Ovalbumin-induced fusion of acidic phospholipid vesicles at low pH

The interactions of ovalbumin (OA) with large unilamellar vesicles (LUV) of phosphatidylserine (PS) and PS/phosphatidylethanolamine (PE) were studied. It was observed that OA induces aggregation, destabilization, and fusion of these LUV composed of acidic phospholipids at low pH levels. The fusion of LUV by OA was monitored by measuring the intermixing of internal aqueous contents of vesicles, by resonance energy transfer assay which follows the mixing of the membrane components, and by thin-sectioning electron microscopy. The pH profile of fusion was found to be similar to the pH-dependent binding of OA to the same phospholipid vesicles. Proteolytic digestion and hydrophobic labeling with dansyl chloride and photoreactive phosphatidylcholine (PC) of the OA-vesicle complex showed that a segment of OA with a molecular weight of approximately 2,500 penetrates the bilayer. The amino acid composition of this segment indicated that it is the 291-322 fragment and not the putative signal sequence.     

Fusion of phospholipid vesicles induced by alpha-lactalbumin at acidic pH

Alpha-Lactalbumin (alpha-LA), lysozyme, and ribonuclease are found to induce fusion of phosphatidylserine/phosphatidylethanolamine vesicles at low pH. The fusogenic behavior and the binding to phospholipid vesicles of one of these proteins, alpha-LA, are studied at a wide range of conditions. The initial rate of fusion in the presence of alpha-LA increases with increasing acidity below pH 6, and the extent of alpha-LA binding to the vesicles is also found to increase with decreasing pH. Once bound to the vesicles in acidic media, the neutralization to pH 7 fails to dislodge the alpha-LA from the vesicles, and this irreversible binding also increases with decreasing pH. A segment of alpha-LA is found to be resistant to the proteolytic digestion when initially incubated with the vesicles at low pH. The amino acid composition of this fragment was determined, and from this the sequence of alpha-LA fragment, which appears to be inserted into the bilayer, is deduced. Hydrophobic labeling with dansyl chloride renders support that this segment indeed penetrates into the hydrophobic interior of bilayer. Since both the N-terminal and the C-terminal of this vesicle-bound protein are accessible to the externally added proteolytic enzymes, it is concluded that a loop of the polypeptide segment goes into the bilayer. These observations, taken together, suggest a possibility that the penetration by a loop of alpha-LA segment into the phospholipid bilayer is responsible for the fusion.     

Insulin-mediated fusion of negatively charged phospholipid vesicles at low pH

Fusion of negatively charged phospholipid vesicles by bovine insulin was studied. The fusion induced by the hormone was demonstrated by resonance energy transfer, sepharose chromatography, light scattering and electron microscopy. The insulin effect was more effective when the pH was in the range of 3.6 - 3.9. The action of insulin also depends on the phosphatidylcholine: phosphatidic acid molar ratio, and buffer and vesicles concentration. At optimal conditions, half-maximal effect was obtained at 2 X 10(-8)M. The insulin-mediated fusion is non specific. The potential importance of these studies is discussed.     

Substrate-induced deformation and adhesion of phospholipid vesicles at the main phase transition

The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T(m). In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T(m) and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 microm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.     

Conformational status of apomyoglobin in the presence of phospholipid vesicles at neutral pH

The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near- and far-UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed.     

Encapsulation of hemoglobin in phospholipid vesicles

Hemoglobin has been encapsulated in phospholipid vesicles by extrusion of hemoglobin/lipid mixtures through polycarbonate membranes. This technique avoids the use of organic solvents, sonication, and detergents which have proven deleterious to hemoglobin. The vesicles are homogeneous, with a mean size of 2400 A as determined by photon correlation spectroscopy. The encapsulated hemoglobin binds oxygen reversibly and the vesicles are impermeable to ionic compounds. Hemoglobin encapsulated in egg phosphatidylcholine vesicles converts to methemoglobin within 2 days at 4 degrees C. By contrast, when a mixture of dimyristoyl phosphatidylcholine, cholesterol and dicetyl phosphate is used there is no acceleration in methemoglobin formation, and the preparation is stable for at least 14 days at 4 degrees C.     

Fusion and fragmentation of phospholipid vesicles by apohemoglobin at low pH.

Human apohemoglobin in acidic media was found to induce fusion of phosphatidylcholine/phosphatidylserine (1:1) vesicles at low protein concentration but to fragment the same vesicles to form micellar complex at high protein concentration. The fusion was demonstrated by size increase, vesicle content mixing, lipid mixing, and electron microscopy. The micellization of phospholipid vesicles was observed by light scattering, gel filtration, and electron microscopy. The hydrophobic labeling of the apohemoglobin/vesicle complex followed by CNBr cleavage of apohemoglobin showed that an N-terminal segment of the beta subunit with a molecular weight of approximately 6,000 seems to be mainly involved in the fusion process, but the whole sequences of both alpha and beta chains participate in the micellization process.     

Temperature dependence of the size of phospholipid vesicles

We report measurements of the size of dimyristoyl phosphatidylcholine vesicles over a temperature range that includes the main transition temperature and show that any change in average diameter is less than ±3%.     

Interaction of horse heart and thermus thermophilus type c cytochromes with phospholipid vesicles and hydrophobic surfaces

The binding of horse heart cytochrome c (cyt-c) and Thermus thermophilus cytochrome c(552) (cyt-c(552)) to dioleoyl phosphatidylglycerol (DOPG) vesicles was investigated using Fourier transform infrared (FTIR) spectroscopy and turbidity measurements. FTIR spectra revealed that the tertiary structures of both cytochromes became more open when bound to DOPG vesicles, but this was more pronounced for cyt-c. Their secondary structures were unchanged. Turbidity measurements showed important differences in their behavior bound to the negatively charged DOPG vesicles. Both cytochromes caused the liposomes to aggregate and flocculate, but the ways they did so differed. For cyt-c, more than a monolayer was adsorbed onto the liposome surface prior to aggregation due to charge neutralization, whereas cyt c(552) caused aggregation at a protein/lipid ratio well below that required for charge neutralization. Therefore, although cyt-c may cause liposomes to aggregate by electrostatic interaction, cyt-c(552) does not act in this way. FTIR-attenuated total reflection spectroscopy (FTIR-ATR) revealed that cyt-c lost much of its secondary structure when bound to the hydrophobic surface of octadecyltrichlorosilane, whereas cyt-c(552) folds its domains into a beta-structure. This hydrophobic effect may be the key to the difference between the behaviors of the two cytochromes when bound to DOPG vesicles.     

Cholesterol stabilizes hemifused phospholipid bilayer vesicles

Cholesterol was found to inhibit full fusion of oppositely charged phospholipid bilayer vesicles by stabilizing the contacting membranes at the stage of the hemifused intermediate. Vesicles of opposite charge containing different amounts of cholesterol were prepared using cationic (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and anionic (dioleoylphosphatidylglycerol) phospholipids. Pairwise interactions between such vesicles were observed by fluorescence video microscopy in real time after electrophoretically maneuvering the vesicles into contact. Hemifusion accounted for more than 80% of the observed events when the vesicles contained 33-50 mole% cholesterol. In contrast, vesicles containing only a small proportion of cholesterol (相似文献   

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Ultrasound interaction with large unilamellar vesicles at the phospholipid phase transition: perturbation by phospholipid side chain substitution with deuterium

The ultrasonic absorption, alpha lambda, as a function of temperature and frequency was determined in large unilamellar vesicles (LUVs) in which specific phospholipid side chains were deuterated. Deuteration significantly altered the temperature and frequency dependence of alpha lambda. The frequency change was especially marked, with decreased frequency and broadening of the ultrasound relaxation, even with only minor changes in the phase transition temperature. Deuteration decreased the Tm and enthalpy of the lipid phase transition, as shown by differential scanning calorimetry, whereas electron spin resonance showed that at and above the lipid phase transition, no differences in the mobility as a function of temperature were observed. These results show that the observed increase in ultrasonic absorption in LUVs at the phospholipid phase transition arises from the interaction of ultrasound with the hydrophobic side chains, probably coupling with structural reorganization of small domains of molecules, a process which is maximized at the phase transition temperature.     

Melittin induces fusion of unilamellar phospholipid vesicles

Melittin, the soluble lipophilic peptide of bee venom, causes fusion of phospholipid vesicles when vesicle suspensions are heated or cooled through their thermal phase transition. Fusion was detected using a new photochemical method (Morgan, C.G., Hudson, B. and Wolber, P. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 26–30) which monitors lipid mixing. Electron microscopy and gel filtration confirmed that most of the lipid formed large vesicular structures. Fluorescence experiments with a water-soluble, membrane-impermeable complex of terbium (Wilschut, J. and Papahadjopoulos, D. (1979) Nature 281, 690–692) demonstrate that these ionic contents are released during fusion. The large structures formed by melittin-induced fusion are impermeable to these ions and are resistant to further fusion. This is in contrast to the behavior observed for the cationic detergent cetyltrimethylammonium bromide (CETAB). The large size of the vesicles formed, the extreme speed of the fusion event and the appearance of electron microscope images of the vesicles prior to fusion suggest that the mechanism of the fusion process includes a preaggregation step.     

Generation of multilamellar and unilamellar phospholipid vesicles

Multilamellar and unilamellar vesicles can be generated by a variety of techniques which lead to systems with differing lamellarity, size, trapped volume and solute distribution. The straight-forward hydration of lipid to produce multilamellar vesicles (MLVs) results in systems which exhibit low trapped volumes and where solutes contained in the aqueous buffer are partially excluded from the MLV interior. Large trapped volumes and equilibrium solute distributions can be achieved by freeze-thawing or by ‘reverse phase’ procedures where the lipid is hydrated after being solubilized in organic solvent. Unilamellar vesicles can be produced directly from MLVs by extrusion or sonication or, alternatively, can be obtained by reverse phase or detergent removal procedures. The advantages and limitations of these techniques are discussed.     

Interaction of carrier ionophores with phospholipid vesicles

The interactions of carrier ionophores, nonactin, A23187, and lasalocid A with liposomes formed from the synthetic lipids dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine are investigated by differential scanning calorimetry and 1H and 31P nuclear magnetic resonance techniques. The results indicate that the mode of interaction of these ionophores is dependent on the fluidity of the bilayer and on the chemical nature of these ionophores. The 31P NMR studies are suggestive of the formation of small particles that are probably intervesicular lipid-ionophore aggregates in multilamellar vesicles when they are incorporated with these ionophores at high concentrations. The results are interpreted on the basis of the chemical structure and conformations of the ionophores in membrane mimetic media. The 1H NMR line-width measurements indicate that the aromatic rings containing the carboxyl groups of lasalocid A and A23187 are located near the membrane interface while the rest of the molecule is buried in the membrane interior.     

Reconstitution of rabbit thrombomodulin into phospholipid vesicles

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.     

Spontaneous transfer of gangliotetraosylceramide between phospholipid vesicles

The transfer kinetics of the neutral glycosphingolipid gangliotetraosylceramide (asialo-GM1) were investigated by monitoring tritiated asialo-GM1 movement from donor to acceptor vesicles. Two different methods were employed to separate donor and acceptor vesicles at desired time intervals. In one method, a negative charge was imparted to dipalmitoylphosphatidylcholine donor vesicles by including 10 mol% dipalmitoylphosphatidic acid. Donors were separated from neutral dipalmitoylphosphatidylcholine acceptor vesicles by ion-exchange chromatography. In the other method, small, unilamellar donor vesicles (20-nm diameter) and large, unilamellar acceptor vesicles (70-nm diameter) were coincubated at 45 degrees C and then separated at desired time intervals by molecular sieve chromatography. The majority of asialo-GM1 transfer to acceptor vesicles occurred as a slow first-order process with a half-time of about 24 days assuming that the relative concentration of asialo-GM1 in the phospholipid matrix was identical in each half of the donor bilayer and that no glycolipid flip-flop occurred. Asialo-GM1 net transfer was calculated relative to that of [14C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. A nearly identical transfer half-time was obtained when the phospholipid matrix was changed from dipalmitoylphosphatidylcholine to palmitoyloleoylphosphatidylcholine. Varying the acceptor vesicle concentration did not significantly alter the asialo-GM1 transfer half-time. This result is consistent with a transfer mechanism involving diffusion of glycolipid through the aqueous phase rather than movement of glycolipid following formation of collisional complexes between donor and acceptor vesicles. When viewed within the context of other recent studies involving neutral glycosphingolipids, these findings provide additional evidence for the existence of microscopic, glycosphingolipid-enriched domains within the phospholipid bilayer.     

Monovalent cation-induced fusion of acidic phospholipid vesicles

Fusion of small unilamellar vesicles (SUV) consisting of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and phosphatidylglycerol (PG) from egg yolk, dipalmitoylphosphatidylserine (DPPS) and phosphatidylserine (PS) from bovine brain was studied as a function of monovalent cation concentration. Fusion was detected by measuring the changes in the excimer to monomer fluorescence intensity ratio (IE/M) of pyrene-labeled phospholipid analogues upon fusion of the pyrene-labeled and unlabeled vesicles. No fusion was observed from vesicles consisting of DMPC, PS from bovine brain or PG from egg yolk upon addition of NaCl (up to 1 M). However, considerable fusion was evident for vesicles consisting of DMPG or DPPS upon addition of monovalent cations (300 mM to 1 M). Fusion kinetics were fast reaching a plateau after 5 min of addition of cations. The order of efficiency of different monovalent cations to induce the fusion of DMPG vesicles as judged by the changes of the IE/M ratio was Li+ greater than Na+ greater than K+ greater than Cs+. DSC-scan of sonicated DMPG vesicles showed, in the absence of salt, a phase transition at 19.2 degrees C with enthalpy of 1.1 kcal.mol-1. After incubation in the presence of 600 mM NaCl the DSC scan showed a narrow phase transition at 24.1 degrees C with enthalpy of 6.9 kcal.mol-1 and a pronounced pretransition, both supporting that the fusion of the vesicles had occurred in the presence of NaCl. The results indicate that sonicated vesicles consisting of acidic phospholipids with fully saturated fatty acids fuse in the presence of monovalent cations, whereas those containing unsaturated fatty acids do not.     

Formation and properties of thin-walled phospholipid vesicles

Large numbers of thin-walled vesicles, 0.5 to 10 μ in diameter, can be formed by permitting a thinly spread layer of hydrated phospholipids to swell slowly in distilled water or an aqueous solution of nonelectrolytes. Electron micrographs and phospholipid analyses indicate that the walls consist of a single or a few bilayers. The vesicles can be centrifuged and resuspended in another medium, making them a useful system for studying permeability. The osmolarity of the solution in the interior of the vesicles can be estimated by immersion refractometry. The osmolarity of the internal aqueous phase is linearly related to the osmolarity of the external medium.     

Thermal fluctuations of large cylindrical phospholipid vesicles

The time correlation function of the shape fluctuations of large (greater than 10 micron), cylindrical, hydrated, phospholipid-membrane vesicles consisting of one bimolecular layer was measured. The restoring force of the membrane was due to the excess curvature of a membrane element. A value for the curvature elastic modulus, Kc, was obtained from the mean-square amplitude of the normal modes of the fluctuations using the equipartition theorem. An expression for the correlation time was found by solving the dynamics of the membrane's relaxation against the low Reynolds number viscous drag of the surrounding fluid. The amplitudes and correlation times of the fundamental bending mode of the cylindrical vesicles both yield Kc = 1-2 X 10(-12) ergs.