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

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

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

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

Isolation of two glycolipid transfer proteins from bovine brain: reactivity toward gangliosides and neutral glycosphingolipids

Two glycolipid transfer proteins that catalyze the transfer of gangliosides and neutral glycosphingolipids from phosphatidylcholine vesicles to erythrocyte ghosts have been isolated from calf brain. Purification procedures included differential centrifugation, precipitation at pH 5.1, ammonium sulfate precipitation, and gel filtration on Sephadex G-50 and G-75. The final stage employed fast protein liquid chromatography (Mono S), producing two peaks of activity. Apparent purity of the major peak (TP I) was approximately 85-90%, as judged by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis. That of the minor fraction (TP II) was less. The major band of both fractions had a molecular mass of approximately 20,000 daltons. Both proteins catalyzed the transfer of ganglioside GM1 as well as asialo-GM1, but transfer protein I was more effective with di- and trisialogangliosides. Transfer protein II appeared to be somewhat more specific for neutral glycolipids in that GA1 was transferred more rapidly than any of the gangliosides; however, lactosylceramide transfer was relatively slow. Neither protein catalyzed transfer of phosphatidylcholine.     

Probing for preferential interactions among sphingolipids in bilayer vesicles using the glycolipid transfer protein.

We have investigated the intervesicular transfer of galactosylceramide between unilamellar bilayer vesicles composed of differing sphingomyelin and phosphatidylcholine molar ratios. To monitor glycolipid transfer from donor to acceptor vesicles, we used a fluorescence resonance energy transfer assay involving anthrylvinyl-labeled galactosylceramide (AV-GalCer) and perylenoyl-labeled triglyceride. The transfer was mediated by glycolipid transfer protein (GLTP), purified from bovine brain and specific for glycolipids. The initial transfer rate and the total accessible pool of glycolipid in the donor vesicles were both measured. An increase in the sphingomyelin content of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) vesicles decreased the transfer rate in a nonlinear fashion. Decreased transfer rates were clearly evident at sphingomyelin mole fractions of 0.22 or higher. The pool of AV-GalCer available for GLTP-mediated transfer also was smaller in vesicles containing high sphingomyelin content. In contrast, AV-GalCer was more readily transferred from vesicles composed of POPC and different disaturated phosphatidylcholines. Our results show that GLTP acts as a sensitive probe for detecting interactions of glycosphingolipids with neighboring lipids and that the lateral mixing of glycolipids is probably affected by the matrix lipid composition. The compositionally driven changes in lipid interactions, sensed by GLTP, occur in membranes that are either macroscopically fluid-phase or gel/fluid-phase mixtures. Gaining insights into how changes in membrane sphingolipid composition alter accessibility to soluble proteins with affinity for membrane glycolipids is likely to help increase our understanding of how sphingolipid-enriched microdomains (i.e., "rafts" and caveolae) are formed and maintained in cells.     

Net mass transfer of galactosylceramide facilitated by glycolipid transfer protein from pig brain: a monolayer study

A net mass transfer of galactosylceramide (GalCer) and galactosyldiacylglycerol (GalDG) is catalyzed by the glycolipid transfer protein from pig brain. GalCer and GalDG are transferred from a monolayer to phosphatidylcholine vesicles in the subphase or from a glycolipid monolayer to a phosphatidylcholine monolayer. No transfer of phosphatidylcholine is measured under these conditions. It is found that the glycolipid transfer protein functions as a carrier and that glycolipid is bound to less than 50% of the transfer protein. The presence of lipid-free proteins fits with the proposed mechanism of net mass transfer. The glycolipid transfer is influenced by the fluidity of the lipid interface and by the matrix lipid of the interface. GalCer transfer is stimulated in the presence of GalDG.     

Specificity of the glycolipid transfer protein from pig brain

Lipid specificity has been studied in the lipid transfer reaction facilitated by the glycolipid transfer protein from pig brain. The lipid transfer was measured by determining the transfer of a radioisotopically labeled lipid from donor liposomes to either acceptor liposomes or mitochondria. Whenever possible, the liposomes contained 1 mol % of the lipid whose transfer was under study. The transfer protein accelerates the transfer of glucosylceramide, galactosylceramide (GalCer), lactosylceramide (LacCer), galactosylceramide 3-sulfate, globotriaosylceramide, LacCer sulfate, sialosyl-LacCer, globotetraosylceramide, and globopentaosylceramide. An inverse relationship is found between the length of sugar chains in glycosphingolipids and the transfer rates. In addition to the glycosphingolipids, the transfer protein facilitates the transfer of galactosyldiacylglycerol, digalactosyldiacylglycerol, glucosyldiacylglycerol, and diglucosyldiacylglycerol. The protein does not facilitate the transfer of dimannosyldiacylglycerol. The transfer of periodate-oxidized and subsequently reduced derivatives of GalCer and LacCer is facilitated by the transfer protein. The derivatives of GalCer are transferred at lower rates than GalCer, whereas the derivatives of LacCer are transferred at higher rates than LacCer. The transfer protein does not facilitate the transfer of phosphatidylcholine, phosphatidylinositol, cholesterol, or cholesteryloleate. These results suggest that the glycolipid transfer protein from pig brain has specificity to hydroxyl groups present in the sugar residue directly linked to either ceramide or diacylglycerol. The presence of glucose or galactose linked to these hydrophobic moieties makes the glycolipid transferable by the protein.     

Phosphatidylinositol transfer protein from bovine brain: Substrate specificity and membrane binding properties

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521–528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.     

Phosphatidylinositol transfer protein from bovine brain. Substrate specificity and membrane binding properties

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521-528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Membrane composition modulates thiopental partitioning in bilayers and biomembranes

The nonspecific interaction of thiopental with erythrocyte ghosts, synaptic membranes, microsomes and mitochondria has been measured at 25°C and pH 6.6. In cholesterol-depleted erythrocyte ghosts the partition coefficient decreases with increasing cholesterol content. In sonicated liposomes made from egg lecithin and cholesterol the partition coefficient also decreases with increasing cholesterol content. The dependence of the partition coefficient on cholesterol content in the biological membranes, on average, parallels that in the lipid bilayers. The partition coefficient in lipid bilayers made from lipids extracted from erythrocyte ghosts was comparable to that in the corresponding egg lecithin/cholesterol bilayer. The partition coefficients of all the biomembranes are consistently lower than those in the corresponding egg lecithin/cholesterol bilayer, the free energy of transfer between biomembrane and corresponding bilayer being ?1 kcal/mol.     

Effect of bilayer membrane curvature on activity of phosphatidylcholine exchange protein

Effect of bilayer membrane curvature of substrate phosphatidylcholine and inhibitor phosphatidylserine on the activity of phosphatidylcholine exchange protein has been studied by measuring transfer of spin-labeled phosphatidylcholine between vesicles, vesicles and liposomes, and between liposomes. The transfer rate between vesicles was more than 100 times larger than that between vesicles and liposomes. The transfer rate between liposomes was still smaller than that between vesicles and liposomes and nearly the same as that in the absence of exchange protein. The markedly enhanced exchange with vesicles was ascribed to the asymmetric packing of phospholipid molecules in the outer layer of the highly curved bilayer membrane. The inhibitory effect of phosphatidylserine was also greatly dependent on the membrane curvature. The vesicles with diameter of 17 nm showed more than 20 times larger inhibitory activity than those with diameter of 22 nm. The inhibitory effect of liposomes was very small. The size dependence was ascribed to stronger binding of the exchange protein to membranes with higher curvatures. The protein-mediated transfer from vesicles to spiculated erythrocyte ghosts was about four times faster than that to cup-shaped ghosts. This was ascribed to enhanced transfer to the highly curved spiculated membrane sites rather than greater mobility of phosphatidylcholine in the spiculated ghost membrane.     

Spontaneous and protein-mediated intermembrane transfer of the antiretroviral liponucleotide 3'-deoxythymidine diphosphate diglyceride.

Phospholipid conjugates of antiretroviral nucleosides show activity against the human immunodeficiency virus in vitro [Hostetler, K. Y., Stuhmiller, L. M., Lenting, H. B. M., Van den Bosch, H., & Richman, D. D. (1990) J. Biol. Chem. 265, 6112-6117]. In order to gain insight into the membrane association and the spontaneous and protein-mediated intermembrane transfer of these compounds, we have synthesized the fluorescent analog 3'-deoxythymidine diphosphate 1-myristoyl-2-(10-pyren-1-yl-decanoyl)glycerol. The compound readily incorporated into ethanol-injection vesicles, but the stability of the fluorescent probe (10% of total lipid) in the lipid bilayer was less than that of 1-myristoyl-2-(10-pyren-1-yldecanoyl)phosphatidylcholine. Using a donor-acceptor vesicle assay system, half-times for spontaneous transfer at 25 and 37 degrees C were 20 and 100 min, respectively. The liponucleotide was rapidly transferred between membranes by the nonspecific lipid-transfer protein at a rate at least 10-fold that of the corresponding phosphatidylcholine. Depletion of the liponucleotide from the outer monolayer of vesicles by a large excess of nonspecific lipid-transfer protein indicated a transbilayer distribution similar to the mass distribution of phospholipids. Essentially no flip-flop of the inner monolayer liponucleotide was observed.     

Properties of a specific glycolipid transfer protein from bovine brain

A transfer protein specific for glycolipids has been isolated from bovine brain. As judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, the protein is 68% pure and has a molecular weight of 20 000. Three different assays were employed to study the protein's specificity and glycolipid binding properties. The protein transferred several different neutral glycosphingolipids and ganglioside GM1 equally well, but failed to accelerate phosphatidylcholine or sphingomyelin intervesicular movement. The protein's ability to interact with glycolipids was strongly influenced by the physical properties of the matrix phospholipid in which the glycolipids reside. Both the phase state of the phospholipid matrix and bilayer curvature affected glycolipid intervesicular transfer rates. Protein binding to phospholipid vesicles containing either tritium-labeled or pyrene-labeled glucosylceramide could not be demonstrated by density gradient centrifugation or fluorescence energy transfer measurements, respectively. A specific association of the transfer protein for pyrene-labeled glucosylceramide was found when the fluorescence emission of the pyrene excimer-to-monomer ratio was measured suggesting that a portion of the fluorescent glycolipid was being sequestered from the phospholipid vesicles and was binding to the freely soluble protein.     

Phospholipid transfer between vesicles. Dependence on presence of cytochrome P-450 and phosphatidylcholine-phosphatidylethanolamine ratio

The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicle was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they was transferred to the nonspin-labeled acceptor vesicles. A lower transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles for a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a 31P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.     

The effect of temperature on the agglutinability by lectins of liposomes prepared from total lipids of erythrocytes

Multilamellar liposomes prepared from total lipids of red blood cells are agglutinable by the addition of soybean lectin. At 5 °C the rate of agglutination is significantly slower than at 37 °C, in contrast to erythrocyte ghosts and ghosts sonicated to 1 μ vesicles. The slower lateral mobilities of the lectin glycolipid receptor in the lipid liposomes due to increased microviscosity of the bilayer at the lower temperature, might be one explanation of our agglutination results. However, the opposite temperature dependence seen with ghosts argues for a possible protein modulation of the agglutination reaction.     

Phospholipid transfer activities in toad oocytes and developing embryos

The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing 14C-labeled phospholipids and 3H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.     

Effect of acceptor membrane phosphatidylcholine on the catalytic activity of bovine liver phosphatidylcholine transfer protein

Protein-mediated transfer of phosphatidylcholine (PC) by bovine liver phosphatidylcholine transfer protein (PC-TP) was examined using a vesicle-vesicle assay system. Donor and acceptor membranes were prepared from Escherichia coli phospholipids and limiting amounts of egg yolk PC. PC transfer between vesicles of E. coli lipid/egg PC was markedly higher than transfer of PC from vesicles of E. coli lipid/egg PC to vesicles of E. coli lipid. Kinetic parameters of the interaction between PC-TP and E. coli lipid vesicles with or without PC was investigated. The apparent dissociation constants of the complex formed between PC-TP and these vesicles were determined kinetically and from double-reciprocal plots of intrinsic PC-TP fluorescence intensity increase versus vesicle concentration. The magnitude of the dissociation constant decreased as the PC content of the vesicles increased from 0 to 5 mol%. In addition, kinetic analysis revealed that the presence of PC in acceptor vesicles increased both the association and dissociation of PC-TP from vesicles. The effect of membrane PC molecules on transfer rates was examined using bis-phosphatidylcholine, a dimeric PC molecule which is not transferred by PC-TP. Rates of PC transfer to acceptor vesicles comprised of E. coli lipid/bis-PC were virtually identical to rates observed with acceptors vesicles prepared from E. coli lipid. The results suggest that transfer of PC by PC-TP is enhanced only when insertion of protein-bound PC occurs concurrently with the extraction of a molecule of membrane PC, i.e., a concerted, one-step catalytic mechanism for phospholipid exchange.     

Evidence for the Presence of a Ganglioside Transfer Protein in Brain

An extract from rat brain has been shown to catalyze the transfer of ganglioside GM1 from sonicated vesicles to erythrocyte ghosts. It also enhanced the transfer of GM1 to a crude neuronal membrane preparation, whereas myelin took up only a very limited amount. The transfer activity was heat-labile. Similar transfer activities were found in extracts from bovine gray and white matter, that of the former being comparable to rat brain whereas the latter was greater per milligram protein.     

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.     

Transfer of band 3, the erythrocyte anion transporter, between phospholipid vesicles and cells

Band 3, the anion transport protein of human erythrocyte membranes, can be transferred from cells to liposomes and from liposomes back to cell membranes, retaining function and native orientation. After incubation with cells, sonicated phosphatidylcholine vesicles bind a transmembrane protein that comigrates with band 3 on sodium dodecyl sulfate-polyacrylamide gels. Like native red cell band 3, the vesicle-bound protein is cleaved by chymotrypsin into 65- and 30-kdalton fragments and is not cleaved by trypsin. The protein can be cross-linked by copper-phenanthroline oxidation either before or after transfer to vesicles; in either case, the vesicle fractions contain high molecular weight material that is dissociated into 95-kdalton species by mercaptoethanol. Band 3-vesicle complexes contain no detectable cell lipid and are specifically permeable to anions. Greater than 99% of their anion uptake can be blocked by the band 3 inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). Red cells whose band 3 function has been blocked irreversibly by DIDS or eosin maleimide regain part of their anion permeability upon incubation with band 3-vesicle complexes. Under the conditions employed, an average of one copy of functional band 3 is delivered to half of the cells, increasing by 2.3-fold the number of cells containing functional anion transporters. Incubation of pure lipid vesicles or red cell membrane buds with either normal red cells or eosin maleimide inhibited cells has no detectable effect on the cells' anion permeability.