Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

Comparative lipid analysis and structure of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

The lipid composition and structure of detergent-resistant membrane rafts from human, goat, and sheep erythrocytes is investigated. While the sphingomyelin:cholesterol ratio varied from about 1:5 in human to 1:1 in sheep erythrocytes a ratio of 1:1 was found in all raft preparations insoluble in Triton X-100 at 4 degrees C. Excess cholesterol is excluded from rafts and saturated molecular species of sphingomyelin assayed by gas chromatography-mass spectrometry determines the solubility of cholesterol in the detergent. Freeze-fracture electron microscopy shows that vesicles and multilamellar structures formed by membrane rafts have undergone considerable rearrangement from the original membrane. No membrane-associated particles are observed. Synchrotron X-ray diffraction studies showed that d spacings of vesicle preparations of rafts cannot be distinguished from ghost membranes from which they are derived. Dispersions of total polar lipid extracts of sheep rafts show phase separation of inverted hexagonal structure upon heating and this phase coexists with multilamellar structures at 37 degrees C.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.     

A cytolytic protein from the edible mushroom, Pleurotus ostreatus

Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lytic in vitro for mammalian erythrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists a monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate and sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Effect of Cholesterol Depletion and Temperature on the Isolation of Detergent-Resistant Membranes from Human Erythrocytes

Transient lateral microdomains or lipid rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the nonionic detergents Triton X-100 or octaethylene glycol monododecyl ether (C₁₂E₈) at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C DRMs obtained from cholesterol-depleted versus intact erythrocytes. Flotillin-2, a protein marker of lipid rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C₁₂E₈ DRMs prepared at 4°C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C₁₂E₈ DRMs. However, contrary to the 4°C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C₁₂E₈ treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C₁₂E₈ to the study of DRMs.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions: Effects of local anesthetics,cholesterol and protein

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 °C). Incorporation of cholesterol (30–50%) increased the microviscosity of lipid phases by 200–500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b₅ did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since the latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracaine and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of the anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at the 25 °C varied as follows:polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erytherocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol : phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important fuctional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Incorporation of sphingomyelin increases thermostability of human erythrocyte membrane and resistance of erythrocytes against thermal hemolysis

Erythrocytes of various mammal species differ markedly in their resistance against thermally induced hemolysis that was assumed related to the different sphingomyelin content of their membranes (J. Therm. Biol. 18 (1993) 177). In this work, two choline-containing lipids, sphingomyelin and phosphatidylcholine, were incorporated into the membrane of human erythrocytes and the resulting effect on thermal resistance and membrane thermostability of these cells was studied measuring thermohemolysis upon exposure to constant temperature and electrolyte leakage during transient heating, respectively. While sphingomyelin increased thermal resistance by 56% and increased the inducing temperature T_m of electrolyte leakage by 1±0.2°C, phosphatidylcholine produced practically no effect. The results show that sphingomyelin alone stabilized the structure of plasma membrane providing thermal stability to membrane proteins and in turn to whole cells as well.     

Effect of diamide on protein oxidation and physico-chemical properties of lipids in erythrocyte membranes

The effect of diamide on the physicochemical state of proteins and lipids of human erythrocyte membrane was studied. It was found that diamide at a concentration of 1 mM decreases the content of the SH-groups of membrane proteins by approximately 50%, resulting in enhanced vesiculation of erythrocytes upon metabolic exhaustion of cells. It was shown using fluorescein isothiocyanate-labeled concanavalin A and 4,4'-diisothiocyano-2,2'-stilbene disulfonate that diamide changes the structural state of the main integral protein of erythrocyte membranes, the band 3 protein. Changes in the microviscosity of the membrane lipid bilayer depending on diamide concentration were determined from the changes in the fluorescence parameters of the lipophilic probes (pyrene and 1,6-diphenyl-3,5-hexatriene). The level of lipid peroxidation products in membranes remained unchanged. It follows from these data that the SH-oxidizing agent diamide does not directly interact with the lipid bilayer of membrane and produces changes in the physicochemical state of lipids presumably by disrupting protein-lipid interactions that take place upon oxidation of the SH-groups and cross-linking of membrane proteins.     

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.     

Utilization of membranous lipid substrates by membranous enzymes. Hydrolysis of sphingomyelin in erythrocyte ''ghosts'' and liposomes by the membranous sphingomyelinase of chicken erythrocyte ''ghosts''.

Incubation at 37 degrees C of haemolysed chicken erythrocytes ('chicken erythrocyte ghosts') resulted in hydrolysis of the membrane sphingomyelin, suggesting an activation of a latent sphingomyelinase during the haemolysis procedure. When this incubation was continued for several hours, the entire sphingomyelin of the erythrocyte 'ghosts' was hydrolysed and membranes were obtained that were devoid of sphingomyelin, but had an active sphingomyelinase. Mixing the latter membranes with human erythrocyte 'ghosts' or positively charged liposomes led to hydrolysis of the sphingomyelin in these two membranes. This suggested that, after haemolysis, the activated sphingomyelinase in the membrane of the chicken erythrocyte 'ghosts' could hydrolyse sphingomyelin in its own membrane ('intramembrane utilization') or adjacent membranes ('intermembrane utilization').     

Kinetic attributes of Na+, K+ ATPase and lipid/phospholipid profiles of rat and human erythrocyte membrane

Kinetic properties of Na+, K+ ATPase of membranes from rat and human erythrocytes were examined. The enzyme stability decreased with incubation time. The Vmax of the human enzyme was about 4 times lower than the values of the rat enzyme. However the energies of activation were higher. Phase transition temperature for the rat and the human enzyme was 24 degrees C and 17 degrees C, respectively. The human erythrocyte membranes were characterized by lower total phospholipid and cholesterol contents and were relatively more fluid. The human membranes contained lower proportions of acidic phospholipids which correlated well with the lower Vmax of the enzyme; the proportion of lysophosphoglyceride and sphingomyelin was higher in the human membrane.     

Allometric dependence of the life span of mammal erythrocytes on thermal stability and sphingomyelin content of plasma membranes

Thermal stability of erythrocyte membrane is a measure for its ability to maintain permeability barrier at deleterious conditions. Hence, it could impact the resistance of erythrocytes against detrimental factors in circulation. In this study the thermostability of erythrocyte membranes was expressed by the temperature, T(go), at which the transmembrane gradient of ion concentration rapidly dissipated during transient heating. T(go) is the inducing temperature of the membrane transition that activated passive ion permeability at hyperthermia causing thermal hemolysis. A good allometric correlation of T(go) to the resistance against thermal hemolysis and the life span of erythrocytes were found for 13 mammals; sheep, cow, goat, dog, horse, man, rabbit, pig, cat, hamster, guinea pig, rat, and mouse. For the same group, the values of T(go) were strictly related to the sphingomyelin content of erythrocyte membranes. The residual ion permeability, P, was temperature activated from 38 to 57 degrees C with activation energy of 250+/-15 kJ/mol that strongly differed from that below 37 degrees C. The projected value of P at 37 degrees C was about half that of residual physiological permeability for Na+ and K+ that build ground for possible explanation of the life span vs membrane thermostability allometric correlation.     

A cytolytic protein from the edible mushroom, Pleurotus ostreatus

Aqueous extracts of the edible mushroom, Pleurotus ostreatus, contain a substance that is lytic in vitro for mammalian erthrocytes. The hemolytic agent, pleurotolysin, was purified to homogeneity and found to be a protein lacking seven of the amino acids commonly found in proteins. In the presence of sodium dodecyl sulfate it exists as monomers of molecular weight 12 050 whereas under non-dissociating conditions it appears to exist as dimers. It is isoelectric at about pH 6.4. The sensitivity of erythrocytes from different animals correlates with sphingomyelin content of the erythrocyte membranes. Sheep erythrocyte membranes inhibit pleurotolysin-induced hemolysis and the inhibition is time and temperature dependent. Ability of membranes to inhibit hemolysis is abolished by prior treatment of membranes with specific phospholipases. Pleurotolysin-induced hemolysis is inhibited by liposomes prepared from cholesterol, dicetyl phosphate adn sphingomyelin derived from sheep erythrocytes whereas a variety of other lipid preparations fail to inhibit. It is concluded that sphingomyelin plays a key role in the hemolytic reaction.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

Orientation and motion of spin-labels in rabbit small intestinal brush border vesicle membranes

The temperature dependence of the packing (order) and fluidity (microviscosity) of rabbit small, intestinal brush border vesicle membranes and of liposomes made from their extracted lipids has been investigated by using a variety of lipid spin probes. The lipids in the brush border membrane are present essentially as a bilayer. Compared to other mammalian membranes, the brush border membrane appears to be characterized by a relatively high packing order as well as microviscosity. At body temperature, the lipid molecules undergo rapid, anisotropic motion, which is essentially a fast rotation about an axis approximately perpendicular to the bilayer normal. Both the order (motional anisotropy) and the microviscosity increase with decreasing temperature and with increasing distance from the center of the bilayer. Qualitatively similar motional or fluidity gradients have been reported for other mammalian and bacterial membranes. The liposomes made from the extracted lipids have a somewhat lower packing order and a slightly higher fluidity than brush border vesicle membranes. The differences are, however, small indicating that the packing and the fluidity (microviscosity) of the membrane are primarily determined by the lipid composition. Membrane-associated proteins and cytoskeleton cannot play a dominant role in determining the order and fluidity of the lipid bilayer. Discontinuities are observed in the temperature dependence of various spectral parameters, the order parameter S, the rotational correlation time tau, and 2,2,6,6-tetramethylpiperidinyloxy partitioning. They are assigned to phase transitions and/or phase separations of the membrane lipids. These discontinuities occur at about 30, 20, and 13 degrees C for 5-doxyl-, 12-doxyl-, and 16-doxylstearic acid, respectively. The apparent transition temperature depends on the location of the spin probe along the bilayer normal, being higher the closer the probe is to the membrane surface. This indicates the possibility that chain melting is progressive and spreads with increasing temperature from the center of the membrane outward.     

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.     

Role of phospholipids in the DDT-induced efflux of potassium in human erythrocytes

Incubation of human erythrocytes for 1–2 h at 37°C in a suspension of dipalmitoylphosphatidylcholine (DPPC) liposomes results in a phospholipid enrichment of erythrocyte membranes by 45–55% and a depletion of cholesterol by 19–24%. The enrichment by DPPC was time and concentration dependent. By contrast, dioleoylphosphatidylcholine (DOPC) liposomes were less effective in enriching the membranes with phospholipid and in depleting the membranes of cholesterol. Concomitantly, the DDT-induced efflux of K⁺ was reduced in the case of DPPC-enriched erythrocytes but enhanced in DOPC-enriched erythrocytes. These results suggest that DDT partitions more readily into the unsaturated than the saturated phospholipids of the erythrocyte membrane. It is concluded that the extent to which DDT affects the flux of K⁺ across the membrane is dependent on the fluidity of the lipid phase. We also report here a rapid method for cholesterol depletion of red blood cells in comparison to previously reported methods.