Characterisation of phosphatidylcholine/phosphatidylinositol sonicated vesicles. Effects of phospholipid composition on vesicle size

Phosphatidylinositol (PI), dipalmitoylphosphatidylcholine (DPPC) and mixed lipid (DPPC plus PI) sonicated vesicles have been prepared covering a range of composition. The vesicles were characterised by gel filtration, electron microscopy and photon correlation spectroscopy. The dimensions of the vesicles as measured by electron microscopy were in good accord with those obtained from photon correlation spectroscopy measurements. The number average diameters of the vesicles increase on increasing the PI content and range from approx. 30–80 nm as the weight % of PI is increased from 0 to 100. Gel filtration on Sepharose 4B columns gave anomalous results indicating that PI-containing vesicles were retarded on the gel possibly due to an interaction between the inositol headgroup and the gel matrix. Electrophoretic measurements on multilamellar vesicles show that the surface charge density increases with the PI content of the vesicles upto 50 weight % PI and remains constant thereafter. The radii of sonicated vesicles also increase with PI content which reflects a decreasing liposome curvature with increasing surface charge density.     

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Using dynamic light scattering we have been able to determine precisely the hydrodynamic radius of l-α-dimyristoylphosphatidylcholine (DMPC) vesicles as a function of temperature. We have detected a sharp, thermally reversible change in the vesicle radius at a phase transition temperature 24°C, corresponding to an approximate 11% increase in surface are. In the range 10–20°C, the change in radius is less than 1%.     

Proton-induced membrane fusion role of phospholipid composition and protein-mediated intermembrane contact

Glycolipid-phospholipid vesicles containing phosphatidate and phosphatidylethanolamine were found to undergo proton-induced fusion upon acidification of the suspending medium from pH 7.4 to pH 6.5 or lower, as determined by an assay for lipid intermixing based on fluorescence resonance energy transfer. Lectinmediated contact between the vesicles was required for fusion. Incorporation of phosphatidylcholine in the vesicles inhibited proton-induced fusion. Vesicles in which phosphatidate was replaced by phosphatidylserine underwent fusion only when pH was reduced below 4.5, while no significant fusion occured (pH ? 3.5) when the anionic phospholipid was phosphatidylinositol. It is suggested that partial protonation of the polar headgroup of phosphatidate and phosphatidylserine, respectively, causes a sufficient reduction in the polarity and hydration of the vesicle surface to trigger fusion at sites of intermembrane contact.     

The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.     

The effect of surface charge density on valinomycin-K+ complex formation in model membranes

The model membrane approach was used to investigate the surface charge effect on the ion-antibiotic complexation process. Mixed monolayers of valinomycin and lipids were spread on subphases containing K⁺ or Na⁺. The surface charge density was modified by spreading ionizable valinomycin analogs on aqueous subphases of different pH or by changing the nature of the lipid (neutral, negatively charged) in the mixed film. Surface pressure and surface potential measurements demonstrated that a neutral lipid (phosphatidylcholine) or positively charged valinomycin analogs didn't enhance the antibiotic complexing capacity. However, a maximal complexation is reached for a critical lipid concentration in the valinomycin-phosphatidylserine mixed film. The role of the surface charge on the valinomycin complexing properties was examined in terms of the Gouy-Chapman theory. As a consequence of the negative charge of the lipid monolayer, the K⁺ concentration near the surface is larger than the bulk concentration, by a Boltzmann factor. A good agreement was observed between the experimental results and the theoretical predictions. Conductance measurements of asymmetric bilayers containing a neutral lipid (egg lecithin) on one side and a negatively charged lipid (phosphatidylserine) on the other, confirm the role of the surface charge. Indeed, addition of K⁺ to the neutral side of the bilayer containing valinomycin had no effect on the conductance whereas addition of K⁺ to the charged side of the bilayer caused a 80-fold conductance increase.     

Ionic charge on phospholipids and their interaction with the mitochondrial adenosine triphosphatase

The activity of the lipid-depleted, oligomycin-sensitive mitochondrial ATPase has been measured in the presence of liposomes prepared from mixtures of phosphatidylglycerol and phosphatidylglycerol lysine. Enzyme activity increased linearly with an increase in the negative charge of liposomes prepared from the phosphatidylglycerol-phosphatidylglycerol lysine mixtures. The electrophoretic mobility and activating capacity of liposomes of several other phospholipids were determined. A linear relationship between electrophoretic mobility of the liposomes and oligomycin-sensitive activity was again apparent. These observations demonstrate that the activity of the ATPase is directly proportional to the ionic charge on phospholipid activators if the acyl chain composition of the phosphoglycerides is relatively constant.     

Phospholipid composition and external labeling of aminophospholipids of human En(a−) erythrocyte membranes which lack the major sialoglycoprotein (glycophorin a)

Erythrocytes of the rare human blood group En(a?) lack the major sialoglycoprotein, glycophorin A, and the cell population heterozygous for the En(a) antigen contain half the normal amount of glycophorin A. With such cells we have studied whether glycophorin A influences the phospholipid composition and the availability of aminophospholipids to external labeling reagents. We here demonstrate that the amounts of all phospholipids are closely similar in normal and variant membranes. However, using the amino-reactive reagent trinitrobenzenesulfonate, we show that phosphatidylethanolamine is more easily labeled in intact En(a?) cells as compared to normal cells, whereas phosphatidylethanolamine shows an intermediate labeling in En(a) heterozygous cells.     

Clustering of fatty acids in phospholipid bilayers

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at $\text{pH}\text{>11}$ is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

II. Phospholipid exchange and size enlargement in sonicated vesicles induced by a ‘critical’ fatty acid concentration

Size enlargement of dipalmitoyl phosphatidylcholine vesicles was greatly accelerated in the range of the phase-transition temperatures, when fatty acid concentration was above a threshold level (‘critical’ concentration). This ‘critical’ concentration varied with the length of the fatty acid chain. The size enlargement process had second-order kinetics dependent on the vesicle concentration. Alkaline pH and low ionic strength inhibited the rate of size enlargement.Phospholipid exchange between dimyristoyl and dipalmitoyl phosphatidylcholine vesicles increased abruptly above a ‘critical’ fatty acid concentration. The donor vesicles were those vesicles in which fatty acids reached the ‘critical’ concentration. The phospholipid exchange occurred both in fluid- and in solid-state vesicles. The ‘critical’ fatty acid concentration accelerating the phospholipid exchange process was lower than that accelerating the size enlargement process.The phospholipid exchange process explained in terms of a diminished hydrophobic attraction among the phospholipid molecules of the bilayer occurs via a free phospholipid molecule transfer through the aqueous phase. The size enlargement process is interpreted in terms of high fatty acid concentration in the membrane fluid domains. The membrane structure is locally perturbed inducing vesicle sticking after collision.