g(m1)-ganglioside and 1-palmitoyl-2-oleoylphosphatidylcholine

β-Amyloid (Aβ) is a 39-43 residue peptide involved in the pathogenesis of Alzheimer's disease. Aβ deposits onto the cells and gives rise to the plaques that are characteristic of the disease. In an effort to understand the molecular mechanism of plaque formation, we have examined the interaction of Aβ42, considered to be the most pathogenic of the peptides, with lipid bilayers consisting of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to which small amounts of GM1 ganglioside (1-5 mol%) were incorporated. POPC bilayers exist in the fluid, or liquid-disordered state at room temperature, mimicking the fluidity of cell membranes. An Aβ42 preparation consisting essentially of peptide monomers was used. A combination of molecular dynamics (MD), isothermal calorimetry and Langmuir balance measurements was applied. Our results show that Aβ binds POPC bilayers, and that binding increases (ΔG of binding decreases) with GM1, but only up to 3 mol% of the ganglioside, larger concentrations appearing to have a lower effect. MD and Langmuir balance measurements concur in showing that the peptide adsorbs onto the bilayer surface, but does not become inserted into it at surface pressures compatible with the cell membrane conditions. Thioflavin T measurements agree with MD in revealing a very low degree of peptide oligomerization/aggregation under our conditions. This is in contrast with previous studies showing peptide aggregation and insertion when interacting with membranes in the liquid-ordered state. The present contribution underlines the importance of bilayer lipid composition and properties for Aβ plaque formation.

Topics: Adsorption; Amyloid beta-Peptides; Benzothiazoles; Calorimetry; Cell Membrane; G(M1) Ganglioside; Humans; Lipid Bilayers; Molecular Dynamics Simulation; Peptide Fragments; Phosphatidylcholines

Biomembranes are built up from lipid bilayers with two leaflets that typically differ in their lipid composition. Each lipid molecule stays within one leaflet of the bilayer before it undergoes a transition, or flip-flop, to the other leaflet. The corresponding flip-flop times are very different for different lipid species and vary over several orders of magnitude. Here, we use molecular dynamics simulations to elucidate the consequences of this separation of time scales for compositionally asymmetric bilayers. We first study bilayers with two lipid components that do not undergo flip-flops on the accessible time scales. In such a situation, one must distinguish a bilayer state in which both leaflets have the same preferred area from another state in which each leaflet is tensionless. However, when we add a third lipid component that undergoes frequent flip-flops, the bilayer relaxes toward the state with tensionless leaflets, not to the state with equal preferred leaflet areas. Furthermore, we show that bilayers with compositional asymmetry acquire a significant spontaneous curvature even if both leaflets are tensionless. Our results can be extended to lipid bilayers with a large number of lipid components provided at least one of these components undergoes frequent flip-flops. For cellular membranes containing lipid pumps, the leaflet tensions also depend on the rates of protein-induced flip-flops.

Topics: Cell Membrane; G(M1) Ganglioside; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC

Topics: 2-Naphthylamine; Carbocyanines; Fluorescent Dyes; G(M1) Ganglioside; Laurates; Lipid Bilayers; Microscopy, Fluorescence; Molecular Structure; Phosphatidylcholines; Sulfoglycosphingolipids; Unilamellar Liposomes

Multivalent receptor-mediated interactions between virions and a lipid membrane can be weakened using competitive nonpathogenic ligand binding. In particular, the subsequent binding of such ligands can induce detachment of bound virions, a phenomenon of crucial relevance for the development of new antiviral drugs. Focusing on the simian virus 40 (SV40) and recombinant cholera toxin B subunit (rCTB), and using (monosialotetrahexosyl)ganglioside (GM1) as their common receptor in a supported lipid bilayer (SLB), we present the first detailed investigation of this phenomenon by employing the quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) microscopy assisted 2D single particle tracking (SPT) techniques. Analysis of the QCM-D-measured release kinetics made it possible to determine the binding strength of a single SV40-GM1 pair. The release dynamics of SV40, monitored by SPT, revealed that a notable fraction of SV40 becomes mobile just before the release, allowing to estimate the distribution of SV40-bound GM1 receptors just prior to release.

Topics: Animals; Cattle; Cholera Toxin; G(M1) Ganglioside; Kinetics; Ligands; Lipid Bilayers; Phosphatidylcholines; Simian virus 40; Virion; Virus Attachment

Glycosphingolipids are minor yet essential components of eukaryotic cell membranes and are involved in a variety of cellular processes. Although glycosphingolipids such as GM1 have been previously reported to influence the function of G protein-coupled receptors (GPCRs), the molecular mechanism remains elusive. In this paper, we have explored the interaction of GM1 with the serotonin

Topics: Binding Sites; Cholesterol; G(M1) Ganglioside; Humans; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Protein Binding; Protein Interaction Domains and Motifs; Receptor, Serotonin, 5-HT1A

The influence of the glycolipid GM1 on the physical properties of POPC membranes was studied systematically by using different methods applied to giant and large unilamellar vesicles. The charge per GM1 molecule in the membrane was estimated from electrophoretic mobility measurements. Optical microscopy and differential scanning calorimetry were employed to construct a partial phase diagram of the GM1/POPC system. At room temperature, phase separation in the membrane was detected for GM1 fractions at and above ∼5 mol %, whereby GM1-rich gel-like domains were observed by fluorescent microscopy. Fluctuation analysis, vesicle electrodeformation, and micropipette aspiration were used to assess the bending rigidity of the membrane as a function of GM1 content. In the fluid phase, GM1 was shown to strongly soften the bilayer. In the region of coexistence of fluid and gel-like domains, the micropipette aspiration technique allowed measurements of the bending rigidity of the fluid phase only, whereas electrodeformation and fluctuation analysis were affected by the presence of the gel-phase domains. The observation that GM1 decreased the bilayer bending rigidity is important for understanding the role of this ganglioside in the flexibility of neuronal membranes.

Topics: G(M1) Ganglioside; Membrane Microdomains; Phosphatidylcholines; Temperature; Unilamellar Liposomes

β2 Adrenergic receptor (β2AR) is a kind of G-protein coupled receptors (GPCRs) which transduce a wide range of extracellular signals into intracellular messages responsible for the regulation of diverse cell functions. Because of their functional ubiquity, GPCR is one of the most important drug targets in pharmaceutical industry. Although recent crystallographic studies provided both the active and the inactive states of some families of GPCRs, the influence of lipid composition of bilayer membrane on their activation is still poorly understood. In this work, we address the influence of lipid composition on the structural stability of GPCR, performing molecular dynamics simulations of three kinds of states: apo-, and agonist epinephrine-, or antagonist alprenolol-bound β2AR. These three kinds of β2ARs were embedded in four types of lipid membranes: (i) pure palmitoyl-oleoyl-phosphatidyl-choline (POPC), (ii) POPC/cholesterol (CHL), (iii) POPC/CHL/GM1 (GM1 ganglioside), (iv) POPC/palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE)/CHL/sphingomyeline (SM). The side chains of Lys267(6.29) and Asp331(7.58) showed different conformations among the three states in all types of lipid membranes. The distances between Lys267(6.29) and Asp331(7.58) of apo- and alprenolol-bound β2ARs are smaller than that of the epinephrine-bound β2AR. In contrast, β2ARs in POPC/CHL bilayer were unstable in which the salt bridge; i.e., ionic lock, was not formed between Arg131(3.50) and Glu268(6.30). We have also examined the distribution of lipid molecules. A stable hydrophobic interaction between CHL and β2AR was observed at transmembrane helix5 in POPC/CHL/GM1 and POPC/POPE/CHL/SM membranes. These results suggest that the lipid composition strongly affects the conformation of GPCR and essentially concerns the GPCR activation.

Topics: Adrenergic beta-2 Receptor Agonists; Adrenergic beta-2 Receptor Antagonists; Alprenolol; Binding Sites; Cholesterol; Epinephrine; G(M1) Ganglioside; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Protein Binding; Protein Stability; Protein Structure, Tertiary; Receptors, Adrenergic, beta-2

Interactions of amyloid-β (Aβ) with neuronal membrane are associated with the progression of Alzheimer's disease (AD). Ganglioside GM1 has been shown to promote the structural conversion of Aβ and increase the rate of peptide aggregation; but the exact nature of interaction driving theses processes remains to be explored. In this work, we have carried out atomistic-scale computer simulations (totaling 2.65 µs) to investigate the behavior of Aβ monomer and dimers in GM1-containing raft-like membrane. The oligosaccharide head-group of GM1 was observed to act as scaffold for Aβ-binding through sugar-specific interactions. Starting from the initial helical peptide conformation, a β-hairpin motif was formed at the C-terminus of the GM1-bound Aβ-monomer; that didn't appear in absence of GM1 (both in fluid POPC and liquid-ordered cholesterol/POPC bilayers and also in aqueous medium) within the simulation time span. For Aβ-dimers, the β-structure was further enhanced by peptide-peptide interactions, which might influence the propensity of Aβ to aggregate into higher-ordered structures. The salt-bridges and inter-peptide hydrogen bonds were found to account for dimer stability. We observed spontaneous formation of intra-peptide D(23)-K(28) salt-bridge and a turn at V(24)GSN(27) region - long been accepted as characteristic structural-motifs for amyloid self-assembly. Altogether, our results provide atomistic details of Aβ-GM1 and Aβ-Aβ interactions and demonstrate their importance in the early-stages of GM1-mediated Aβ-oligomerisation on membrane surface.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Cholesterol; G(M1) Ganglioside; Humans; Hydrogen Bonding; Lipid Bilayers; Molecular Dynamics Simulation; Molecular Sequence Data; Peptide Fragments; Phosphatidylcholines; Protein Binding; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Secondary

GM1 gangliosides form a microdomain with sphingomyeline (SM) and cholesterol (Chol) and are deeply involved in the aggregation of amyloid beta (Aβ) peptides on neural membranes. We performed molecular dynamics simulations on two kinds of lipid bilayers containing GM1 ganglioside: GM1/SM/Chol and GM1/POPC. Both 10 and 100 ns simulations and another set of 10 ns simulations with different initial lipid arrangement essentially showed the same computational results. GM1 molecules in the GM1/SM/Chol membrane were condensed, whereas those in GM1/POPC membrane scattered. That is, the formation of GM1 cluster was observed only on the GM1/SM/Chol mixed membrane. There appeared numerous hydrogen bonds among glycan portions of the GM1 clusters due to the condensation. A comparison in distribution of lipid molecules between the two kinds of membranes suggested that cholesterol had important roles to prevent the membrane from interdigitation and to stabilize other lipids for interacting with each other. This property of cholesterol promotes the formation of GM1 clusters.

Topics: Amyloid beta-Peptides; Cholesterol; Cluster Analysis; G(M1) Ganglioside; Hydrogen Bonding; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Sphingomyelins

Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.

Topics: Cell Membrane; Escherichia coli Proteins; G(M1) Ganglioside; Immunoprecipitation; Membrane Proteins; Membranes, Artificial; Nanostructures; Phosphatidylcholines; Protein Binding; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surface Plasmon Resonance

The behavior of the amyloid β-peptide (Aβ) within a membrane environment is integral to its toxicity and the progression of Alzheimer's disease. Ganglioside GM1 has been shown to enhance the aggregation of Aβ, but the underlying mechanism is unknown. Using atomistic molecular dynamics simulations, we explored the interactions between the 40-residue alloform of Aβ (Aβ(40) ) and several model membranes, including pure palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylserine (POPS), an equimolar mixture of POPC and palmitoyloleoylphosphatidylethanolamine (POPE), and lipid rafts, both with and without GM1, to understand the behavior of Aβ(40) in various membrane microenvironments. Aβ(40) remained inserted in POPC, POPS, POPC/POPE, and raft membranes, but in several instances exited the raft containing GM1. Aβ(40) interacted with GM1 largely through hydrogen bonding, producing configurations containing β-strands with C-termini that, in some cases, exited the membrane and became exposed to solvent. These observations provide insight into the release of Aβ from the membrane, a previously uncharacterized process of the Aβ aggregation pathway.

Topics: Alzheimer Disease; Amyloid beta-Peptides; G(M1) Ganglioside; Hydrogen Bonding; Membranes, Artificial; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines

The differential organization of lipid components in a multicomponent membrane leads to formation of domains having diverse composition and size. Cholesterol and glycosphingolipids are known to be important components of such lateral assembly. We report here the ordering of cholesterol around ganglioside GM1 and the nature of the cluster from an all-atom simulation of a ternary lipid system. The results are compared with a binary bilayer and a pure phospholipid bilayer. The difference in molecular rearrangements in ternary and binary lipid mixture shows the role of GM1 in the rearrangement of cholesterol. Calculation of the radial distribution function, rotational reorientation, and residence time analysis of cholesterol shows that cholesterol is preferentially accumulating near gangliosides, while the lateral translational motion, rotational diffusion, and order parameter of phospholipids characterize the amount of rigidity imparted on the phospholipid bilayer.

Topics: Cholesterol; Computational Biology; Computer Simulation; Diffusion; G(M1) Ganglioside; Glycosphingolipids; Lipid Bilayers; Lipids; Models, Chemical; Models, Statistical; Molecular Conformation; Phosphatidylcholines; Software; Surface Properties; Time Factors

Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.

Topics: Cholera Toxin; Dimyristoylphosphatidylcholine; G(M1) Ganglioside; Galactosylceramides; Lipid Bilayers; Microscopy; Phase Transition; Phosphatidylcholines

There is increasing evidence that a class of cell membrane glycolipids, gangliosides, can mediate the fibrillogenesis and toxicity of Alzheimer's disease amyloid-beta peptide (Abeta). Using lipid monolayers and vesicles as model membranes, we measured the insertion of Abeta into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-ganglioside GM1 monolayers to probe Abeta-GM1 interactions, imaged the effects of Abeta insertion on monolayer morphology, and measured the rate of Abeta fibril formation when incubated with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-GM1 vesicles. Furthermore, the location of Abeta association in the monolayer was assessed by dual-probe fluorescence experiments. Abeta exhibited direct and favorable interactions with GM1 as Abeta insertion monotonically increased with GM1 concentration, despite increases in monolayer rigidity at low GM1 levels. At low GM1 concentrations, Abeta preferentially inserted into the disordered, liquid expanded phase. At higher GM1 concentrations, Abeta inserted more uniformly into the monolayer, resulting in no detectable preferences for either the disordered or condensed phase. Abeta insertion led to the disruption of membrane morphology, specifically to the expansion of the disordered phase at low GM1 concentrations and significant disruption of the condensed domains at higher GM1 concentrations. During incubation with POPC vesicles containing physiological levels of GM1, the association of Abeta with vesicles seeded the formation of Abeta fibrils. In conclusion, favorable interactions between Abeta and GM1 in the cell membrane may provide a mechanism for Abeta fibrillogenesis in vivo, and Abeta-induced disruption of the cell membrane may provide a pathway by which Abeta exerts toxicity.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Amyloid; Amyloid beta-Peptides; G(M1) Ganglioside; Lipid Bilayers; Membranes, Artificial; Microscopy, Fluorescence; Phosphatidylcholines

Phase separation of glycolipids in lipid mono- and bilayers is of great interest for the understanding of membrane function. The distribution of the ganglioside GM1 in sphingomyelin (SM)/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), SM/1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DOPC) and SM/cholesterol/POPC Langmuir-Blodgett (LB) monolayers transferred at 36 mN/m has been studied by scanning force microscopy. Besides lateral organization of the glycolipid in LB monolayers as deduced from topography, material properties have been investigated by phase imaging, pulsed force mode and force modulation microscopy. It was shown that GM1 preferentially clusters in an ordered lipid matrix, i.e. the SM phase in the case of the SM/POPC and SM/DOPC mixture or in the ordered phase of POPC/SM/cholesterol monolayers. At higher local concentrations, three-dimensional protrusions enriched in GM1 occur, which may represent a precursor for the formation of micelles budding into the aqueous subphase. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00249-002-0232-4.

Topics: Brain; Cholesterol; G(M1) Ganglioside; Lipid Bilayers; Lipids; Membranes, Artificial; Microscopy, Atomic Force; Phosphatidylcholines; Sphingomyelins

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Animals; Cholesterol; Fluorescent Dyes; G(M1) Ganglioside; Kidney Cortex; Laurates; Lipid Bilayers; Membrane Lipids; Microscopy, Fluorescence; Microvilli; Models, Biological; Models, Molecular; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Rats; Rats, Sprague-Dawley; Sphingomyelins

Microsphere-based flow cytometric detection of cholera toxin (CT) through distance-dependent fluorescence resonant energy transfer (FRET) has been developed. Simultaneous double-fluorescence changes induced by multivalent interactions between CT and fluorophore (both fluorescence donor and acceptor)-labeled ganglioside GM1 on a biomimetic membrane surface (supported bilayers of phospholipids) can be measured by a commercial flow cytometer, providing a convenient and sensitive detection method for CT. The flow cytometry-based biosensor is capable of detecting less than 10 pM CT within 30 min. The signal generation strategy coupled with flow cytometry also provides a convenient method for kinetic studies of multivalent interactions. The surface density and the ratio of donor/acceptor-labeled GM1 on the surfaces of phospholipid bilayers are optimized to achieve high sensitivity.

Topics: Biosensing Techniques; Boron Compounds; Cholera Toxin; Flow Cytometry; Fluorescent Dyes; G(M1) Ganglioside; Humans; Kinetics; Lipid Bilayers; Phosphatidylcholines; Proteins; Sensitivity and Specificity; Serum Albumin; Time Factors

Three highly sensitive, selective, and reagent-free optical signal transduction methods for detection of polyvalent proteins have been developed by directly coupling distance-dependent fluorescence self-quenching and/or resonant-energy transfer to the protein-receptor binding events. The ganglioside GM1, as the recognition unit for cholera toxin (CT), was covalently labeled with fluorophores and then incorporated into a biomimetic membrane surface. The presence of CT with five binding sites for GM1 causes dramatic change for the fluorescence of the labeled GM1. (1) In the scheme using fluorescence self-quenching as a signal-transduction mechanism, the fluorescence intensity drops significantly as a result of aggregation of the fluorophore-labeled GM1 on a biomimetic surface. (2) By labeling GM1 with a fluorescence energy transfer pair, aggregation of the labeled GM1 results in a decrease in donor fluorescence and an increase in acceptor fluorescence, providing a unique signature for selective protein-receptor binding. (3) In the third scheme, using the biomimetic surface as part of signal transduction and combining both fluorescence self-quenching and energy-transfer mechanisms to enhance the signal transduction, a signal amplification was achieved. The detection systems can reliably detect less than 0.05 nM CT with fast response (less than 5 min). This approach can easily be adapted to any biosensor scheme that relies on multiple receptors or co-receptors. The methods can also be applied to investigate the kinetics and thermodynamics of the multivalent interactions.

Topics: Albumins; Binding Sites; Boron Compounds; Cholera Toxin; Contrast Media; Energy Transfer; Fluorescein; Fluorescent Dyes; G(M1) Ganglioside; Lipid Bilayers; Membranes, Artificial; Optics and Photonics; Phosphatidylcholines; Proteins; Sensitivity and Specificity; Signal Transduction; Spectrometry, Fluorescence; Toxins, Biological

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Chromatography, Liquid; Freeze Fracturing; G(M1) Ganglioside; Lipid Bilayers; Micelles; Microscopy, Electron; Phosphatidylcholines

The transfer kinetics of the negatively charged glycosphingolipid II3-N-acetylneuraminosyl-gangliotetraosylceramide (GM1) were investigated by monitoring tritiated GM1 movement between donor and acceptor vesicles. After appropriate incubation times at 45 degrees C, donor and acceptor vesicles were separated by molecular sieve chromatography. Donors were small unilamellar vesicles produced by sonication, whereas acceptors were large unilamellar vesicles produced by either fusion or ethanol injection. Initial GM1 transfer to acceptors followed first-order kinetics with a half-time of about 40 h assuming that GM1 is present in equal mole fractions in the exterior and interior surfaces of the donor vesicle bilayer and that no glycolipid flip-flop occurs. GM1 net transfer was calculated relative to that of [14C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. Factors affecting the GM1 interbilayer transfer rate included phospholipid matrix composition, initial GM1 concentration in donor vesicles, and the GM1 distribution in donor vesicles with respect to total lipid symmetry. The findings provide evidence that GM1 is molecularly dispersed at low concentrations within liquid-crystalline phospholipid bilayers.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Brain; Cattle; Cholesterol Esters; Chromatography, Gas; G(M1) Ganglioside; Kinetics; Lipid Bilayers; Liposomes; Phosphatidylcholines; Tritium