Labeling of human erythrocyte membrane proteins by photoactivatable radioiodinated phosphatidylcholine and phosphatidylserine. A search for the aminophospholipid translocase

We have synthesized radioiodinated photoactivatable phosphatidylcholine (125I-N3-PC) and phosphatidylserine (125I-N3-PS). After incubation with red blood cells in the dark, the labeled PC could be extracted but not the corresponding PS molecule, indicating that the latter was transported by the aminophospholipid translocase, but not the former. When irradiated immediately after incorporation, N3-PS, but not N3-PC, partially blocked subsequent translocation of spin-labeled aminophospholipids. Analysis of probe distribution by SDS-polyacrylamide gel electrophoresis revealed that 125I-N3-PS labeled seven membrane bound components with molecular masses between 140 and 27 kDa: one (or several) of these components should correspond to the aminophospholipid translocase.     

The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Vesicles have been prepared from 18 : 1_c/18 : 1_c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, ³¹P-NMR and K⁺ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.     

Glycolipid transfer proteins and membrane interaction

The glycolipid transfer protein is found from animals and fungi to plants and red micro-alga. Some eukaryotes that do not encode the glucosylceramide synthase like the yeast Schizosaccharomyces pombe and Saccharomyces cerevisiae do neither produce glycolipid transfer like proteins. On the other hand yeast like Eremothecium gossypii that do synthesize glucosylceramide also express glycolipid transfer protein. Based on this novel genetic relationship it is not far fetched to assume that there must be a strong correlation between the synthesis of the glycolipid precursor and the glycolipid transfer protein. Because the glycolipid transfer protein is localized in the cytosol it is unlikely that it would participate in events associated with lipid rafts or caveolar structures, since they are found on the outer leaflet of the plasma membrane. Rather, GLTP is likely to be involved in events at the cytosolic side of the plasma membrane or the endoplasmic reticulum, maybe function as a reporter or sensor of glycolipid levels. A similar function has been proposed for other proteins with affinity for lipids like the oxysterol binding proteins and phosphatidylinositol transfer proteins that are thought to be able act as lipid sensors. Recent discoveries in the glycolipid transfer protein field are discussed.     

Separation and some properties of the major proteins of the human erythrocyte membrane

A fractionation procedure is described which allows the isolation of three major human erythrocyte membrane proteins. Their isolation involves three sequential extraction procedures followed by gel filtration in 1% sodium dodecyl sulphate and preparative gel electrophoresis. All three proteins can be isolated from a single preparation. One of the proteins is the erythrocyte sialoglycoprotein, for which no C- or N-terminal residues were found. The other two proteins, which have not previously been isolated, have subunit molecular weights of 74000 and 93000 and contain 9 and 7% carbohydrate respectively. These glycoproteins have blocked N-terminal residues and show similarities in their chemical properties. Preparations derived from blood-group O erythrocytes contain no N-acetylgalactosamine, but similar preparations from blood-group A erythrocytes do contain this sugar. These three proteins cannot easily be solubilized by gentle aqueous procedures and represent about half of the erythrocyte ;ghost' protein. They carry a large proportion of the cell-surface carbohydrate.     

Labelling of egg phosphatidylcholine vesicles and myelin membrane with a photoreactive lipophilic reagent.

The preparation and isolation of [3H]phenyl azide, a photosensitive non-polar probe, is reported. The reagent partitions into the lipid bilayer of egg phosphatidylcholine vesicles and bovine myelin membranes. On photoactivation to generate the nitrene grouping, as much as 90% of the covalently attached label is associated with the fatty acyl residues of the constituent phospholipid molecules. The remainder is found in the polar head groups. The cholesterol component of myelin membranes is also heavily labelled. These results suggest that such reagents may be used to probe the hydrophobic regions of natural membranes.     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

Melittin: structure, properties, interaction with a membrane

The paper is concerned with the analysis of the present data as to the structure and properties of melittin, a biologically active peptide of bee venom, able to be inserted spontaneously into the natural and synthetic membranes and to destruct cells in micromolar concentrations. This property is a result of the peptide structure peculiarities. Attention is focused on the mechanism of melittin insertion into the phospholipid bilayer as well as on the possibility of its use to study the nature of protein-lipid interactions and formation of ionic channels.     

Studies on the interaction of human erythrocyte band 3 with membrane lipids using deuterium nuclear magnetic resonance and differential scanning calorimetry

Human erythrocyte band 3, reconstituted into large unilamellar phospholipid vesicles, has been used as a model system for studying the interactions between membrane lipids and large transmembrane glycoproteins. Both 2H-nuclear magnetic resonance (2H-NMR) and differential scanning calorimetric techniques have been used to probe dimyristoylphosphatidylcholine-band 3 interactions over the temperature range 4-32 degrees C. Analysis of 2H-NMR spectra allowed the assignment of liquid crystal, gel phase and two-phase regions for several protein/lipid mole fractions in the range (1-20) X 10(-4). Sample size was limited by the amount of available glycoprotein and this precluded exact determination of the phase boundaries for this system. The sharp discontinuity in the spectral first moment, M1, seen at the phase transition of the pure phospholipid is progressively diminished by addition of protein, and at the highest protein concentration the first moment varies smoothly between the two phases. For T greater than 26 degrees C or less than 16 degrees C, the moments are relatively insensitive to protein concentration, while between 20 and 26 degrees C the moments increase with protein concentration up to the boundary of the two-phase region. Beyond this boundary, they remain constant or decrease slightly with increasing amount of protein. A preliminary phase diagram for band 3 in this lipid system is presented, based on 2H-NMR data. Differential scanning calorimetry (DSC) showed that addition of glycoprotein dramatically alters the scan shape and tends to extend the coexistence of two phases to higher temperatures.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

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.     

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.     

Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: characterization of interaction of NSAIDs with phosphatidylcholine

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most widely consumed pharmaceuticals, yet both the mechanisms involved in their therapeutic actions and side-effects, notably gastrointestinal (GI) ulceration/bleeding, have not been clearly defined. In this study, we have used a number of biochemical, structural, computational and biological systems including; Fourier Transform InfraRed (FTIR). Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) spectroscopy, and cell culture using a specific fluorescent membrane probe, to demonstrate that NSAIDs have a strong affinity to form ionic and hydrophobic associations with zwitterionic phospholipids, and specifically phosphatidylcholine (PC), that are reversible and non-covalent in nature. We propose that the pH-dependent partition of these potent anti-inflammatory drugs into the phospholipid bilayer, and possibly extracellular mono/multilayers present on the luminal interface of the mucus gel layer, may result in profound changes in the hydrophobicity, fluidity, permeability, biomechanical properties and stability of these membranes and barriers. These changes may not only provide an explanation of how NSAIDs induce surface injury to the GI mucosa as a component in the pathogenic mechanism leading to peptic ulceration and bleeding, but potentially an explanation for a number of (COX-independent) biological actions of this family of pharmaceuticals. This insight also has proven useful in the design and development of a novel class of PC-associated NSAIDs that have reduced GI toxicity while maintaining their essential therapeutic efficacy to inhibit pain and inflammation.     

Direct interaction of mepacrine with erythrocyte and platelet membrane phospholipid

Mepacrine has been used as an inhibitor of the activation of endogenous phospholipases in many systems. These endogenous phospholipases are important in the modification of the lipid environment of membrane proteins and in the release of locally active oxygenated arachidonic acid metabolites. In both human platelets and erythrocytes, mepacrine blocks the release of fatty acid from phospholipid by endogenous phospholipases. However, mepacrine also interacts directly with membrane phospholipids, primarily phosphatidylethanolamine, to form less polar derivatives. This interaction occurs rapidly and is maximal at concentrations of mepacrine greater than 0.2 mM. Such drug-phospholipid interaction may perturb membrane architecture and function and be responsible for the inhibitory effects of mepacrine on cellular responses observed in many systems. Since the alteration in membrane phospholipid composition occurs under the same conditions as phospholipase inhibition, it is not possible to be certain that the inhibition of cellular responses by mepacrine is due to inhibition of phospholipases rather than to direct perturbation of the membrane. It is also possible that inhibition of phospholipase action by mepacrine is in part a consequence of the change in phospholipid composition. These results indicate that caution should be exercised in the interpretation of results obtained using mepacrine and that the usefulness of this compound for the investigation of the biological importance of phospholipase activation is limited.     

Malarial proteins that interact with the erythrocyte membrane and cytoskeleton

Several distinct classes of Plasmodium proteins have been proposed to interact with the submembrane skeleton of the erythrocyte based upon differential solubility and subcellular localization studies. That the parasite affects the erythrocyte membrane by interacting with the submembrane skeleton is an attractive hypothesis since the membrane skeleton likely regulates many aspects of membrane topography and function. The precise interactions between host and parasite proteins at the molecular level and how the parasite proteins are transported to the erythrocyte membrane are not completely understood. Experiments addressing these questions are under way, and such studies will provide valuable information about the host-parasite interface. In addition, the characterization of the interaction of Plasmodium proteins with the host erythrocyte membrane may also provide new insight into the structure and function of the erythrocyte membrane or membranes in general.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

The anion permeability of vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Band 3 protein was reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 2500:1 phospholipid:protein molar ratio by means of a Triton X-100/beads method. The SO2-4 permeability of the resulting vesicles was measured using an influx assay procedure in which the vesicles were sampled and subsequently eluted over Sephadex columns at appropriate time intervals. The accuracy of the assay was greatly increased by using an internal standard in order to correct for vesicle recovery. In agreement with previous work, it could be demonstrated that incorporation of band 3 in the vesicles caused an increase in SO2-4 permeability, which could be (partially) inhibited by high concentrations of DIDS or a competitive anion such as thiocyanate. However, the magnitude of the increased SO2-4 permeability was highly variable, even when vesicles were reconstituted using band 3 isolated from one batch of ghosts. In addition, the SO2-4 influx curves showed complex kinetics. These results are related to the existence of vesicle heterogeneity with respect to protein content and vesicle size as revealed by stractan density gradient centrifugation and freeze-fracture electron microscopy. Band 3 incorporation also increased the L-glucose permeability of the vesicles which could also be inhibited by DIDS. Glycophorin, which has no known transport function, reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 400:1 phospholipid:protein molar ration increased the bilayer permeability towards SO2-4 as well as towards L-glucose. Surprisingly, the SO2-4 permeability in the vesicles could also be inhibited by DIDS and thiocyanate. It is concluded that the use of DIDS and a competitive anion, thiocyanate, in order to prove that band 3 is functionally reconstituted, is highly questionable. The increased SO2-4 and L-glucose permeability of band 3-lipid as well as glycophorin-lipid vesicles and the inhibitory action of DIDS are discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce the formation of pores. Since the band 3-lipid vesicles are more permeable for SO2-4 than for L-glucose, in contrast to the glycophorin-containing vesicles, it is suggested that some anion specificity of the increased bilayer permeability in the band 3-lipid vesicles is still preserved.