Micropolarities of lipid bilayers and micelles 5. Localization of pyrene in small unilamellar phosphatidylcholine vesicles

The excimer/monomer ratio of emission intensities (I_E/I_M) and the enhancement of the 0-0 vibronic transition in the fluorescence spectra of pyrene (PY) and 16-(1-pyrenyl)hexadecanoic acid (C₁₆PY) were used to investigate the localization of PY in the bilayers of small unilamellar vesicles constituted of phosphatidylcholine (SUV-PC). First, from comparison of the fluorescence characteristics of PY in water with those of PY incorporated into the SUV-PC membranes, we concluded that the probe is incorporated preferentially in the lipid phase of the vesicles and not in the bulk aqueous phase. In addition, we found that, contrary to what happens with the pyrenyl moiety of C₁₆PY the location of PY varies with its relative concentration in the membrane space. The critical concentration was observed to be around 1.0 mol% of incorporated PY. At concentrations below this value, PY is located in the hydrocarbon core of the lipid bilayers. Above 1.0 mol%, the PY molecules reside preferentially in the neighbourhood of the glyceryl moiety region of the PC vesicles.     

Influence of the shape of phospholipid vesicles on the measurement of their size by photon correlation spectroscopy

The influence of shape transformation of large unilamellar vesicles (LUV) on their size measurement by photon correlation spectroscopy (PCS) has been investigated. The experimental size of vesicles after hyperosmotic contractions of increasing intensities have been compared to the theoretical volume decrease determined by applying Boyle Van't Hoff's law. The main observation is that PCS size measurement gives overestimated values when LUV have been subjected to a volume decrease of more than 20% of their initial volume. The PCS size overestimation is related to the influence of the shape transformation of the vesicles on their diffusion coefficient (D) as shown by modelling the evolution of D of a sphere which is transformed into an ellipsoid by internal volume reduction under constant area. Received: 4 December 1997 / Revised version: 2 March 1998 / Accepted: 15 April 1998     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 μm diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca²⁺ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca²⁺ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca²⁺ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg²⁺ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg²⁺ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca²⁺ and also enhanced     

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (T_c) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [¹⁴C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle: (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the T_c of the lipid and increases with temperature above the T_c. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.     

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.     

Transfer properties of the bovine brain phospholipid transfer protein. Effect of charged phospholipids and of phosphatidylcholine fatty acid composition

The monolayer technique has been used to study the transfer of [¹⁴C]phosphatidylinositol from the monolayer to phosphatidylcholine vesicles. An equivalent transfer rate was found for egg phosphatidylcholine, dioleoylphosphatidylcholine, dielaidoylphosphatidylcholine and dipalmitoylphosphatidylcholine. A reduced transfer rate was found for a shorter-chain derivative, dimyristoylphosphatidylcholine, and for species with two polyunsaturated fatty acid chains such as dilinoleoylphosphatidylcholine, diheptadecadienoylphosphatidylcholine, dilinolenoylphosphatidylcholine and diether and dialkyl derivatives. No activity was found for 1,3-dipalmitoylphosphatidylcholine. The presence of up to 5 mol% phosphatidylinositol in egg phosphatidylcholine vesicles had no effect on the transfer rate. Introduction of more than 5 mol% phosphatidylinositol or phosphatidic acid into the phosphatidylcholine vesicles gradually decreased the rate of phosphatidylinositol transfer from the monolayer. 20 mol% acidic phospholipid was nearly completely inhibitory. Transfer experiments between separate monolayers of phosphatidylcholine and phosphatidylinositol showed that the protein-bound phosphatidylcholine is readily exchanged for phosphatidylinositol, but the protein-bound phosphatidylinositol exchange for phosphatidylcholine occurs at a 20-times lower rate. The release of phosphatidylinositol is dependent on the lipid composition and the concentration of charged lipid in the acceptor membrane, but also on the ratio between donor and acceptor membranes. The main transfer protein from bovine brain which transfer phosphatidylinositol and phosphatidylcholine transfers also phosphatidylglycerol, but not phosphatidylserine or phosphatidic acid. The absence of significant changes in the surface pressure indicate that the phosphatidylinositol and phosphatidylcholine transfer is not accompanied by net mass transfer.     

Determination of the equilibrium constant for the binding of ferricytochrome c to phospholipid vesicles and the effect of binding on the reduction rate of cytochrome c

The rate of reduction of cytochrome c by 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine was examined as a function of binding to liposomes prepared from mixed soybean phospholipids, asolectin, and from various purified phospholipids. Binding of cytochrome c to asolectin liposomes caused an increase in the rate of reduction by the pteridine derivative from 2900 to 16 000 M^?1 · s^?1 at pH 7. At low ionic strength (0.003 M) the binding stoichiometry between cytochrome c and asolectin vesicles is 15 ± 2 phosphospolipid/cytochrome c (mole ratio), determined by monitoring the change in reduction rate of cytochrome c by pteridine as cytochrome c is bound to the vesicles. A stoichiometry of 14 phospholipid/cytochrome c was obtained from gel filtration studies. Equilibrium association constants for the binding of cytochrome c to sites on the asolectin vesicles varied from 2.2 · 10⁶ to 1.8 · 10³ M^?1 between 0.02 and 0.10 M ionic strength, respectively. In general, liposomes prepared from purified phospholipids resulted in less binding of cytochrome c per mole of phospholipid and lower reduction rates than those prepared from asolectin.     

The influence of Ca2+ on the lateral lipid distribution and phase transition in phosphatidylethanolamine/phosphatidylserine vesicles

The lateral lipid distribution within dipalmitoylphosphatidylethanolamine (DPPE)/dipalmitoylphosphatidylserine (DPPS) vesicle membranes was investigated under the influence of Ca²⁺ using a lipid cross-linking method. To characterize the phase transition in DPPE/DPPS vesicles and to correlate the different phase states of the membrane lipids with the obtained lipid distribution ESR measurements using a fatty acid spin label were carried out. It is shown that Ca²⁺ has a significant influence on the lateral lipid distribution within the fluid phase of the membrane lipids; instead of a slight alternating lipid arrangement in absence of Ca²⁺ due to the electrostatic interaction between the DPPS headgroups after addition of Ca²⁺ a lateral cluster structure is characteristic of the fluid phase.     

Effect of succinylphosphatidylcholine on phosphatidylcholine vesicles. Structural studies by gel chromatography,electron microscopy and nuclear magnetic resonance

The effect of an aqueous dispersion of succinylphosphatidylcholine on an aqueous suspension of phosphatidylcholine vesicles was studied by gel chromatography, freeze-fracture electron microscopy and proton nuclear magnetic resonance with Mn²⁺ (broadening paramagnetic reagent). Total phospholipid concentrations were in the range 10–20 mM.Succinylphosphatidylcholine is in micellar form and behaves as a detergent. The structures obtained depend on the molar percentage of succinylphosphatidylcholine.Above a succinylphosphatidylcholine molar percentage of 60%, mixed micelles are formed, assumed to be essentially spherical.Below a succinylphosphatidylcholine molar percentage of 30%, principally mixed vesicles are observed, with an external diameter of 215–240 Å, and an almost constant internal volume.Between 30 and 60% of succinylphosphatidylcholine, a mixture of these structures is obtained; rod-shaped profiles are also observed in electron microscopy, which may correspond to sections of leaky vesicles or to a new kind of cylindrical micelle.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(1) ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C₁₂E₈) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K⁺-stimulated Na⁺-influx is found to be about 35 μmol Na⁺ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na⁺ transported per ATP molecule hydrolyzed.     

The relatioship between plasma membrane lipid composition and physical-chemical properties II. Effect of phospholipid fatty acid modulation on plasma membrane physical properties and enzymatic activities

The fatty acid composition of plasma membrane phospholipids of the murine T lymphocyte tumor EL4 were systematically modified in an attempt to understand the relationship between lipid bilayer composition and plasma membrane physical and biological properties. Two plasma membrane enzyme activities, adenylate cyclase and ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase, were measured in normal and fatty acid-substituted EL4 plasma membrane fractions. The fatty acid effect on enzyme activities was similar to previously reported effects of fatty acids on cytotoxic T cell function. The activity of both enzymes was inhibited by saturated fatty acids, while unsaturated fatty acids had a moderate enhancing effect on both enzyme activities. Using two different nitroxide derivatives of stearic acid, the order parameter and approximate rotational correlation times were calculated from ESR spectra of normal and fatty acid-modified plasma membranes. No significant difference was found in either parameter in these membranes. These results, in conjunction with earlier data from our laboratory and others, suggest that caution should be exercised in inferring changes in membrane ‘fluidity’ based on lipid modulation of biological membranes.     

Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles

The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.