The role of amphiphiles in model and cell membrane fusion.
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Animals↗
Biomedical subjects
F M Goñi
Publications and source records attributed to F M Goñi.
Surfactant enhancement of polyethyleneglycol-induced cell fusion.
B16 mouse melanoma cells in monolayers may be satisfactorily fused with 50% PEG 1500. However, pre-treatment with detergents in solution at low concentrations significantly increases PEG fusion, up to 8-fold in some instances, without impairing cell viability. The practical and mechanistical implications of this finding are discussed.
Animals↗
Liposome fusion catalytically induced by phospholipase C.
Large unilamellar vesicles composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (50:25:25 mole ratio) were treated with phospholipase C. The early stages of phospholipid cleavage are accompanied by mixing of bilayer lipids (monitored by dequenching of octadecylrhodamine fluorescence) and leakage-free mixing of vesicle contents [measured by using 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylenebis(pyridinium bromide) (DPX)]. These results are interpreted in terms of vesicle fusion induced by the catalytic activity of phospholipase C. The use of sonicated unilamellar vesicles decreases the lag time, but does not modify the amplitude, of the fusion process. The presence of both phosphatidylethanolamine and cholesterol appears to be essential for measurable fusion effects to occur with low levels of phospholipid hydrolysis. Optimal fusion rates are observed with about 10-20 enzyme molecules per large unilamellar vesicle. This system of catalytically induced liposome fusion may be of relevance for the interpretation of physiological membrane fusion processes.
Cholesterol↗
Membrane solubilization by the non-ionic detergent triton X-100. A comparative study including model and cell membranes.
The solubilizing effects of the non-ionic detergent Triton X-100 have been examined on three membranous systems, namely rabbit sarcoplasmic reticulum, Halobacterium purple membrane and gramicidin A-phosphatidylcholine liposomes. The loss of membrane structure has been assessed through changes in suspension turbidity, while chemical analysis has revealed the differential solubilization of proteins and lipids. Solubilization data obtained on the above three systems are compared with previously published values concerning other membrane preparations. Also, solubilization of sarcoplasmic reticulum by Triton X-100 is monitored by Fourier-transform infrared spectroscopy and, similarly, purple membrane-surfactant interaction is studied using visible spectroscopy. The biochemical and spectroscopic data may be rationalized assuming a three-stage model of membrane-detergent interaction, incorporation of surfactant monomers into the membrane; disruption of the bilayer into mixed micelles, and separation of lipid and protein.
Animals↗
Lipid-protein interactions. The mitochondrial complex III-phosphatidylcholine-water system.
Bovine heart mitochondrial complex III (ubiquinol-cytochrome-c reductase) has been reconstituted into phosphatidylcholine bilayers and the effect of varying lipid/protein ratios on the structure and function of the protein has been examined. Electron microscopy, differential scanning calorimetry and Arrhenius plots of enzyme activity provide evidence that the protein is incorporated in an active conformation into pure phosphatidylcholine bilayers. At low lipid/protein ratios (e.g. 80:1 molar ratio) the protein exists in the form of aggregates. As the lipid proportion is increased, electron microscopy reveals the gradual formation of lipid bilayers; structures with the appearance of closed vesicles are seen at or above 300:1 phospholipid/protein molar ratios. Changes in enzyme activity as a function of lipid contents reveal a progressive increase in activity as more lipid is added, with a tendency to reach a saturation point. From the experimental data, a kinetic model is proposed, according to which the protein has an indefinite number of unspecific, independent and identical binding sites for phospholipids, the latter acting as essential enzyme activators. Varying lipid/protein ratios induce structural changes in complex III; visible spectra indicate changes in the polarity of the heme group environment, while Fourier-transform infrared spectroscopy suggests a change in the secondary structure of the protein as the lipid proportion is increased.
Animals↗
Lipid-protein interaction. The incorporation of myelin proteolipid apoprotein into phosphatidylcholine bilayers.
Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide, perturbing the phospholipids close to it, but allowing rapid (greater than 10(4) s-1) exchange with all the lipid molecules in the system.
Apoproteins↗
Structural changes induced by Triton X-100 on sonicated phosphatidylcholine liposomes.
Solubilization of sonicated unilamellar vesicles by Triton X-100 is a complex process. Solubilization starts at low detergent concentrations, as compared to the case of large vesicles, and is accompanied by the simultaneous rapid formation of large multilamellar liposomes. Measurements of lipid and detergent distribution indicate that, at a 1:1 lipid:detergent mole ratio, about one-third of the lipid, with most of the detergent, is solubilized in the form of mixed micelles. The remaining two-thirds are in the form of multilamellar liposomes, virtually free of detergent. Higher detergent concentrations also bring about the solubilization of these liposomes.
Calorimetry↗
Sublytic and lytic effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethylammonio)-1-propanesulfonate on phosphatidylcholine liposomes.
The effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethyl-ammonio)-1-propanesulfonate (Chaps) on multilamellar phosphatidylcholine liposomes have been characterized. When the surfactant is added to preformed liposome suspensions, equilibrium is attained in less than 6 h. Fifty percent solubilization, as measured by analysis of lipid P in supernatants after solubilization, occurs at a 0.32 lipid/detergent mole ratio for a 1 mM phospholipid concentration. Fifty percent release of entrapped glucose occurs at the same detergent concentration, suggesting that, in this system, no increase in permeability occurs prior to solubilization. A linear relationship is found between phospholipid concentration and amount of surfactant producing 50% solubilization. No lytic effect of Chaps is seen below 2 mM surfactant, this being probably near the critical micellar concentration of the amphiphile under our conditions. In the sublytic range of detergent concentrations, Chaps binds the lipid bilayers with high affinity, so that, at least at 1 mM phospholipid, the amount of free Chaps is negligible; solubilization starts when about two surfactant molecules are incorporated per phospholipid molecule. Differential scanning calorimetry shows that incorporation of Chaps into saturated phosphatidylcholine bilayers, even at concentrations below those producing solubilization, causes a decrease in the Tc gel-to-liquid crystalline main transition temperature of the phospholipid, and a decrease in the transition enthalpy; at the same time, a "shoulder" appears on the low-temperature side of the main endotherm. The ensemble of our data suggests that the behavior of Chaps toward phospholipid bilayers is intermediate between that of the natural bile derivatives and that of some well-known nonionic synthetic surfactants.
Calorimetry, Differential Scanning↗
Surfactant-induced release of liposomal contents. A survey of methods and results.
A systematic approach to the phenomenon of surfactant-dependent release of liposomal contents has been attempted. A variety of methods have been comparatively studied. The influence of the size of the entrapped molecule, nature of the surfactant, composition of bilayers and sonication of liposomes have been considered separately. In order to compare different results, a parameter has been defined, R50, as the phospholipid/surfactant mole ratio producing 50% release of the entrapped solute. This parameter appears to be, to a large extent, independent of time and liposome concentration. Surfactant-induced release of liposomal contents does not occur as a result of breakdown of phospholipid bilayers, but is rather a different phenomenon, occurring at detergent concentrations substantially lower (2-5 times) than solubilization. The required amount of surfactant appears to increase with the size of the entrapped solute. R50 depends clearly on the nature of the soluble amphiphile, but there is no obvious relationship with its critical micellar concentration. Liberation of vesicle content also depends on bilayer composition: phospholipids have various effects on the stability of the membrane, while the hydrophobic peptide, gramicidin A, appears to have little influence. Cholesterol is interesting, since at equimolar proportions with phosphatidylcholine, it decreases the stability of bilayer towards Triton X-100, while increasing it in the presence of cholate. Sonication also exerts an influence on the surfactant-dependent release of vesicle contents; it appears to decrease the bilayer stability, so that lower detergent concentrations are required to liberate the entrapped solutes. Finally, it should be noted that, although the decrease in self-quenching of 6-carboxyfluorescein is a convenient method for the study of solute liberation, glucose release, as detected by enzymatic methods, may be more reliable for accurate measurements.
Fluoresceins↗
Surfactant-induced liposome fusion: molecular mechanisms and biotechnological applications.
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Calorimetry, Differential Scanning↗
Micellar properties of the zwitterionic bile derivative CHAPS.
The critical micellar concentration (cmc) of the zwitterionic bile derivative CHAPS has been measured by fluorescence, light scattering and vapour pressure osmometry methods. The latter technique has been found most convenient and reliable. At 25 degrees C, in pure water, a cmc near 9 mM is found. Temperature decreases only slightly the cmc, from 9 to 7 mM upon heating from 25 to 50 degrees C. pH, in the range between 4 and 11, does not affect the cmc of CHAPS. Ionic strength appears to induce a marked decrease in cmc: measurements in 40 mM KCl give values about one-half those found for CHAPS in pure water.
Cholic Acids↗
The influence of membrane composition on the solubilizing effects of Triton X-100.
Multilamellar liposomes containing pure phosphatidylcholine (PC) or mixtures of PC with cholesterol, cholesteryl palmitate, beta-carotene, cardiolipin, phosphatidylethanolamine or gramicidin A have been treated with the detergent Triton X-100. Solubilization has been monitored as a decrease in turbidity of the liposome suspension, and also by determination of bilayer components in the solubilized fraction. The same solubilization pattern is found for unsaturated (egg yolk) or saturated (dimyristoyl) PC. Similar results are also found when dimyristoyl PC is solubilized above or below its gel-to-fluid transition temperature. Cholesterol solubilizes in parallel with PC; gramicidin A is solubilized preferentially to this phospholipid and the non-polar lipids cholesteryl palmitate or beta-carotene remain insoluble at detergent concentrations producing complete PC solubilization. Addition of cardiolipin or phosphatidylethanolamine does not seem to alter the general pattern of PC solubilization. Phosphatidylethanolamine is less soluble than PC, while cardiolipin solubilizes at the same detergent concentrations than PC. These results are considered in relation to previous studies with natural membranes.
Cholesterol↗
Kinetic studies on the interaction of phosphatidylcholine liposomes with Triton X-100.
Sonicated unilamellar and large multilamellar liposome suspensions have been treated with the non-ionic detergent Triton X-100, and the subsequent changes in turbidity have been studied as a function of time. Sonicated liposome suspensions exhibit an increase in turbidity that takes place in two stages, a fast, low-amplitude one is completed in less than 100 ms, and a slow large-amplitude one occurs in 20-40 s. The first increase in turbidity is associated to detergent incorporation into the bilayer, and the second one, to vesicle fusion. The fast stage may be detected at all detergent concentrations, while the slow one is only seen above the critical micellar concentration of Triton X-100. Both processes may be interpreted in terms of first-order kinetics. Studies of the variation of kexp with lipid and detergent concentration suggest a complex multi-step mechanism. In the case of multilamellar liposomes, a fast increase in turbidity is also seen after detergent addition, which is followed by a slow (20-60 s) decrease in turbidity and a very slow (up to 12 h) large scale decrease in turbidity. These processes do not conform to single-exponential patterns. The fast stage is also thought to reflect surfactant incorporation, while the decrease in turbidity is interpreted as bilayer solubilization starting with the outer bilayer (slow stage) and proceeding through the remaining ones (very slow stage).
Kinetics↗
Tryptophan fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers.
The tryptophan intrinsic fluorescence of mitochondrial complex III reconstituted in phosphatidylcholine bilayers was examined at different temperatures. Absorption and emission maxima occur at 277 and 332 nm, irrespective of temperature or lipid:protein ratio even if there are indications (from fluorescence quenching) of protein conformational changes as a function of lipid:protein ratio. Low values of Trp fluorescence quantum yield in complex III (0.008-0.010) are probably due to the neighborhood of the heme groups. The temperature-dependent decrease of fluorescence intensity is nonlinear; the corresponding Arrhenius plots show "breaks" or discontinuities that could be interpreted as thermally dependent changes in protein conformation. However, no temperature-dependent changes in fluorescence quenching have been observed that may be related to protein conformational changes. In addition, Arrhenius plots of the fluorescence intensity of simple molecules, such as Trp or 1-anilino-8-naphthalene sulfonate in the presence of aqueous phospholipid dispersions, also show breaks in the same temperature range. Stern-Volmer plots of acrylamide and iodide quenching were also nonlinear, indicating large differences in quenching constants for the various tryptophanyl residues. The quenching results also suggest that, at high lipid:protein ratios, the microviscosity of the protein matrix is higher than that in lipid-poor systems. Comparison of quenching efficiencies of iodide and acrylamide suggest that no significant fraction of the fluorophores occurs in the neighborhood of charged residues.
Animals↗
Cell membrane proteins. An overview.
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Animals↗
The interaction of phosphatidylcholine bilayers with Triton X-100.
The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and 2H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.
1,2-Dipalmitoylphosphatidylcholine↗
Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles.
Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The effects of surfactant removal do not appear to be readily reversible upon readdition of Triton X-100.
Acrylamide↗
A study of phospholipid phosphate groups in model membranes by Fourier transform infrared spectroscopy.
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Fourier Analysis↗