Amplitude Hierarchy of Vesicle Shapes

Shapes of fluid lipid vesicles are governed by the bending elasticity of their membrane as described by the Area-Difference-Elasticity (ADE) model. These shapes can be quantified using a suitable modal representation of the vesicle contour. Prolate vesicles are characterized by a hierarchy in their shape amplitudes. Experimentally, we find an ordering of the amplitudes with mode number both in large (100 nm) as well as giant (10 m) unilamellar vesicles. Mean shapes are found only within the small energetically stable region of the prolate phase. Our study demonstrates that bending energy concepts may be quantitatively used on cellular length scales ranging from the size of organelles to the plasma membrane.     

Gramicidin induced aggregation and size increase of phosphatidylcholine vesicles

To investigate the role of membrane proteins in the fusion process, linear hydrophobic polypeptide gramicidin was used as fusogenic agent in small unilamellar vesicles (SUV) constituted of saturated lecithins. It was found that gramicidin, externally added to a suspension of vesicles, induces a reversible vesicles aggregation. When incorporated into the bilayer, gramicidin induces increase in vesicle size. The vesicle size increase was monitored by column chromatography and transmission electron microscopy. The process of vesicle size increase occurs only when the lipid membrane is in the gel state. A maximum is observed in the kinetics at a temperature of approx. 25 degrees C lower than the phase transition temperature of lipids. Higher rates of vesicle size increase are obtained as the lipid chain length increases. The process is accompanied by a release of internal vesicle content and by membrane lipid mixing.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Lipid miscibility and size increase of vesicles composed of two phosphatidylcholines

The size increase of small unilamellar vesicles composed of binary mixtures either of saturated fatty acid phosphatidylcholines with different chain lengths or of saturated and unsaturated phosphatidylcholines was found to depend on the miscibility properties of the lipid components. No size increase was detected in vesicles formed by two miscible phosphatidylcholines. In vesicles composed of two lipids which are partially immiscible in the gel state, a size increase was observed at temperatures which mainly overlapped the range of temperatures of the lipid phase transition. The rate of size increase of vesicles composed of two lipids which are immiscible in the gel state was faster than that of vesicles composed of two partially immiscible phosphatidylcholines, and the process occurred not only at the temperature ranges of the lipid phase transition, but also when both lipids were in the gel state. The vesicle size increase process occurred without the mixing of the internal content of the vesicles. A model is proposed in which the presence of 'fractures' between membrane regions of different fluidity and/or lipid composition controls the rate of this process.     

Role of oxysterol structure on the microdomain-induced microsolubilization of phospholipid membranes by apolipoprotein A-I

Oxygenated derivatives of cholesterol, oxysterols, have different physicochemical properties and three-dimensional shapes. The kinetics of microsolubilization of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles by apolipoprotein A-I (apoA-I) to form discoidal high-density lipoproteins (rHDL) was dramatically affected by oxysterol chemical structure. Under the experimental conditions of varying oxysterol chemical structure, sterol concentration, and the lipid phase state of DMPC, the kinetics varied over 3 orders of magnitude. Some oxysterols behaved similarly to cholesterol and increased the rate of microsolubilization; however, they were not as effective as cholesterol. Other oxysterols greatly inhibited this process. In general, there was no correlation of the rates with membrane fluidity as measured by fluorescence polarization. The rate of DMPC microsolubilization by apoA-I is highly dependent upon the presence of lattice defects in the membrane surface that occur due to imperfect packing of coexisting lipid phases. The differential ability of various oxysterols to induce the formation of an ordered lipid phase is the probable basis for their effects on the rates of DMPC microsolubilization. There was no effect of oxysterol chemical structure on the structure of the equilibrium rHDL products; however, there was a dramatic effect of sterol concentration on rHDL particle size. Different oxysterols regulate the kinetics of apoA-I membrane association by altering structural microheterogeneity at the membrane surface. However, once the kinetic barrier is overcome, the particle sizes of rHDL products formed are determined solely by the amount of sterol presence.     

Conversion between two cytochalasin B-binding states of the human GLUT1 glucose transporter.

Two cytochalasin B-binding states of the human red blood cell facilitative glucose transporter GLUT1 were studied, one exhibiting one cytochalasin B-binding site on every second GLUT1 monomer (state 1) and the other showing one site per monomer (state 2). Quantitative affinity chromatography of cytochalasin B was performed on (a) biotinylated red blood cells, (b) cytoskeleton-depleted red blood cell membrane vesicles, and (c) GLUT1 proteoliposomes. The cells were adsorbed on streptavidin-derivatized gel beads, and the vesicles and proteoliposomes entrapped in dextran-grafted agarose gel beads. Cytochalasin B binding to free vesicles and proteoliposomes was analyzed by Hummel and Dreyer size-exclusion chromatography and ultracentrifugation. Analysis of the biotinylated cells indicated an equilibrium between the two GLUT1 states. GLUT1 in free membrane vesicles attained state 2, but was converted into state 1 on entrapment of the vesicles. Purification of GLUT1 in the presence of non-ionic detergent followed by reconstitution produced GLUT1 in state 1. This state was maintained after entrapment of the proteoliposomes. Finally, GLUT1 showed slightly higher affinity for cytochalasin B in state 1 than in state 2. In summary, the cytochalasin B-binding state of GLUT1 seemed to be affected by (a) biotinylation of the cell surface, (b) removal of the cytoskeleton at high pH and low ionic strength, (c) interaction between the dextran-grafted agarose gel matrix and the membrane vesicles, and (d) reconstitution to form proteoliposomes.     

Kinetics of cholesterol and phospholipid exchange between Mycoplasma gallisepticum cells and lipid vesicles. Alterations in membrane cholesterol and protein content

The kinetics of exchange of radiolabeled cholesterol and phospholipids between intact Mycoplasma gallisepticum cells and unilamellar lipid vesicles were investigated over a wide range of cholesterol/phospholipid molar ratio. The change in cholesterol/phospholipid molar ratio was achieved by adapting the sterol-requiring M. gallisepticum to grow in cholesterol-poor media, providing cells with decreased unesterified cholesterol content. At least 90% of the cholesterol molecules in unsealed M. gallisepticum membranes underwent exchange at 37 degrees C as a single kinetic pool in the presence of albumin (2%, w/v). However, we observed biphasic exchange kinetics with intact cells, indicating that cholesterol translocation from the inner to outer monolayers was rate-limiting in the exchange process. Approximately 50% of the cholesterol molecules were localized in each kinetic pool, independent of the cholesterol/phospholipid molar ratio in the cells and vesicles. A striking change in the kinetic parameters for cholesterol exchange occurred between 20 and 26 mol % cholesterol; for example, when the cholesterol/phospholipid molar ratio was decreased from 0.36 to 0.25, the half-time for equilibration of the two cholesterol pools at 37 degrees C decreased from 4.6 +/- 0.5 to 2.5 +/- 0.1 h. Phospholipid exchange rates were also enhanced on decreasing the membrane cholesterol content. The ability of cholesterol to modulate its own exchange rate, as well as that of phospholipids, is suggested to arise from the sterol's ability to regulate membrane lipid order. Extensive chemical modification of the membrane surface by cross-linking of some of the protein constituents with 1,4-phenylenedimaleimide decreased the cholesterol exchange rate. Depletion of membrane proteins by treatment of growing cultures with chloramphenicol increased the cholesterol exchange rate, possibly because of removal of some of the protein mass that may impede lipid translocation. The observations that phospholipid exchange was one order of magnitude slower than cholesterol exchange and that dimethyl sulfoxide, potassium thiocyanate, and potassium salicylate enhanced the cholesterol exchange rate are consistent with a mechanism involving lipid exchange by diffusion through the aqueous phase.     

Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase

The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of ≥60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter ≈100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.     

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)     

Unravelling the molecular basis of the selectivity of the HIV-1 fusion inhibitor sifuvirtide towards phosphatidylcholine-rich rigid membranes

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel HIV-1 fusion inhibitor with improved antiretroviral activity. In this work we evaluated the physical chemistry foundation of the interaction of sifuvirtide with biomembrane model systems. Since this peptide has aromatic residues, fluorescence spectroscopy techniques were mostly used. The interaction was assessed by partition and quenching experiments. Results showed no significant interaction with large unilamellar vesicles composed by sphingomyelin and ceramide. In contrast, sifuvirtide presented selectivity towards vesicles composed by phosphatidylcholines (PC) in the gel phase, in opposition to fluid phase PC vesicles. The interaction of this peptide with gel phase PC membranes (K_p = 1.2 × 10²) is dependent on the ionic strength, which indicates the mediation of electrostatic interactions at an interfacial level. The effects of sifuvirtide on the lipid membranes' structural properties were further evaluated using dipole-potential membrane probes, zeta-potential, dynamic light scattering and atomic force microscopy measurements. The results show that sifuvirtide does not cause a noticeable effect on lipid bilayer structure, except for membranes composed by cationic phospholipids. Altogether, we can conclude that sifuvirtide presents a specific affinity towards rigid PC membranes, and the interaction is mediated by electrostatic factors, not affecting the membrane architecture.     

The membrane modulates internal proton transfer in cytochrome c oxidase

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O(2) reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pK(a) in the pH dependence profile of the proton-uptake rate. This pK(a) change resulted in decreased proton-uptake rates in the pH range of ~6.5-9.5, which is explained in terms of lowering of the pK(a) of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal Cu(A)-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.     

Giant Unilamellar Vesicles Formed by Hybrid Films of Agarose and Lipids Display Altered Mechanical Properties

Giant unilamellar vesicles (GUVs) are presumably the current most popular biomimetic membrane model. Preparation of GUVs in physiological conditions using the classical electroformation method is challenging. To circumvent these difficulties, a new method was recently reported, by which GUVs spontaneously swell from hybrid films of agarose and lipids. However, agarose is left encapsulated in the vesicles in different amounts. In this work, we thoroughly characterize the mechanical properties of these agarose-GUVs in response to electric pulses, which induce vesicle deformation and can lead to membrane poration. We show that the relaxation dynamics of deformed vesicles, both in the presence and absence of poration, is significantly slowed down for agarose-GUVs when compared to agarose-free GUVs. In the presence of poration, agarose polymers prevent complete pore closure and lead to high membrane permeability. A fraction of the vesicles were found to encapsulate agarose in the form of a gel-like meshwork. These vesicles rupture and open up after electroporation and the meshwork is expelled through a macropore. When the agarose-GUVs are heated above the melting temperature of agarose for 2 h before use, vesicle response is (partially) recovered due to substantial release of encapsulated agarose during temperature treatment. Our findings reveal potential artifactual behavior of agarose-GUVs in processes involving morphological changes in the membrane as well as poration.     

The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase.

Horse kidney brush border membrane proteins were incorporated into phosphatidylcholine vesicles. Structural analysis of proteoliposomes prepared with various lipid:protein ratios showed that: (a) only a few of the proteins present in the crude brush border extract are integrated, (b) all known membrane hydrolases are integrated, and (c) these proteoliposomes are homogeneous vesicles. Papain solubilization of brush border membrane hydrolases, i.e. aminopeptidase M, neutral alpha-glucosidase, gamma-glutamyltransferase and alkaline phosphatase, performed in parallel on native membrane vesicles and proteoliposomes, revealed similar kinetics. Analysis of membrane vesicles and proteoliposomes on sucrose density gradients either without any treatment, or after papain treatment showed that: (a) in proteoliposomes, neutral alpha-glucosidase is associated with radiolabelled phosphatidylcholine, and (b) papain-treated vesicles and proteoliposomes released enzyme activity in the same way. These results suggest that the integration mechanism of brush border membrane proteins may be similar in proteoliposomes and native membrane vesicles. Transport experiments under equilibrium exchange conditions showed that the uptake properties of proteoliposomes are similar to those of brush border membrane vesicles.     

Electrostatic effects on the kinetics of electron transfer reactions of cytochrome c caused by binding to negatively charged lipid bilayer vesicles.

The effect of binding reduced tuna mitochondrial cytochrome c to negatively charged lipid bilayer vesicles at low ionic strength on the kinetics of electron transfer to various oxidants was studied by stopped-flow spectrophotometry. Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenanthroline ion, whereas the rate of reaction with the negatively charged ferricyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants such as benzoquinone and Rhodospirillum rubrum cytochrome c2 were unaffected by binding to vesicles, suggesting little or no effect of membrane association on cytochrome structure or accessibility of the heme center. The kinetic effects were largest at lower cytochrome c to vesicle ratios, where there was a greater degree of exposure of negatively charged regions on the membrane. The reduction of cobalt phenanthroline and ferricyanide by bound cytochrome c proceeded by nonexponential kinetics, as compared with the monophasic kinetics observed in the absence of vesicles. This was probably due to the heterogeneous distribution of vesicle sizes which exists at a given lipid to protein ratio. Nonlinear oxidant concentration dependencies were observed for cobalt phenanthroline oxidation of membrane-bound cytochrome c, consistent with a (minimal) two-step kinetic mechanism involving association of the oxidant with the membrane followed by electron transfer. Based on a comparison of second-order rate constants as a function of lipid to protein mole ratio, binding of cytochrome c to the bilayer increased the efficiency of the cobalt phenanthroline reaction by a factor of approximately 500 at the highest lipid:protein ratio used. The results suggest a mechanism involving attractive and repulsive electrostatic interactions between the negatively charged bilayer and the electrically charged oxidants, which increase or decrease their effective concentrations at the membrane surface.     

Interaction of piroxicam and meloxicam with DMPG/DMPC mixed vesicles: anomalous partitioning behavior

Small unilamellar vesicles (SUVs) formed from a mixture of dimyristoylphosphatidylcholine (zwitterionic lipid with bulkier headgroup) and dimyristoylphosphatidylglycerol (anionic lipid with relatively smaller headgroup) allows better modulation of the physical properties of lipid bilayers compared to SUVs formed by a single type of lipid, providing us with a better model system to study the effect of membrane parameters on the partitioning of small molecules. Membrane parameter like packing of the vesicles is more pronounced in the gel phase and hence the study was carried out in the gel phase. Mixed vesicles formed from DMPG and DMPC with the mole percent ratio of 100:0, 90:10 and 80:20 were used for this study. As examples of polar solutes, piroxicam and meloxicam, two Non Steroidal Anti-inflammatory Drugs (NSAIDs) were chosen. The pH was adjusted to 2.8 in order to eliminate the presence of anionic forms of the drugs that would not approach the vesicles containing negatively charged DMPG (50% deprotonated at pH 2.8). Surface potential measured by using TNS (2,6-p-toluidinonaphthalene sulfonate, sodium salt) as surface charge sensitive probe showed no significant changes in the surface electrostatics in increasing DMPC content from 0 to 20%. Transmission electron microscopy (TEM) was used to characterize SUVs of different composition at pH 2.8. The average diameter of the mixed vesicles was found to be smaller than that formed by DMPG and DMPC alone. Partition coefficient (K(P)) of piroxicam and meloxicam was measured using intrinsic fluorescence of these molecules. K(P) value of piroxicam decreases with increase in DMPC content whereas it increases with DMPC content in case of meloxicam. This anomalous behavior of partitioning is unexpected since there was no significant change in surface pH of the vesicles and has been explained in terms of lipid packing and water penetration in the lipid bilayer.     

Structural studies on reconstituted reaction center-phosphatidylcholine membranes.

Reaction center protein, isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26 mutant, was incorporated into phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. Cytochrome c, added to preformed reaction center-phosphatidylcholine vesicles, rapidly reduced up to 90% of the laser-generated (BChl)2+ of the reaction center (with kinetics of electron transfer similar to those in the chromatophore membrane) which suggests that the portion of the reaction center which accommodates functional cytochrome c binding sites is exposed predominantly on the exterior of the vesicles. Unit cell electron density profiles were derived from lamellar X-ray diffraction from oriented reaction center-phosphatidylcholine membrane multilayers at varying lipid/protein ratios. The analysis of these profiles showed that the reaction center protein incorporates into the phosphatidylcholine membrane with unique sidedness and that the profile of the reaction center protein itself is asymmetric and spans the membrane.     

Folding kinetics of an alpha helical membrane protein in phospholipid bilayer vesicles

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.     

Size effects on diffusion processes within agarose gels

To investigate diffusion processes in agarose gel, nanoparticles with sizes in the range between 1 and 140 nm have been tested by means of fluorescence correlation spectroscopy. Understanding the diffusion properties in agarose gels is interesting, because such gels are good models for microbial biofilms and cells cytoplasm. The fluorescence correlation spectroscopy technique is very useful for such investigations due to its high sensitivity and selectivity, its excellent spatial resolution compared to the pore size of the gel, and its ability to probe a wide range of sizes of diffusing nanoparticles. The largest hydrodynamic radius (R(c)) of trapped particles that displayed local mobility was estimated to be 70 nm for a 1.5% agarose gel. The results showed that diffusion of particles in agarose gel is anomalous, with a diverging fractal dimension of diffusion when the large particles become entrapped in the pores of the gel. The latter situation occurs when the reduced size (R(A)/R(c)) of the diffusing particle, A, is >0.4. Variations of the fractal exponent of diffusion (d(w)) with the reduced particle size were in agreement with three-dimensional Monte Carlo simulations in porous media. Nonetheless, a systematic offset of d(w) was observed in real systems and was attributed to weak nonelastic interactions between the diffusing particles and polymer fibers, which was not considered in the Monte Carlo simulations.     

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane fusion due to dehydration by polyethylene glycol, dextran, or sucrose

To determine whether polyethylene glycol (PEG) causes growth of liposomes by affecting them directly or indirectly, vesicles composed of phosphatidylcholine were exposed to increasing concentrations of Mr 15 000-20 000 PEG or Mr 40 000 dextran either by direct mixing or across a dialysis membrane. After incubation at room temperature and dilution below at least 5% (w/w) polymer, the vesicles were monitored for fluorescence energy transfer and for absorbance at 400 nm. PEG induced the same levels of dequenching or lipid mixing and increased turbidity, regardless of whether the vesicles had been mixed directly with or dialyzed against PEG. These changes occurred within 5-15 min of polymer application. It is concluded that the increased lipid mixing and/or increased turbidity, indicating vesicle growth, resulted from an indirect effect of PEG on the vesicles--most likely dehydration. Dextran, in contrast to PEG, induced less dequenching and/or less turbidity increase when vesicles were directly mixed with, as opposed to dialyzed against, dextran. Although dextran not in contact with vesicles and with osmotic activity comparable to PEG was able to cause a degree of membrane fusion similar to that of PEG, therefore, the dehydrating effect of dextran could be mitigated if it were allowed to interact with vesicles. In further support of membrane dehydration as a precursor to membrane fusion, lipid mixing among sonicated and sonicated, frozen-thawed vesicles dialyzed against sucrose increased as a function of sucrose concentration. Vesicle morphology generally determined the maximal degree of membrane fusion inducible by the polymers.(ABSTRACT TRUNCATED AT 250 WORDS)