On the mechanism of drug-induced acceleration of phospholipid translocation in the human erythrocyte membrane

Small amphiphilic compounds (M_r<200 Da) such as anaesthetics and hexane derivatives with different polar groups produced a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labelled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration characteristic for each compound, the flip rate gradually increased at increasing concentrations in the medium. For compound concentrations required to produce a defined flip acceleration, corresponding membrane concentrations were estimated using reported octanol/water partition coefficients. The effective threshold membrane concentrations (50–150 mmol l^?1) varied in the order: hexylamine>isoflurane=hexanoic acid>hexanol=chloroform>hexanethiol=1,1,2,2-tetrachloroethane>chlorohexane. Apolar hexane, which mainly distributes in the apolar membrane core, was much less effective and supersaturating concentrations were required to enhance flip. Localization of the drug at the lipid–water interface seems to be required for flip acceleration. Such a localization may increase the lateral pressure in this region and the bilayer curvature stress with concomitant decrease of order and rigidity at the interface. This unspecific bilayer perturbation is proposed to enhance the probability of formation of hydrophobic defects in the bilayer, facilitating penetration of the polar head group of the phospholipid into the apolar membrane core.     

On the mechanism of drug-induced acceleration of phospholipid translocation in the human erythrocyte membrane

Small amphiphilic compounds (M(r)<200 Da) such as anaesthetics and hexane derivatives with different polar groups produced a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labelled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration characteristic for each compound, the flip rate gradually increased at increasing concentrations in the medium. For compound concentrations required to produce a defined flip acceleration, corresponding membrane concentrations were estimated using reported octanol/water partition coefficients. The effective threshold membrane concentrations (50-150 mmol l(-1)) varied in the order: hexylamine>isoflurane=hexanoic acid>hexanol=chloroform>hexanethiol=1,1,2,2-tetrachloroethane>chlorohexane. Apolar hexane, which mainly distributes in the apolar membrane core, was much less effective and supersaturating concentrations were required to enhance flip. Localization of the drug at the lipid-water interface seems to be required for flip acceleration. Such a localization may increase the lateral pressure in this region and the bilayer curvature stress with concomitant decrease of order and rigidity at the interface. This unspecific bilayer perturbation is proposed to enhance the probability of formation of hydrophobic defects in the bilayer, facilitating penetration of the polar head group of the phospholipid into the apolar membrane core.     

Possible relationship between membrane proteins and phospholipid asymmetry in the human erythrocyte membrane.

After incubation of human erythrocytes at 37 degrees C in the absence of glucose (A) for 24 h, (B) for 4 h with 8 mM hexanol or (C) for 3 h with SH reagents, phosphatidylethanolamine becomes partly susceptible to hydrolysis by phospholipase A2 from Naja naja. The presence of glucose during the pretreatments suppresses this effect, except in the case of SH reagents that inhibit glycolysis. After incubation with tetrathionate, up to 45% of the phosphatidylethanolamine is degraded by the enzyme, an amount considerably in excess of the 20% attacked in fresh erythrocytes. Pancreatic phospholipase A2, an enzyme unable to hydrolyse the phospholipids of intact erythrocytes, partially degrades phosphatidylcholine and phosphatidylethanolamine of erythrocytes pretreated with hexanol or SH reagents. Reagents capable of oxidizing SH groups to disulfides (tetrathionate, o-iodosobenzoate and hydroquinone) even render susceptible to pancreatic phospholipase A2 phosphatidylserine, a phospholipid supposed to be entirely located in the inner lipid layer of the membrane. Alkylating or acylating SH reagents have no such effect. It is postulated that disulfide bond formation between membrane protein SH groups leads to an alteration in protein-phospholipid interactions and consequently induces a reorientation of phospholipids between the inner and the outer membrane lipid layer.     

Polyamine regulation of plasma membrane phospholipid flip-flop during apoptosis.

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.     

Possible relationship between membrane proteins and phospholipid asymmetry in the human erythrocyte membrane

After incubation of human erythrocytes at 37 °C in the absence of glucose (A) for 24 h, (B) for 4 h with 8 mM hexanol or (C) for 3 h with SH reagents, phosphatidylethanolamine becomes partly susceptible to hydrolysis by phospholipase A₂ from Naja naja. The presence of glucose during the pretreatments suppresses this effect, except in the case of SH reagents that inhibit glycolysis. After incubation with tetrathionate, up to 45% of the phosphatidylethanolamine is degraded by the enzyme, an amount considerably in excess of the 20% attacked in fresh erythrocytes.Pancreatic phospholipase A₂, an enzyme unable to hydrolyse the phospholipids of intact erythrocytes, partially degrades phosphatidylcholine and phosphatidylethanolamine of erythrocytes pretreated with hexanol or SH reagents. Reagents capable of oxidizing SH groups to disulfides (tetrathionate, $\text{o-}\text{iodosobenzoate}$ and hydroquinone) even render susceptible to pancreatic phospholipase A₂ phosphatidylserine, a phospholipid supposed to be entirely located in the inner lipid layer of the membrane. Alkylating or acylating SH reagents have no such effect. It is postulated that disulfide bond formation between membrane protein SH groups leads to an alteration in protein-phospholipid interactions and consequently induces a reorientation of phospholipids between the inner and the outer membrane lipid layer.     

Acceleration of phospholipid flip-flop in the erythrocyte membrane by detergents differing in polar head group and alkyl chain length

The detergents, alkyltrimethylammonium bromide, N-alkyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (zwittergent), alkane sulfonate, alkylsulfate, alkyl-beta-D-glucopyranoside, alkyl-beta-D-maltoside, dodecanoyl-N-methylglucamide, polyethylene glycol monoalkyl ether and Triton X-100, all produce a concentration-dependent acceleration of the slow passive transbilayer movement of NBD-labeled phosphatidylcholine in the human erythrocyte membrane. Above a threshold concentration, which was well below the CMC and characteristic for each detergent, the flip rate increases exponentially upon an increase of the detergent concentration in the medium. The detergent-induced flip correlates with reported membrane-expanding effects of the detergents at antihemolytic concentrations. From the dependence of the detergent concentration required for a defined flip acceleration on the estimated membrane volume, membrane/water partition coefficients for the detergents could be determined and effective detergent concentrations in the membrane calculated. The effective membrane concentrations are similar for most types of detergents but are 10-fold lower for octaethylene glycol monoalkyl ether and Triton X-100. The effectiveness of a given type of detergent is rather independent of its alkyl chain length. Since detergents do not reduce the high temperature dependence of the flip process the detergent-induced flip is proposed to be due to an enhanced probability of formation of transient hydrophobic structural defects in the membrane barrier which may result from perturbation of the interfacial region of the bilayer by inserted detergent molecules.     

Spontaneous, reversible protein cross-linking in the human erythrocyte membrane. Temperature and pH dependence.

Changes in pH significantly affect the morphology and physical properties of red cell membranes. We have explored the molecular basis for these phenomena by characterizing the pattern of protein disulfide cross-linkages formed spontaneously in ghost exposed to acid pH or elevated temperature (37 degrees C). Protein aggregation was analyzed by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. incubation of ghosts at pH 4.0 to 5.5 (0-4 degrees C) yielded (i) complexes of spectrin and band 3, (ii) complexes of actin and band 3, (iii) band 3 complexes, i.e. dimer and trimer, and (iv) heterogeneous aggregates involving spectrin, band 3, band 4.2, and actin in varying proportions. Aggregation was maximal near the isoelectric points of the major membrane proteins, and appeared to reflect (i) the aggregation of intramembrane particles including band 3 and (ii) more intimate contact between spectrin-actin meshwork and band 3.     

Topological asymmetry of phospholipid metabolism in rat erythrocyte membranes. Evidence for flip-flop of lecithin.

1. The distribution of phospholipids between inside and outside of rat erythrocyte membranes was studied by incubating the cells with phospholipase A2 from Naja naja venom and sphingomyelinase from Staphylococcus aureus. 2. Choline-containing phospholipids were found to comprise the majority of the outer layer of the membrane. 3. The incorporation of radioactive fatty acids into phospholipids occurred predominantly at the inside of the membrane. 4. Exchange of phospholipids between red cell membranes and plasma lipoproteins occurred at the outside of the membrane. 5. Indications were found for a rather slow flip-flop of lecithin across the membrane.     

Effect of benzyl alcohol on phospholipid transverse mobility in human erythrocyte membrane.

The effect of benzyl alcohol on the transverse mobility and repartition of phospholipids in the human erythrocyte membrane was investigated using electron spin resonance and morphological modification of red blood cells. Transmembrane internalization rates and equilibrium distribution in red blood cells of short-chain spin-labeled phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were strongly modified by treatment with 10-70 mM benzyl alcohol. A dual effect was observed: (a) at 4 degrees C and 37 degrees C there was an N-ethylmaleimide-sensitive, long lasting and fully reversible increase in the spin-labeled phosphatidylserine and phosphatidylethanolamine internalization rate; (b) at 37 degrees C, an enhancement of N-ethylmaleimide-insensitive fluxes of all the labeled phospholipids through the membrane occurred. Both effects were dose-dependent. Erythrocytes submitted to benzyl alcohol incubation also showed dose-dependent shape changes: an immediate one from discocytes to echinocytes, followed by a slower N-ethylmaleimide- and ATP-dependent change to stomatocytes. Moreover, benzyl alcohol treatment was shown to lead to enhanced hydrolysis of intracellular ATP. All the effects of benzyl alcohol can be described as an accumulation of labeled phosphatidylethanolamine (and labeled phosphatidylcholine at 37 degrees C) in the inner leaflet. This can be interpreted as a perturbation of the erythrocyte membrane, leading to an energy-consuming specific increase in aminophospholipid translocase activity, in addition to a slow and passive bidirectional flux of all phospholipids at 37 degrees C.     

Spectrin as a stabilizer of the phospholipid asymmetry in the human erythrocyte membrane

After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A₂ cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B. (1976) Biochim. Biophys. Acta 436, 353–365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys. Acta 323, 178–193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of < 10⁶ dalton.Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents.It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.     

Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain

Three-dimensional crystals were obtained for the membrane domain of the human erythrocyte anion exchanger (AE1, Band 3). Protein homogeneity and stability and the delicate balance between the detergent used and the amount of phospholipids copurifying are critical to the formation of three-dimensional crystals of the AE1 membrane domain. While deglycosylation improved the protein homogeneity, its stability was significantly increased by inhibitor binding. Size-exclusion chromatography showed that the protein was monodisperse in detergents with acyl chains of 10-12 carbons over a pH range of 5.5-10.0. This pH range and the detergents that retained the protein's monodispersity were used for crystallization screening. Crystals were obtained with the protein purified in C(12)E(8), dodecylmaltoside, decylthiomaltoside, and cyclohexyl-hexylmaltoside. Five to 13 lipid molecules per protein were required for the protein crystal formation. Those crystals grown in dodecylmaltoside diffracted X-rays to 14 A. With these factors taken into consideration, ways to further improve the crystal quality are suggested.     

HgCl2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers

The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.     

Partial reversible inactivation of enzymes due to binding to the human erythrocyte membrane

Summary Hypotonic human erythrocyte ghosts, devoid of the original glyceraldehyde-3-phosphate dehydrogenase content of the red cell, bind added glyceraldehyde-3-phosphate dehydrogenases, isolated from human erythrocytes, rabbit and pig muscle, as well as rabbit muscle aldolase. There are only slight differences in the affinities towards the various glyceraldehyde-3-phosphate dehydrogenases. On the other hand, glyceraldehyde-3-phosphate dehydrogenases are bound much stronger than aldolase; in an equimolar mixture the former can prevent the binding of the latter, or replace previously bound aldolase at the membrane surface. Binding is always accompanied by the partial inactivation of enzymes, which can be reverted by desorption. Unwashed ghosts rich in hemoglobin seem to have a more pronounced inactivating effect on bound glyceraldehyde-3-phosphate dehydrogenase. In isotonic media ghosts, whether white or unwashed, reseal and do not interact with the enzymes.     

The anticancer drug chlorambucil interacts with the human erythrocyte membrane and model phospholipid bilayers

The plasma membrane has gained increasing attention as a possible target of antitumor drugs. It has been reported that they act as growth factor antagonists, growth factor receptor blockers, interfere with mitogenic signal transduction or exert direct cytotoxic effects. Chlorambucil (4-[p-(bis[2-chloroethyl]amino)phenyl]butyric acid) is an alkylating agent widely used in the treatment of chronic lymphocytic leukaemia. Contradictory reports have been published concerning its interaction with cell membranes. Whereas a decrease in the fluidity of Ehrlich ascite tumor cells has been adduced, no evidences were found that chlorambucil changes membrane lipid fluidity and alkylating agents had effects in these systems even at highly toxic concentrations. Our results showed that chlorambucil at a dose equivalent to its therapeutical concentration in the plasma (3.6 microM) caused the human erythrocyte membrane to develop cup-shaped forms (stomatocytes). Accordingly to the bilayer couple hypothesis, this means that the drug is inserted into the inner monolayer of the erythrocyte membrane, a conclusion supported by X-ray diffraction performed on multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. It is concluded that the cytotoxic effect of chlorambucil might be due to alteration of the structure and therefore of the physiological properties of cell membranes such as fluidity, permeability, receptor and channel functions.     

Arsenite interactions with phospholipid bilayers as molecular models for the human erythrocyte membrane

There are scanty reports concerning the effects of arsenic compounds on the structure and functions of cell membranes. With the aim to better understand the molecular mechanisms of the interaction of arsenite with cell membranes we have utilized bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of arsenite to perturb the bilayer structures was determined by X-ray diffraction and fluorescence spectroscopy, whilst the modification of their thermotropic behaviour was followed by differential scanning calorimetry (DSC). The experiments carried out by X-ray diffraction and calorimetry clearly indicated that NaAsO(2) interacted with DMPE and modified its thermotropic behaviour. No such information has been so far reported in the literature.     

Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, and a tyrosine kinase, p72syk. cdb3 exists in a reversible, pH-dependent conformational equilibrium characterized by large changes in Stokes radius (11 A) and intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibrium might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have mutated both donor (W105L) and acceptor (D316A) residues of a prominent H bond that bridges the above two domains and have examined the effect on the resulting conformational equilibrium. Analysis of the intrinsic fluorescence, Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data further reveal that cdb3 exists in three native pH-dependent conformations and that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mid-pH conformation to the high pH conformation would now appear to involve structural changes primarily in the peripheral protein binding domain. Because ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.     

Abnormal erythrocyte membrane phospholipid organisation in chronic myeloid leukaemia

The membrane phospholipid organisation in the red cells of humans suffering from chronic myeloid leukaemia has been analysed using the amino-group labelling reagent trinitrobenzenesulphonic acid and the fluid-sensing fluorophore, Merocyanine 540. Unlike the normal human erythrocytes, trinitrobenzenesulphonic acid in intact chronic myeloid leukaemia erythrocytes modified about 30% phosphatidylserine, under controlled conditions. Also, the chronic myeloid laukaemia red cells, but not the normal cells, were found to bind the fluorescent dye Merocyanine 540. These results demonstrate that loss of the transmembrane phospholipid asymmetry in chronic myeloid leukaemia erythrocytes is accompanied by an enhancement in the outer surface fluidity and, therefore, suggest that the red cells membrane phase-state asymmetry originates probably from the asymmetric arrangements of phospholipids across the membrane bilayer.     

竹红菌甲素对红细胞膜蛋白及膜磷脂的光敏损伤

In this paper, using human erythrocyte membrane, the photodamage of Hypocrellin A to membrane protein and phospholipid was studied by measuring the lipid peroxidation, the damage of phospholipid, the change of protein secondary structure, the endogenous fluorescence change and SDS-polyacrylamide gel electrophoresis analysis. These results showed that illumination of erythrocyte membrane in presence of Hypocrellin A can cause lipid peroxidation producing fluorescence adduct and MDA, decomposing in phospholipid composition in which PE and PS were more sensitive than others. Meanwhile, the secondary structure of membrane protein was destroyed and endogenous fluorescence decreased. The photodamage on phospholipid and spectrin occurred more seriously in the case they were embedded in membrane than they were in isolated form. So we suggest that they are interactions existing between proteins and phospholipids to enhance the damage on protein and phospholipid during the HA-sensitized photodamage on membrane.