Gel filtration of egg phosphatidylcholine vesicles

The abilities of Sepharose 2b (Pharmacia), Controlled Pore Glass (Electro-Nucleonics) and Bio-Gel A150m (Bio-Rad) to purify small unilamellar vesicles prepared by sonication and the ethanol-injection methods were compared. The Bio-Gel causes complete aggregation of the sonicated vesicles and partial aggregation of the ethanol-injection vesicles. Both Sepharose and Controlled Pore Glass are acceptable for purifying vesicles from multilamellar liposomes; however, neither will separate the vesicles from sonication by-products which might be formed.     

Ca2+-induced fusion of cardiolipin/phosphatidylcholine vesicles monitored by mixing of aqueous contents

The kinetics of Ca²⁺-induced fusion of large (0.1 μm) unilamellar cardiolipin/phosphatidylcholine (1:1) vesicles have been investigated by continuous monitoring of the mixing of the aqueous vesicle contents. In parallel, release of vesicle contents to the external medium has been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents being largely a secondary phenomenon. The minimal Ca²⁺ concentration required for fusion in this system is approx. 9 mM. At higher Ca²⁺ concentrations fusion is extremely fast, occurring on the time scale of seconds.     

Origin of the latency phase during the action of phospholipase A2 on unmodified phosphatidylcholine vesicles

The reaction progress curve for the action of pig-pancreatic phospholipase A₂ on dimyristoylphosphatidylcholine vesicles is characterized under a variety of conditions. The factors that regulate the rate of hydrolysis during the presteady-state phase determine the latency period. The results demonstrate that the accelerated hydrolysis following the latency phase of the reaction progress curve is due to the product-assisted binding of the enzyme to the substrate bilayer by chaning the number of bindings sites and therefore the binding equilibrium. A critical mole fraction of products appears to be formed in the substrate bilayers before the steady-state phase of hydrolysis begins. The latency phase shows a minimum at the phase-transition temperature of the substrate vesicles; however, we did not observe a significant binding of the enzyme to pure substrate bilayers even at the phase-transition temperature. The rate of binding of the enzyme is found to be fast and the rate of desorption of the bound enzyme is very slow compared to the latency phase. The rate of redistribution of products between substrate bilayers is rather slow. These observations demonstrate that during the latency phase of the action of phospholipase A₂, a critical mole fraction of products is formed in the substrate bilayer.     

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.     

Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the reaction rates of vitamin E (α-, β-, γ-, δ-tocopherols, TocH) and tocol with aroxyl radical (ArO) as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry. Egg yolk phosphatidylcholine (EYPC) vesicles were used as a membrane model. EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H₂O = 7:3, v/v) that gave the lowest turbidity in samples. The second-order rate constants (k_s) for α-TocH in MeOH/H₂O solution with EYPC vesicles were apparently 3.45 × 10⁵ M⁻¹ s⁻¹, which was about 8 times higher than that (4.50 × 10⁴ M⁻¹ s⁻¹) in MeOH/H₂O solution without EYPC vesicles. The corrected k_s of α-TocH in vesicles, which was calculated assuming that the concentration of α-TocH was 133 times higher in membranes of 10 mM EYPC vesicles than in the bulk MeOH/H₂O solution, was 2.60 × 10³ M⁻¹ s⁻¹, which was one-seventeenth that in MeOH/H₂O solution because of the lower mobility of α-TocH in membranes. Similar analyses were performed for other vitamin E analogues. The k_s of vitamin E in membranes increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH. There was not much difference in the ratios of reaction rates in vesicles and MeOH/H₂O solution among vitamin E analogues [k_s(vesicle)/k_s (MeOH/H₂O) = 7.7, 10.0, 9.5, 7.4, and 5.1 for α-, β-, γ-, δ-TocH, and tocol, respectively], but their reported ratios in solutions of micelles and ethanol were quite different [k_s(micelle)/k_s(EtOH) = 100, 47, 41, 15, and 6.3 for α-, β-, γ-, δ-TocH, and tocol, respectively]. These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes, which increased in the order of tocol < δ-TocH < γ-TocH ∼ β-TocH < α-TocH.     

Transbilayer movement of 24Na in sonicated phosphatidylcholine vesicles exposed to frequency-modulated microwave radiation

Sonicated egg phosphatidylcholine vesicles loaded with ²⁴Na⁺ were exposed at 20mW to a frequency-modulated (3 Hz) microwave field in the range of 2350 to 2550 MHz, or at 80 mW to a 2450-MHz CW (continuous wave) field, in a waveguide. The vesicle suspension absorbed microwaves at about 1 mW/ml and 25 mW/ml (CW experiment). The average temperature change of the irradiated suspension was < 0.1 °C from ambient. Leakage of ²⁴Na⁺ from the vesicles for up to 19 hours was measured. No difference was noted in the movement of ²⁴Na⁺ from the vesicles in the irradiated and control dispersions.     

Lysis of egg phosphatidylcholine vesicles by tricyclic carboxamide antitumor agents

The stability of small unilamellar vesicles formed by egg phosphatidylcholine has been examined in the presence of 38 tricyclic carboxamide DNA-intercalating agents (19 phenylquinolines, 17 phenylbenzimidazoles, an acridine and an anthracene). Lysis of the vesicular membrane is time-dependent and also dependent on the concentration of the cytotoxic agent. The relative concentration of agent to cause a fixed degree of lysis in a fixed time, as measured by the release of encapsulated 6-carboxyfluorescein, is directly related to the relative hydrophobicity of the agents.     

Ca 2+ outflow from sarcoplasmic reticulum vesicles during changes in membrane potential, Ca2+ concentration and pH

The concentration gradient Ca2+ outflux from the vesicles of the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied under conditions of the induced membrane potential, the concentrations of Ca2+ and H+ in the medium washing over the vesicles being different. The Ca2+ outflux from vesicles is shown to be the same with a decrease of the membrane potential from--80 down to -10 mV and gets higher with the zero and subsequent positive values of the latter. A significant intensification of the Ca2+ outflux from vesicles under the effect of external-vesicular Ca2+ has been observed at its concentration of 10(-5) M. Against this background of external-vesicular Ca2+ and zero value of the membrane potential either exogenous AMP or the pH increase from 6.5 up to 7.8 favour a release of more than 70% of passively accumulated Ca2+. The pH effect grows with a decrease in the external-vesicular concentration of Ca2+. A conclusion is drawn on the significance of protons in the regulation of the Ca2+ release from the sarcoplasmic reticulum.     

Uptake of Ca2+ mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles

The (Ca2+ + Mg2+)-ATPase was purified from skeletal muscle sarcoplasmic reticulum and reconstituted into sealed phospholipid vesicles by solution in cholate and deoxycholate followed by detergent removal on a column of Sephadex G-50. The level of Ca2+ accumulated by these vesicles, either in the presence or absence of phosphate within the vesicles, increased with increasing content of phosphatidylethanolamine in the phospholipid mixture used for the reconstitution. The levels of Ca2+ accumulated in the absence of phosphate were very low for vesicles reconstituted with egg yolk phosphatidylcholine alone at pH 7.4, but increased markedly with decreasing pH to 6.0. Uptake was also relatively low for vesicles reconstituted with dimyristoleoyl- or dinervonylphosphatidylcholine, and addition of cholesterol had little effect. The level of Ca2+ accumulated increased with increasing external K+ concentration, and was also increased by the ionophores FCCP and valinomycin. Vesicle sizes changed little with changing phosphatidylethanolamine content, and the sidedness of insertion of the ATPase was close to random at all phosphatidylethanolamine contents. It is suggested that the effect of phosphatidylethanolamine on the level of Ca2+ accumulation follows from an effect on the rate of Ca2+ efflux mediated by the ATPase.     

The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]), if added before GTP, blocks both Ca2+ efflux promoted by GTP and the effect of GTP on enhancement of inositol 1,4,5-triphosphate (IP3)-promoted Ca2+ release from preloaded microsomal vesicles. If, however, GTP[S] is added after GTP, it does not reverse the Ca2+ efflux promoted by GTP, nor does it inhibit IP3-promoted Ca2+ release. 2. The effect of GTP in enhancing IP3-promoted Ca2+ release is maintained after washing the microsomal vesicles free of added GTP. After this treatment, enhancement of IP3-promoted Ca2+ efflux can be observed in the absence of poly(ethylene glycol). 3. Electron microscopy shows that during GTP treatment of microsomal vesicles there is rapid production of very large vesicular structures, apparently produced by fusion of smaller vesicles. 4. Light-scattering changes are detectable during the fusion process. 5. Both Ca2+ efflux promoted by GTP and the enhancement of IP3-promoted Ca2+ release seen in the presence of GTP can probably be attributed to GTP-dependent vesicle fusion.     

Interdependence of H+ and K+ fluxes during the Ca2+-pumping activity of sarcoplasmic reticulum vesicles

The release of H⁺ during the oxalate-supported Ca²⁺ uptake in sarcoplasmic reticulum vesicles is kinetically coincident with the initial phase of Ca²⁺ accumulation. The Ca²⁺ uptake is increased and the H⁺ release is decreased in the presence of KCl and other monovalent chloride salts as expected for a H⁺-monovalent cation exchange. The functioning of the Ca²⁺-pump is disturbed by the presence of potassium gluconate and, to a lesser extent, of choline chloride. These salts do not inhibit the ATPase activity of Ca²⁺-permeable vesicles, suggesting a charge imbalance inhibition which is specially relevant in the case of gluconate. Therefore, K⁺, and also Cl^–, appear to be involved in secondary fluxes during the active accumulation of Ca²⁺. The microsomal preparation seems homogeneous with respect to the K⁺-channel, showing an apparent rate constant for K⁺ release of approximately 25 s^–1 measured with the aid of⁸⁶Rb⁺ tracer under equilibrium conditions. A Rb⁺ efflux, sensitive to Ca²⁺-ionophore, can be also detected during the active accumulation of Ca²⁺. The experimental data suggest that both monovalent cations and anions are involved in a charge compensation during the Ca²⁺ uptake and H⁺ release. Fluxes of these highly permeable ions would contribute to cancel the formation of a resting membrane potential through the sarcoplasmic reticulum membrane.     

Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles

Ca2+-ATPase from sarcoplasmic reticulum was reconstituted into phospholipid/cholesterol (9:1) vesicles (RO). Sucrose density gradient centrifugation of the RO vesicles separated a light layer (RL) with a high lipid/protein ratio and a heavy layer (RH). RH vesicles exhibited a high rate of Ca2+-dependent ATP hydrolysis but did not accumulate Ca2+. RL vesicles, on the other hand, showed an initial molar ratio of Ca2+ uptake to ATP hydrolysis of approximately 1.0. Internal trapping of transported Ca2+ facilitated studies over periods of several minutes. Ca2+ transport and ATP hydrolysis declined concomitantly, reaching levels near 0 with external Ca2+ concentrations less than or equal to 2 microM. Ca2+ uptake was inhibited by the Ca2+ ionophore A23187, the detergent Triton X-100, and the metabolic inhibitor quercetin. Ca2+ transport generated a transient electrical potential difference, inside positive. This finding is consistent with the hypothesis that the Ca2+ pump is electrogenic. Steady state electrical potentials across the membrane were clamped by using potassium gradients and valinomycin, and monitored with voltage-sensitive dyes. Over a range of +50 to -100 mV, there was an inverse relationship between the initial rate of Ca2+ uptake and voltage, but the rate of ATP hydrolysis was nearly constant. In contrast, lowering the external Ca2+ concentration depressed both transport and ATP hydrolysis. These findings suggest that the membrane voltage influences the coupling between Ca2+ transport and ATP hydrolysis.     

Effect of Ca2+ on thermotropic properties of saturated phosphatidylcholine liposomes

The effect of various concentrations of calcium ion (Ca²⁺) on dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC) and mixed DPPC/DSPC (1:1) liposomes was studied by differential scanning calorimetry. Ca²⁺ concentrations of 10 mM and above split the main transition peak of DPPC into two distinguishable components, and, at 30 mM and above, also resulted in the disappearance of a pre-transition peak. The effect of Ca²⁺ on DSPC liposomes was even more dramatic in that it induced a more definitive split in the main transition peak and caused the loss of the pretransition peak at only 10 mM concentration. The thermograms of the DPPC/DSPC mixed liposomes were unaltered in the presence of Ca²⁺, even at a concentration of 50 mM. Whether or not Ca²⁺ is able to alter thermograms of phosphatidylcholine liposomes appears to be dependent on the degree of molecular order of the bilayer prior to interaction with Ca²⁺.     

Specific interaction of arabinose residue in ginsenoside with egg phosphatidylcholine vesicles

The interaction of the specific sugar residue in ginsenosides with egg phosphatidylcholine vesicles was investigated by ESR spectrometry using phosphatidic acid spin-labeled at the polar head groups. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue and agglutinability toward egg yolk phosphatidylcholine vesicles (Fukuda, K. et al. (1985) Biochim. Biophys. Acta 820, 199-206), caused the restriction of the segmental motion of spin-labeled phosphatidic acid in egg phosphatidylcholine vesicles, indicating that the saponin interacted with the polar head groups of vesicles. Other ginsenosides-Rb2, Rb1, Rd and p-nitrophenyl glycoside derivatives which have less or no agglutinability were also investigated in the same manner. Only ginsenoside-Rb2 and p-nitrophenyl alpha-L-arabinofuranoside which have the specific sugar residue (arabinose) showed a strong interaction with the polar head groups of vesicles. To gain an insight into the mechanism of agglutination by ginsenoside-Rc, the interaction with the fatty acyl groups was also studied by using phosphatidylcholine spin-labeled at the fatty acyl groups. Ginsenoside-Rc increased the order parameter of the spin-labeled phosphatidylcholine, indicating that the saponin was inserted into lipid bilayers. In other saponins investigated, only ginsenoside-Rb2 interacted with the fatty acyl part of vesicles. The process of expression of agglutination by ginsenoside-Rc was discussed on the basis of the ESR studies.     

Timemresolved fluorescence of S-100a protein: effect of Ca, Mg and unilamellar vesicles of egg phosphatidylcholine

Phase-modulation fluorescence lifetime measurements were used to study the single Trp residue of the Ca²⁺-binding protein S-100a both in the absence and in the presence of Ca²⁺ and/or Mg²⁺. Trp fluorescence decay for the protein was satisfactorily described by Lorentzian lifetime distributions centered around two components (approximately 4 ns and 0.5 ns). Lifetime values were unchanged by 2 mM Ca²⁺, but the fractional intensity associated with longer lifetime increased up to 75%. In the presence of Mg²⁺, the Ca²⁺ induced increase of the fractional intensity associated with longer lifetime was only 57%. For the protein in buffer, about the 85% of the recovered anisotropy was associated to a rotational correlation time of 6.7 ns. After the addition of Ca²⁺, this value was increased to 16.08 ns. In the presence of Mg²⁺, Ca⁺² increased the rotational correlation time to 33.75 ns. Similar studies were performed with S-100a interacting with egg phosphatidylcholine vesicles (SUV). Our data suggest that the conformation of the protein may be influenced by structural features of the lipidic membrane. Moreover, data obtained in the presence of Mg²⁺ indicate some interaction between lipids and S-100, likely mediated by this ion.     

Effect of encapsulated Ca2+ on the surface properties of curved phosphatidylcholine bilayers

In this paper, the influence of Ca2+ on the adsorption properties of 1,8-anilinonaphthalenesulfonate (ANS) and analogous probes to sonicated vesicles of phosphatidylcholine was studied by means of spectrofluorometry. The fluorescence of ANS added to the vesicle dispersion increases with the Ca2+ concentration in the inner media but remains constant when Ca2+ concentration is changed in the outside solution. However, the fluorescence decreases when large anions such as ClO4- are present in the external solution. Ca2+ inside large liposomes promotes a similar behaviour to that found in sonicated vesicles when they are osmotically contracted in hypertonic media. The results can be interpreted in terms of Ca2+ adsorption on the inner interface and a cooperative interaction between the monolayers.     

13C NMR studies on [4-13C] cholesterol incorporated in sonicated phosphatidylcholine vesicles

1. 90.5 MHz 13C NMR linewidth measurements were performed on mixed sonicated [4-13C] cholesterol/phosphatidylcholine vesicles of different fatty acid composition. 2. From the Dy3+ -induced shift of the C4 resonance of cholesterol it suggested that this part of the molecule is localized in the ester bond region of the bilayer. 3. The local motion of the cholesterol ring system is restricted and independent of fatty acid composition. 4. At cholesterol concentrations below 30 mol percent the ring system becomes more immobilised when the fatty acids of the phosphatidylcholine molecules enter the gel state.     

Ca2+-induced phosphatidylcholine vesicle aggregation in the presence of ferricyanide

The titration of sonicated vesicles of egg phosphatidylcholine with ferricyanide in the presence of Ca2+ results in the formation of aggregates. The turbidity increase caused by these aggregates cannot be reversed by EDTA treatment. In addition, no rearrangement of the bilayer structure has been found in this process, either measuring leakage of vesicle content or exchange of lipids among the bilayers themselves. The aggregation is dependent on the Ca2+ content of the vesicles, the outer Ca2+ and Fe(CN)3-(6) concentration and the order of addition of Ca2+ and ferricyanide. The results can be explained by a specific adsorption of Fe(CN)3-(6) to bilayers of sonicated vesicles, in contrast to other multivalent anions. In contrast to the stability found with sonicated vesicles, the aggregation causes a leakage of the internal solution when multilamellar liposomes are titrated with Fe(CN)3-(6).     

A role of H+ flux in active Ca2+ transport into sarcoplasmic reticulum vesicles. II. H+ ejection during Ca2+ uptake

Proton efflux during Ca2+ transport into sarcoplasmic reticulum vesicles was examined. Although a rapid H+ ejection was observed during the initial phase of Ca2+ uptake and the amount of the liberated H+ was more than that due to hydrolysis of ATP, generation of a pH difference as a result of the H+ efflux could not be detected by direct pH measurement with a pH meter. Alkalinization of the inside of the vesicles during Ca2+ uptake was more precisely examined by flow dialysis assay and a significant uptake of acetate or salicylate into the vesicles was found, suggesting the generation of a small pH difference across the SR membrane. From these results, it was concluded that counter-transport of H+ was operative in Ca2+ uptake but that only a relatively small pH difference was generated as a result of the H+ efflux. The intrinsic buffering capacity of sarcoplasmic reticulum vesicles was measured and a relatively large value (130 nmol H+/pH unit/mg at pH 6.2) was obtained.