Phospholipases. III. Effects of ionic surfactants on the phospholipase-catalyzed hydrolysis of unsonicated egg lecithin liposomes.

Apparent values of Km and Vmax have been measured for catalysis of hydrolysis of unsonicated egg lecithin liposomes, activated through addition of 0.4 M n-hexanol, by phospholipases A2 from bee and snake venoms and by phospholipase C from Clostridium welchii as a function of the concentration of three surfactants: hexadecylamine, hexadecyltrimethylammonium bromide, and dihexadecyl phosphate. For all three enzymes, values of Km and Vmax show little or no dependence on the concentration of these ionic surfactants, demonstrating that the liposomal surface charge is not a crucial factor in determining susceptibility to phospholipase-catalyzed hydrolysis.     

Thermotropic behavior of liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and their mixtures

The thermotropic properties of multilamellar liposomes from egg yolk lecithin, hydrogenized egg yolk lecithin and several mixtures of these two lipids were studied with the application of excimer--forming optical probe pyrene and microcalorimetry. It was discovered that when the proportion of the egg yolk lecithin in the lipid mixture was raised the temperature of the main phase transition reduced. For all this, independent of the lipid mixture composition when the temperature was raised, apparently, polarity of pyrene microenvironment in the liposomes bilayers decreased. On the basis of the analysis of solidus and liquidus curves obtained from calorimetric studies of the lipid mixtures and bend points of Arrhenius anamorphose obtained during the pyrene excimer formation measurements some conclusions were made about the role of unmodified and hydrogenized egg yolk lecithin cluster formation in the determination of thermotropic properties of the liposomes from the above two lipids mixtures. High temperature phase transition discovered for the egg yolk lecithin while measuring the pyrene excimer formation is proposed to be closely connected with temperature-dependent changes in the organization of phospholipid heads on the interphase bilayer/H2O solution.     

Tin compounds interaction with membranes of egg lecithin liposomes

This work is a continuation of earlier research concerning the influence of tin compounds on the dynamic properties of liposome membranes produced with lecithin hen egg yolks (EYL). The experiments were carried out at room temperature (about 25 degrees C). Four tin compounds were chosen, including three organic ones, (CH3)4Sn, (C2H5)4Sn and (C3H7)3SnCl, and one inorganic, SnCl2. The investigated compounds were admixed to water dispersions of liposomes. The content of the admixture changed within the range 0 mol-% to 11mol-% in proportion to EYL. Two spin probes were used in the experiment: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 2-ethyl-2-(15-methoxy-15-oxopentadecyl)-4,4-dimethyl-3-oxazolidinyloxyl (16-DOXYL-stearic acid), which penetrated through different areas of the membrane. It was found that tin compounds containing chlorine were the most active in interaction with liposome membranes. In the case of (C3H7)3SnCl, after exceeding 4% admixture content, an additional line appeared in the spectrum of the TEMPO probe which can be a result of formation of domain structures in the membranes of the studied liposomes. Compounds containing chlorine are of ionized form in water solution. The obtained results can thus mean that the activity of admixtures can be seriously influenced by their ionic character. In case of an admixture of non-ionic compounds the compound with a longer hydrocarbon chain displayed a slightly stronger effect on the spectroscopic parameters of the probes.     

Interaction between RNAs from different sources and egg lecithin liposomes

The interaction of RNAs of molecular weight ranging between 15,000 and 30,000 with egg lecithin large and small unilamellar liposomes has been investigated. Torula and tRNA proved to be incorporated to a similar extent but no linear relationship between molecular weight and percent of incorporation was demonstrated. A relationship between percent of secondary structure of RNA and its ability to be trapped in SUV liposomes is suggested.     

Permeability properties of liposomes prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin

Liposomes have been prepared from dipalmitoyllecithin, dimyristoyllecithin, egg lecithin, rat liver lecithin and beef brain sphingomyelin.Permeability properties of liposomes thus prepared were studied toward glucose. The glucose permeability of liposomes with saturated lecithins (dipalmitoyllecithin and dimyristoyllecithin) and sphingomyelin appears to be more strongly temperature dependent than that of liposomes with lecithin containing unsaturated fatty acyl chains (egg and rat liver lecithins). The permeability of glucose through vesicles of dipalmitoyllecithin or dimyristoyllecithin was enhanced drastically at their transition temperatures, while the incorporation of about 25 mole% of egg lecithin into liposomes of saturated lecithins suppressed the enhanced permeation rates of glucose above the transition temperatures.The incorporation of small amounts of cholesterol enhanced the temperature-dependent permeability of glucose through the bilayer of saturated lecithins or sphingomyelin. This tendency was best shown in the case of dipalmitoyl-lecithin, in which 20 mole% of cholesterol had the most stimulating effect on the temperature-dependent permeability. The introduction of more than 33 mole% of cholesterol showed, however, reduced effects on the temperature-dependent permeability through liposomes with saturated lecithins or sphingomyelin. It was also shown that cholesterol had a much larger effect on the regulation of the temperature-dependent permeability of liposomes prepared with saturated lecithins or sphingomyelin than on that of liposomes prepared with phospholipids containing unsaturated fatty acids.     

Use of bromthymol blue for studying the thermotropic properties of the liposomes from egg lecithin

The dependence of the turbidity changes at 615 nm monobilayers liposomes from egg yolk lecithin in the presence of bromothymol blue on temperature and storage conditions has been investigated. It is established that the thermotropic properties of liposomes change irregularly and depend on the storage conditions. Sharp release of the bound dye at temperature above 35-37 degrees C is associated with thermotropic change in liposomes and the detected effects, with the change of orientation of the phosphorylcholine group.     

Studies on spin-labelled egg lecithin dispersions

ESR spectra of egg lecithin dispersions labelled with 5-nitroxide stearic acid are recorded with a 50 G field sweep, and also with a new technique which “expands” the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The expansion technique improves the precision of the order parameters determined from the hyperfine splitting measurements, and may prove useful in future spin label membrane studies.Approximate order parameters are derived to describe the fluidity of fatty acid spin-labelled membranes in those cases where either the inner or outer hyperfine extrema are not well defined. The ability of these expressions to measure the fluidity of labelled egg lecithin dispersions for the temperature range 14–42° C is examined.     

Liposomal membranes. VII. Fusion and aggregation of egg lecithin liposomes as promoted by polysaccharides

Pullulan, one of polysaccharides, induces aggregation of egg lecithin multilamellar liposomes as detected by turbidity of the solution. The turbidity disappears upon treatment with pullulanase, indicating that the aggregation induced by pullulan is reversible. On the other hand, the turbidity induced on single-walled liposomes is not completely reversed by incubation with the enzyme. The phenomenon is interpreted as the involvement of irreversible fusion of liposomes. Thus, the enzyme digestion method provides a novel simple means to distinguish fusion of liposomes from aggregation.     

Studies on spin-labelled egg lecithin dispersions.

ESR spectra of egg lecithin dispersions labelled with 5-nitroxide stearic acid are recorded with a 50 G field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The expansion technique improves the precision of the order parameters determined from the hyperfine splitting measurements, and may prove useful in future spin label membrane studies. Approximate order parameters are derived to describe the fluidity of fatty acid spin-labelled membranes in those cases where either the inner or outer hyperfine extrema are not well defined. The ability of these expressions to measure the fluidity of labelled egg lecithin disperions for the temperature range 14-42 degrees C is examined.     

The interaction of NADH-cytochrome b5 reductase and cytochrome b5 bound to egg lecithin liposomes.

Incubation of liposomes prepared by sonication of egg lecithin with the amphipathic form of cytochrome b5 results in the binding of a maximum of 244 molecules of cytochrome b5 per liposomal vesicle. Interactions of the phospholipid with the hydrophobic segment of cytochrome b5 are involved in this binding which does not disrupt the liposome. When a small amount of NADH-cytochrome b5 reductase is bound liposomes simultaneously with cytochrome b5, the two proteins catalyze the reduction of cytochrome c by NADH. A qualitative kinetic analysis reveals that all of the cytochrome b5 interacts with reductase, a result consistent with these protein undergoing translational diffusion in the plane of the membrane. This system and the purified stearyl coenzyme A desaturase provide a model to study the dynamics of protein andlipid interactions in this membrane-bound oxidative sequence.     

Interaction of aldolase A with lecithin liposomes

The aldolase A binding to the lecithin liposomes (Kd = 2.4 +/- 0.1 X 10(-3) M) has been shown by the fluorescence and tryptophan phosphorescence at the room temperature. The interaction is accompanied by an increase in the phospholipid bilayer microviscosity, and some conformational changes in the hydrophobic part of the enzyme, pronouncing themselves in Trp-147 environment rigidity, decrease. The observation of membrane viscosity vs. incubation time revealed practically instant enzyme-membrane interaction and no gradual incorporation. The accessibility of the NAD-binding domain of aldolase for NADH in the liposome presence remains unaltered.     

Adsorption of lecithin liposomes to acid clay

The interaction between lecithin liposomes and acid clay was investigated to clarify the mechanism for liposome adsorption to the clay. It was found that the multilamellar vesicular structure of the liposomes was broken as a result of primary adsorption. The acid clay particles aggregated and were eventually covered by the lecithin layer structure. In the case of kaolin, on the other hand, the liposomes were weakly adsorbed to the clay and maintained the vesicular structure. The amount of primary adsorption to the clay surface, which was estimated from the adsorption isotherm, was more for acid clay than for kaolin, and the total amount adsorbed to the acid clay was also more than to kaolin. This result can be explained by the much higher density of the negative charge on the acid clay surface than that for kaolin. The liposomes are therefore considered to be adsorbed to the acid clay mainly by the choline positive charge residing at the end of the lecithin molecule, although this is of no net charge as a whole.     

Magnetic anisotropy of egg lecithin membranes.

Magnetic realignment and rotational diffusion of cylindrical egg lecithin vesicles were measured under a phase contrast microscope. The anisotropy of magnetic susceptibility times membrane thickness was calculated from the data for several thin-walled vesicles. The resulting values were assigned to discrete numbers of bilayers. The difference between the susceptibilities parallel and perpendicular to the long axes of the lecithin molecules is deduced to be X parallel - X perpendicular = -(0.28 +/- 0.02) . 10(-8) cgs at 23 degrees C, if a bilayer thickness of 60 A is assumed.     

Solubilization of multilamellar liposomes of egg yolk lecithin by the bile salt sodiumtaurodeoxycholate and the effect of cholesterol--a rapid-ultrafiltration study

The solubilization of multilamellar egg yolk lecithin liposomes by sodiumtaurodeoxycholate in aqueous phase was studied by ultrafiltration as a function of time, bile salt and cholesterol concentration. The corresponding equilibrium states were analysed. Complete solubilization was achieved at total bile salt/lecithin molar mixing ratios of approximately 5. The minimum ratio to start solubilization was 0.1, corresponding to a free bile salt concentration of only 5% of the critical micelle concentration (CMC). Mean equilibrium constants for the partition of bile salts between non-filterable aggregates and filterable mixed micelles and also the free bile salt concentration were determined. Sodiumtaurodeoxycholate had a higher affinity for small mixed micelles than for lamellar mixed aggregates especially in the presence of cholesterol, which reduces the degree and rate of the solubilization process. A non-homogeneous distribution of bile salts in the lipid phase was detected at low bile salt concentrations.     

Effect of cholesterol on sensitivity of lecithin liposomes to surface-active substances

The effect of cholesterol incorporation into multilamellar egg lecithin liposomes on the liposomes sensitivity toward N-acyl derivatives of amino acids was examined. Free energy of intermolecular interaction between lecithin head groups in the bilayer is estimated as 3.8 ± 0.1 kcal/mol.