The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4′-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent ‘donor’ and 1-(4′-carboxyethyl)-6-(4″-nitro)diphenyl-trans-1,3,5-hexatriene as ‘acceptor’. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20°C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of l-α-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with < 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.     

Aggregation and fusion of unilamellar vesicles by poly(ethylene glycol)

Various aspects of the interaction between the fusogen, poly(ethylene glycol) and phospholipids were examined. The aggregation and fusion of small unilamellar vesicles of egg phosphatidylcholine (PC), bovine brain phosphatidylserine (PS) and dimyristoylphosphatidylcholine (DMPC) were studied by dynamic light scattering, electron microscopy and NMR. The fusion efficiency of Dextran, glycerol, sucrose and poly(ethylene glycol) of different molecular weights were compared. Lower molecular weight poly(ethylene glycol) are less efficient with respect to both aggregation and fusion. The purity of poly(ethylene glycol) does not affect its fusion efficiency. Dehydrating agents, such as Dextran, glycerol and sucrose, do not induce fusion. 31P-NMR results revealed a restriction in the phospholipid motion by poly(ethylene glycol) greater than that by glycerol and Dextran of similar viscosity and dehydrating capacity. This may be associated with the binding of poly(ethylene glycol) to egg PC, with a binding capacity of 1 mol of poly(ethylene glycol) to 12 mol of lipid. Fusion is greatly enhanced below the phase transition for DMPC, with extensive fusion occurring below 6% poly(ethylene glycol). Fusion of PS small unilamellar vesicles depends critically on the presence of cations. Large unilamellar vesicles were found to fuse less readily than small unilamellar vesicles. The results suggest that defects in the bilayer plays an important role in membrane fusion, and the 'rigidization' of the phospholipid molecules facilitates fusion possibly through the creation of defects along domain boundaries. Vesicle aggregation caused by dehydration and surface charge neutralization is a necessary but not a sufficient condition for fusion.     

Effect of poly(ethylene glycol) on the polarity of aqueous solutions and on the structure of vesicle membranes

The partitioning of TEMPO into phosphatidylcholine vesicle membranes is reduced upon addition of poly(ethylene glycol). This is caused by reduced polarity of the aqueous phase as well as decreased membrane fluidity in the presence of poly(ethylene glycol). The isotropic hyperfine splitting of TEMPO in aqueous poly(ethylene glycol) solutions was used as a measure of solvent polarity. The alterations of the membrane fluidity were detected by means of two different fatty acid spin labels. The influences of physicochemical properties of an aqueous poly(ethylene glycol) phase on the membrane structure of cells and vesicles are discussed in the light of membrane fusion.     

Non-covalent cross-linking of lipid bilayers by myelin basic protein: a possible role in myelin formation.

Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, L-alpha-dimyristoyl phosphatidylcholine and DL-alpha-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15-18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associated species. A similar interaction was observed with diacyl phosphatidylserine vesicles. These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myelin, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.     

Effects of proteins on phospholipid vesicle aggregation and lipid vesicle-monolayer interactions

The effects of proteins on divalent cation-induced phospholipid vesicle aggregation and phospholipid vesicle-monolayer membrane interactions (fusion) were examined. Glycophorin (from human erythrocytes) suppressed the membrane interactions more than N-2 protein (from human brain myelin) when these proteins were incorporated into acidic phospholipid vesicle membranes. The threshold concentrations of divalent cations which induced vesicle aggregation were increased by protein incorporation, and the rate of vesicle aggregation was reduced. A similar inhibitory effect by the proteins, incorporated into lipid vesicle membranes, was observed for Ca²⁺-induced lipid vesicle-monolayer interactions. However, when these proteins were incorporated only in the acidic phospholipid monolayers, the interaction (fusion) of the lipid vesicle-monolayer membranes, induced by divalent cations, was not appreciably altered by the presence of the proteins.In contrast to these two proteins, the presence of synexin in the solution did enhance the Ca²⁺-induced aggregation of phosphatidylserine vesicles, but did not seem to affect the degree of Ca²⁺-induced fusion between phosphatidylserine/phosphatidylcholine (1:1) and phosphatidylserine vesicles and monolayer membranes.     

Distribution of phospholipids around gramicidin and D-beta-hydroxybutyrate dehydrogenase as measured by resonance energy transfer

A resonance energy transfer method was developed to study the distribution of phospholipids around integral membrane proteins. The method involved measuring the extent of energy transfer from tryptophan residues of the proteins to different phospholipids labeled with a dansyl moiety in the fatty acid chain. No specific interactions were observed between gramicidin and dansyl-labeled phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The results were consistent with a random distribution of each phospholipid in the bilayer in the presence of gramicidin. However, a redistribution of both gramicidin and dansyl-labeled phospholipids was easily observed when a phase separation was induced by adding Ca2+ to vesicles made up of phosphatidylcholine and phosphatidic acid. Polarization measurements showed that in the presence of Ca2+ a rigid phosphatidic acid rich region and a more fluid phosphatidylcholine-rich region were formed. Energy-transfer measurements from gramicidin to either dansylphosphatidylcholine or dansylphosphatidic acid showed gramicidin preferentially partitioned into the phosphatidylcholine-rich regions. Energy-transfer measurements were also carried out with D-beta-hydroxybutyrate dehydrogenase reconstituted in a vesicle composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Although the enzyme has a specific requirement for phosphatidylcholine for activity, the extent of energy transfer decreased in the order dansylphosphatidic acid, dansylphosphatidylcholine, dansylphosphatidylethanolamine. Thus, the enzyme reorganized the phospholipids in the vesicle into a nonrandom distribution.     

The influence of poly(ethylene glycol) 6000 on spermine-induced aggregation of liposomes.

Poly(ethylene glycol) 6000 affected the aggregation of mixed liposomes induced by spermine. It lowered the concentration of spermine causing 50% maximal aggregation, accelerated the rate and increased the extent of aggregation. The effect was inversely proportional to the density of the acidic phospholipid in the vesicles. These effects were not due either to poly(ethylene glycol) 6000-induced permanent structural modification of the liposome or increased binding of spermine to the vesicles. These findings are discussed in relation to a decreased hydration force caused by the ability of poly(ethylene glycol) 6000 to alter the water of hydration of the phospholipid polar groups in the liposome.     

Effect of poly(ethylene glycol) on phospholipid hydration and polarity of the external phase

The hydration properties of phosphatidylcholine (PC)/water dispersions on the addition of poly(ethylene glycol) were studied by means of ²H-NMR. The quadrupole splittings and their temperature dependences correspond to measurements of PC/water dispersions at low water content. It is concluded that the bound water is partly extracted by poly(ethylene glycol) but the binding properties of the water in the inner hydration shell of about five water molecules are not changed. The ability of some phospholipid/water dispersions to undergo phase transitions to nonlamellar structures upon dehydration is discussed. Dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylcholine do not form nonlamellar structures on addition of purified poly(ethylene glycol), as was demonstrated by means of ³¹P-NMR. Poly(ethylene glycol) decreases the polarity of the aqueous phase and the partition of hydrophobic molecules between the membrane and the external phase is changed. This was demonstrated using the excimer fluorescence of pyrene in a ghost suspension. It is suggested that the changes in polarity and hydration on the addition of poly(ethylene glycol) can contribute to the alterations in the membrane surface observed under conditions of membrane contact and fusion.     

Fusion of lipid vesicles with ascites tumor cells and their lipid-depleted variants. Studies with radioactive- and fluorescent-labeled vesicles

Cultured ascites tumor cells and their lipid-depleted variants, which contained 35-40% less membrane phospholipid and cholesterol, were used for fusion experiments with unilamellar lipid vesicles which were between 300 and 600 nm in diameter. Vesicle-cell interaction was followed by tracer studies using vesicles double-labeled in the lipid moiety, by vesicle-encapsulated [3H] dextran, and by measurements of energy transfer between N-(10-[1-pyrene]decanoyl)sphingomyelin-labeled vesicles and alpha-parinaric acid-labeled cells in the presence of poly(ethylene glycol) (PEG) as fusogen. The reaction rates measured with the radiolabeled vesicles were found to follow patterns similar to those obtained with the resonance energy transfer assay. This latter method revealed a vesicle-cell membrane fusion reaction, which was substantiated by radiolabeling the internal cellular compartment after treatment of the cells with [3H]dextran-encapsulated vesicles as shown by electron microscopic autoradiography on semi-thin sections. Endocytosis as a reaction mechanism can be excluded, since no energy transfer was observed at 25 degrees C in the absence of PEG. Investigations of vesicle bilayer order and fluidity on vesicle-cell interaction revealed optimal reactivity, with intermediate fluidity corresponding to cholesterol/phospholipid ratios between 0.7 and 1.0 and fluorescence depolarization (P) values of 0.18 and 0.21. Lipid depletion decreased the reaction velocity between cells and vesicles by about 20%, exhibiting V values of 33.2 mumol/min, as compared to the control of 41.4 mumol/min determined for 10(7) cells. The affinity constants for vesicle lipid were affected only slightly with Km values of 0.195 mM (0.210 mM). The activation energies for the reaction were calculated to give values of EA = 22.44 kJ/mol for the control and of EA = 20.4 kJ/mol for the modified cells. These data indicate that the decrease in membrane lipid content apparently has no major influence on the extent of the interaction.     

Osmoelastic coupling in biological structures: decrease in membrane fluidity and osmophobic association of phospholipid vesicles in response to osmotic stress

Poly(ethylene glycol)- (PEG-) induced change in membrane fluidity and aggregation of phospholipid vesicles were studied. A threshold concentration of PEG was required to induce the aggregation. This concentration increased with a decrease in the molecular weight of PEG, e.g., from 5% (w/w) with PEG 6000 (PEG with an average molecular weight of 7500) to more than 30% (w/w) with PEG 200. The aggregation was reversible upon dilution of PEG if the initial PEG concentration was smaller than a certain value, e.g., 22% (w/w) for PEG 6000. Addition of PEG caused a decrease in membrane fluidity of the vesicles detected by fluorescence anisotropy of diphenylhexatriene and by electron spin resonance of a spin-labeled fatty acid. The anisotropy change of diphenylhexatriene fluidity change had an inflection point at approximately 5% (w/w) of PEG 6000, which might suggest that the aggregation would make the decrease of membrane fluidity smaller. Transfer of lipid molecules between phospholipid vesicles was enhanced by the PEG-induced aggregation. The enhancement occurred not only upon direct addition of PEG to the suspending medium, but also upon dialysis of the vesicle suspension against a high concentration of PEG. All these features are consistent with osmoelastic coupling in the phospholipid membranes and the subsequent osmophobic association of the vesicles. The imbalance of osmolarity between the region adjacent to the vesicle surface (exclusion layer) and the bulk aqueous phase, which results from the preferential exclusion of PEG from the exclusion layer in the case of direct addition of PEG, exerts an osmotic stress on the vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion.

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.     

A new fluorimetric method to measure protein-catalyzed phospholipid transfer using 1-acyl-2-parinaroylphosphatidylcholine

A new, simple and versatile method to measure phospholipid transfer has been developed, based on the use of a fluorescent phospholipid derivative, 1-acyl-2-parinaroylphosphatidylcholine. Vesicles prepared of this phospholipid show a low level of fluorescence due to interactions between the fluorescent groups. When phospholipid transfer protein and vesicles consisting of non-labeled phosphatidylcholine are added the protein catalyzes an exchange of phosphatidylcholine between the labeled donor and non-labeled acceptor vesicles. The insertion of labeled phosphatidylcholine into the non-labeled vesicles is accompanied by an increase in fluorescence due to abolishment of self-quenching. The initial rate of fluorescence enhancement was found to be proportional to the amount of transfer protein added. This assay was applied to determine the effect of membrane phospholipid composition on the activity of the phosphatidylcholine-, phosphatidylinositol- and non-specific phospholipid transfer proteins. Using acceptor vesicles of egg phosphatidylcholine and various amounts of phosphatidic acid it was observed that the rate of phosphatidylcholine transfer was either stimulated, inhibited or unaffected by increased negative charge depending on the donor to acceptor ratio and the protein used. In another set of experiments acceptor vesicles were prepared of phosphatidylcholine analogues in which the ester bonds were replaced with ether bonds or carbon-carbon bonds. Assuming that only a strictly coupled exchange between phosphatidylcholine and analogues gives rise to the observed fluorescence increase, orders of substrate preference could be established for the phosphatidylcholine- and phosphatidylinositol transfer proteins.     

Exchangeability and rate of flip-flop of phosphatidylcholine in large unilamellar vesicles, cholate dialysis vesicles, and cytochrome oxidase vesicles.

Three model membrane systems have been characterized in terms of their interaction with phospholipid exchange proteins. Large unilamellar vesicles of phosphatidylcholine prepared by ether vaporization are shown to be homogeneous by gel filtration. Phospholipid exchange proteins from three sources are capable of catalyzing the rapid exchange of approximately half of the phospholipid from these vesicles. The remaining pool of radioactive phospholipid is virtually nonexchangeable (t1/2 of several days). Small unilamellar vesicles of phosphatidylcholine prepared by cholate dialysis also exhibit two pools of phospholipid (65% rapidly exchangable, 35% very slowly exchangeable) when incubated with beef liver phospholipid exchange protein. Cytochrome oxidase vesicles prepared both by a cholate dialysis method and by a direct incorporation method have been fractionated on a Ficoll discontinuous gradient, and tested for interaction with beef heart exchange protein. Two pools of phospholipid are once again observed (70% rapidly exchangable, 30% nonexchangeable), even for vesicles which have incorporated the transmembranous enzyme at a phospholipid to protein weight ratio of 2. The size of the rapidly exchangeable pool of phosphatidylcholine for each of the vesicle systems is consistent with the calculated fraction of phospholipid in the outer monolayer. The extremely slow rate of exchange of the second pool of the second pool of phospholipid reflects the virtual nonexistence of phospholipid flip-flop in any of these model membranes.     

Membrane proteins exposed on the external side of the intestinal brush-border membrane have fusogenic properties

The intestinal brush-border membrane contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompanied by a partial loss of vesicle contents. Proteolytic treatment of the brush-border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush-border membrane. Activation of intrinsic proteinases of the brush-border membrane liberates water-soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush-border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr less than or equal to 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G-75 SF yielding three protein peaks of apparent Mr greater than or equal to 70,000, Mr = 22,000 and Mr = 11,500. All three protein fractions show similar phosphatidylcholine-exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr greater than or equal to 70,000 has the highest fusogenic activity while the protein fraction of apparent Mr = 11,500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable.     

Studies on membrane fusion. 1. Interactions of pure phospholipid membranes and the effect of myristic acid,lysolecithin, proteins and dimethylsulfoxide

The interaction and mixing of membrane components in sonicated unilamellar vesicles and also non-sonicated multilamellar vesicles prepared from highly purified phospholipids suspended in NaCl solutions has been examined. Electron microscopy and differential scanning calorimetry were used to characterize the extent and kinetics of mixing of membrane components between different vesicle populations. No appreciable fusion was detected between populations of non-sonicated phospholipid vesicles incubated in aqueous salt (NaCl) solutions. Mixing of vesicle membrane components via diffusion of phospholipid molecules between vesicles was observed in populations of negatively charged phosphatidylglycerol vesicles but similar exchange diffusion was not detected in populations of neutral phosphatidylcholine vesicles. Incubation of sonicated vesicle populations at temperatures close to or above the phospholipid transition temperature resulted in an increase in vesicle size and mixing of vesicle membrane components as determined by a gradual change in the thermotropic properties of the mixed vesicle population. The interaction of purified phospholipid vesicles was also examined in the presence of myristic acid and lysolecithin. Our results indicate that while these agents enhance mixing of vesicle membrane components, in most cases mixing probably proceeds via diffusion of phospholipid molecules rather than by fusion of entire vesicles. Increased mixing of vesicle membrane components was also produced when vesicles were prepared containing a purified hydrophobic protein (myelin proteolipid apoprotein) or were incubated in the presence of dimethylsulfoxide. In these two systems, however, the evidence suggests that mixing of membrane components results from the fusion of entire vesicles.     

Effective encapsulation of proteins into size-controlled phospholipid vesicles using freeze-thawing and extrusion

We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-l-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 microm diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-)(1) and cooling rate of -140 degrees C min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 +/- 20 nm.     

Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers.

The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37 degrees C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Membrane properties modulate the activity of a phosphatidylinositol transfer protein from the yeast, Saccharomyces cerevisiae

A phospholipid transfer protein from yeast (Daum, G. and Paltauf, F. (1984) Biochim. Biophys. Acta 794, 385-391) was 2800-fold enriched by an improved procedure. The specificity of this transfer protein and the influence of membrane properties of acceptor vesicles (lipid composition, charge, fluidity) on the transfer activity were determined in vitro using pyrene-labeled phospholipids. The yeast transfer protein forms a complex with phosphatidylinositol or phosphatidylcholine, respectively, and transfers these two phospholipids between biological and/or artificial membranes. The transfer rate for phosphatidylinositol is 19-fold higher than for phosphatidylcholine as determined with 1:8 mixtures of phosphatidylinositol and phosphatidylcholine in donor and acceptor membrane vesicles. If acceptor membranes consist only of non-transferable phospholipids, e.g., phosphatidylethanolamine, a moderate but significant net transfer of phosphatidylcholine occurs. Phosphatidylcholine transfer is inhibited to a variable extent by negatively charged phospholipids and by fatty acids. Differences in the accessibility of the charged groups of lipids to the transfer protein might account for the different inhibitory effects, which occur in the order phosphatidylserine which is greater than phosphatidylglycerol which is greater than phosphatidylinositol which is greater than cardiolipin which is greater than phosphatidic acid which is greater than fatty acids. Although mitochondrial membranes contain high amounts of negatively charged phospholipids, they serve effectively as acceptor membranes, whereas transfer to vesicles prepared from total mitochondrial lipids is essentially zero. Ergosterol reduces the transfer rate, probably by decreasing membrane fluidity. This notion is supported by data obtained with dipalmitoyl phosphatidylcholine as acceptor vesicle component; in this case the transfer rate is significantly reduced below the phase transition temperature of the phospholipid.     

1H-NMR Study of the effect of synthetic polymers on the fluidity,transition temperature and fusion of dipalmitoyl phosphatidylcholine small vesicles

The interaction of water-soluble polymers with dipalmitoyl phosphatidylcholine small vesicles and the effect on vesicle fusion were studied by means of ¹H-NMR spectrometry. The motion of dipalmitoyl phosphatidylcholine molecules decreased on interaction with the polymers and was detected as a change in the signal intensity. The interaction behavior of polymers is very sensitive to the chemical structure of the applied polymers. Poly(styrene sulfonic acid) and poly(ethylene glycol) decreased the motion of the choline methyl group, predominantly through coulombic and hydrophobic interaction forces, respectively. For example, in the case of the poly(styrene sulfonic acid)-containing system, the signal intensity of the choline methyl group was decreased about 15% while those of the hydrophobic methylene and terminal methyl groups were scarcely decreased by the addition of polymer to a final concentration of 4.0 · 10^t-2 unit mol/1. These polymers are considered to interact with the surface of the vesicle membrane. On the other hand, poly(l-glutamic acid) and poly($\text{N-}\text{vinyl}\text{-2-}\text{pyrrolidone}$) decreased the signal intensities of not only the choline methyl group, but also those of the hydrophobic methylene and terminal methyl groups. This result suggest that part of these polymers might be incorporated into the hydrophobic region of the vesicle membrane.Addition of the non-ionic polymers inhibited vesicle fusion considerably. This effect was explained by the stabilization of dipalmitoyl phosphatidylcholine vesicles by complexation with these polymers.