent probe. The analyses of literature data, findings of the current study and their comparison with the results of our previous works allow of concluding that the in vitro influence of external electrostatic fields with 200 kV/m tension on the erythrocyte membranes and their ghosts occurs at different sites of membrane. It is shown that the preliminary exposure of erythrocytes in external electrostatic fields leads to the changes of the parameters both of a membrane surface layer and the intra-membrane domains. So, the decrease in the strength of peripheral proteins binding to the erythrocyte membranes and the increase in the microviscosity of the lipid bilayer are observed. The influence of the field on the ghosts of intact erythrocytes results in alterations of the studied parameters only of the membrane surface.     

Modifying effect of concanavalin A and diamide on erythrocyte sensitivity to cold shock]

The temperature (0 degrees C and 37 degrees C) and the medium tonicity (0.15-1.20 M NaCl) were shown to affect erythrocyte agglutination by concanavalin A. Treatment of cells with lectin caused no significant decrease in the erythrocyte hemolysis upon cooling. Diamide, unlike concanavalin A used at concentrations above 2.0 M decreases the cell sensitivity to the cold shock. The changes in the erythrocyte susceptibility to cooling within the temperature range of 37-0 degrees C correlate with changes in the electrophoretic spectrum of membrane proteins. The progressive decrease in the spectrin bands intensity with a simultaneous formation of high molecular weight protein aggregates not included in the gel composition was observed after diamide treatment. The diamide effect depends on the medium tonicity, at which the treatment was performed, being especially well pronounced in hypertonic media with 0.8-1.2 M NaCl concentrations, the maximal spectrin aggregation being observed under these conditions. It is suggested that the main factor of the mechanism underlying the erythrocyte hypertonic cold shock is the increase in the association of peripheral cytoskeleton proteins with plasma membrane in osmotically dehydrated cells which limits the ability of lipids to adapt during cooling and results in the stabilization of defects in the membrane structure at low temperatures. Diamide eliminates these unfavourable changes eventually resulting in the dissociation of peripheral proteins from the cytoplasmic surface of the membrane on the protein aggregation.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Inhibition of protein phosphorylation and induction of protein cross-linking in erythrocyte membranes by diamide

This report presents studies on the effect of diamide on protein phosphorylation in erythrocyte membranes. Diamide, a thiol-oxidizing reagent, nonspecifically inhibits cyclic Amp-dependent and -independent autophosphorylation of red cell memvranes, but not the activity of the solubilized membrane cycle AMP-independent protein kinases. Analysis of diamide-treated membranes by gel electrophoresis indicates that diamide is capable of inducing cross-linking of membrane proteins. The action of diamide, both in the inhibition of membrane autophosphorylation and in the cross-linking of membrane proteins, is very similar to that of Cu2+. o-phenanthroline complex. Our data indicate that diamide inhibits erythrocyte membrane autophosphorylation by perturbing the protein substrates.     

The evaluation of the age-related characteristics of the structural status of the plasma membrane lipid phase in rat fatty tissue by using the method of inductive-resonance energy transfer

Using the method of inductance-resonance energy transfer from tryptophanyl residues to fluorescent pyrene probe the structural state of plasmatic membranes from adipose tissue of different age rats has been studied. The structural heterogeneity of membrane lipid phase has been revealed. The differences in physical properties of annular and bilayer lipids don't depend on age. During aging the membrane lipid viscosity including lipids of near protein area decreases, the conformation of membrane protein components alters during aging as well. The data on various effectiveness of energy transfer from tryptophanyls to pyrene probe in young and aged animals with stable polypeptide composition of membrane proteins indicates that. The structure of membrane lipid phase is suggested to be the main factor affecting the conformational state and functional activity of membrane-bound proteins during aging.     

Phosphorylation and dephosphorylation of membrane proteins as a possible mechanism for structural rearrangement of membrane components.

A correlation was found between dephosphorylation of chicken erythrocyte membrane proteins, aggregation of intramembrane particles, increase in the lipid bilayer phase of the membrane and exposure of membrane phospholipids toward phospholipase A and trinitrobenzene sulfonic acid. Most of the covalently bound phosphate of the membrane proteins turns over and is associated with 5 major bands. It is suggested that phosphorylation and dephosphorylation of these proteins causes changes in their charge and conformation. Such changes might affect the interaction of these proteins with the neighbouring lipids or lipoprotein complexes and results in the aggregation of intramembrane particles and relative increase in the exposed free lipid bilayer phase of the membrane.     

Electron paramagnetic resonance studies of membrane fluidity in ozone-treated erythrocytes and liposomes

Doxyl stearate spin probes which differed in the attachment of the nitroxide free radical to the fatty acid have been used to study membrane fluidity in ozone-treated bovine erythrocytes and liposomes. Analysis of EPR spectra of spin labels incorporated into lipid bilayer of the erythrocyte membranes indicates an increase in the mobility and decrease in the order of membrane lipids. In isolated erythrocyte membranes (ghosts) the most significant changes were observed for 16-doxylstearic acid. In intact erythrocytes statistically significant were differences for 5-doxylstearic acid. The effect of ozone on liposomes prepared from a lipid extract of erythrocyte lipids was marked in the membrane microenvironment sampled by all spin probes. Ozone apparently leads to alterations of membrane dynamics and structure but does not cause increased rigidity of the membrane.     

Regulation of erythrocyte ghost membrane mechanical stability by chlorpromazine

Chlorpromazine (CPZ), a widely used tranquilizer, is known to induce stomatocytic shape changes in human erythrocytes. However, the effect of CPZ on membrane mechanical properties of erythrocyte membranes has not been documented. In the present study we show that CPZ induces a dose-dependent increase in mechanical stability of erythrocyte ghost membrane. Furthermore, we document that spectrin specifically binds to CPZ intercalated into inside-out vesicles depleted of all peripheral proteins. These findings imply that CPZ-induced mechanical stabilization of the erythrocyte ghost membranes may be mediated by direct binding of spectrin to the bilayer. Membrane active drugs that partition into lipid bilayer can thus induce cytoskeletal protein interactions with the membrane and modulate membrane material properties.     

Effect of lipid structural modifications on their intermolecular hydrogen bonding interactions and membrane functions

The large number of different membrane lipids with various structural modifications and properties and the characteristic lipid composition of different types of membranes suggest that different lipids have specific functions in the membrane. Many of the varying properties of lipids with different polar head groups and in different ionization states can be attributed to the presence of interactive or repulsive forces between the head groups in the bilayer. The interactive forces are hydrogen bonds between hydrogen bond donating groups such as --P--OH,--OH, and--NH3+ and hydrogen bond accepting groups such as --P--O- and --COO-. These interactions increase the lipid phase transition temperature and can account for the tendency of certain lipids to go into the hexagonal phase and the dependence of this tendency on the pH and ionization state of the lipid. The presence or absence of these interactions can also affect the penetration of hydrophobic substances into the bilayer, including hydrophobic residues of membrane proteins. Evidence for this suggestion has been gathered from studies of the myelin basic protein, a water-soluble protein with a number of hydrophobic residues. In this way the lipid composition can affect the conformation and activity of membrane proteins. Since hydrogen-bonding interactions depend on the ionization state of the lipid, they can be altered by changes in the environment which affect the pK of the ionizable groups. The formation of the hexagonal phase or inverted micelles, the conformation and activity of membrane proteins, and other functions mediated by lipids could thus be regulated in this way.     

Characteristics of the heterogeneity of the physical properties of the lipid phase of synaptic membranes from the rat brain using the fluorescent probe pyrene

A theoretical analysis of the dependence of pyrene excimerization on its concentration in biological membranes was carried out. It was shown that in synaptic membranes the concentration dependencies of pyrene excimerization parameter upon direct stimulation appear as nonlinear, thus being reflective of the heterogeneity of physical properties of the lipid phase. Upon pyrene excitation at the expense of the energy transfer from tryptophanyl residues the dependence is linear, which points to the homogeneity of the anular lipid pool. It was assumed that a comparison of microviscosity of the anular and bilayer lipids requires independent measurements of the coefficient of the probe distribution between the lipids or of the anular lipid content in the membrane.     

Lipid-protein interactions and the function of the Ca2+-ATPase of sarcoplasmic reticulum

Regardless of the nature of the protein constituents of membranes, the molecular arrangement of lipids interacting with them must satisfy hydrophobic, ionic, and steric requirements. Biological membranes have a great diversity of lipid constituents, and this diversity might have functional roles. It has been proposed, for example, that the hydrophobic regions of membrane proteins are stabilized in the membrane through interactions with lipids able to adopt configurations other than the bilayer structure. Progress in understanding at the molecular level how lipid-protein interactions control the properties of membrane proteins has been hindered by the lack of information concerning the structure of the hydrophobic regions of membrane proteins. Nevertheless, there are many examples in the literature describing how changes in the lipid environment affect physical and biochemical properties of membrane proteins. From these studies, discussed in this review, an overall picture of how lipids and proteins interact in membranes is beginning to emerge.     

Structural reorganizations of the lipids and proteins of erythrocyte membranes under the action of low temperatures

The reversible structural rearrangement of lipids and protein oligomerization has been shown to occur during cooling in membranes of model systems (liposome, erythrocyte shadows) and native erythrocytes. Analysing the dependence of Azz in membrane probes (5- or 15-doxylstearic acids) in the Arrhenius plots a conclusion on the structural changes at 13-19 degrees C and within the range of interior water freezing from -17 up to -19 degrees C has been drawn, the last transition is smoothed out in the presence of glycerin. Using diamide joining spectrin and electrophoresis in polyacrylamide gel it has been determined that the low temperatures cause the spatial approach of proteins of spectrin-actinic complex and formation connections between the erythrocyte membrane proteins which aren't destroyed by dodecylsulfate.     

Changes in the physico-chemical properties of synaptosomal membranes induced by phospholipase A2

Using fluorescent and EPR spin probing techniques, the changes in the physico-chemical properties of rat brain synaptosomal membranes induced by phospholipase A2 were studied. It was shown that treatment of synaptosomal membranes with phospholipase A2 leads to their depolarization, increases their surface negative potential and decreases the microviscosity of the membrane lipid bilayer. The observed changes in the physicochemical properties of synaptosomal membranes induced by phospholipase A2 are discussed in terms of a possible regulatory role of lipids in the transmembrane chemical signal transfer.     

Protein modulation of lipids, and vice-versa, in membranes

This review describes: (i) perturbations of the membrane lipids that are induced by integral membrane proteins, and reciprocally, (ii) the effects that the lipids may have on the function of membrane-associated proteins. Topics of the first category that are covered include: stoichiometry and selectivity of the first shell of lipids associated at the intramembranous perimeter of transmembrane proteins; the chain configuration and exchange rates of the first-shell lipids; the effects of transmembrane peptides on transbilayer movement of lipids (flip-flop); the effects of membrane proteins on lipid polymorphism and formation of non-lamellar phases; and the effects of hydrophobic mismatch on lipid chain configuration, phase stability and selectivity of lipid-protein association. Topics of the second category are: the influence of lipid selectivity on conformational changes in the protein; the effects of elastic fluctuations of the lipid bilayer on protein insertion and orientation in membranes; the effects of hydrophobic matching on intramembrane protein-protein association; and the effects of intrinsic lipid curvature on membrane integration, oligomer formation and activity of membrane proteins.     

The involvement of cytoskeletal proteins in the maintenance of phospholipid topology in renal brush-border membranes

When incubated for 14 h at 37°C in the absence of energy supply, brush-border membrane vesicles from rabbit kidney cortex maintain, as judged by the use of sphingomyelinase and trinitrobenzene sulfonate as membrane probes, their highly asymmetrical phospholipid distribution. In particular, sphingomyelin still accounts for 75% of the phospholipids present on the outer membrane leaflet. Pretreatment of the vesicles with 5 mM diamide resulted in extensive crosslinking of membranous and cytoskeletal proteins. Although it had no immediate effect on the topology of phospholipids, this crosslinking resulted in a limited but significant increase in the amount of aminophospholipids present on the outer membrane leaflet after 14-h incubations. Degradation of aminophospholipids, upon incubation with hog pancreas and bee venom phospholipases A₂, was also enhanced by diamide. However, this enhanced hydrolysis was observed immediately after the diamide treatment. A similar increase in degradation of aminophospholipids was obtained when vesicles were incubated with dihydrocytochalasin B. Our results strongly suggest that cytoskeletal proteins, via interactions with aminophospholipids, stabilize the lipid bilayer of the brush-border membrane. It is also suggested that, due to a low transbilayer migration rate, sphingomyelin may play an important role in the maintenance of the lipid asymmetry in these membranes.