Physical properties of isolated complexes of human and bovine A-I apolipoproteins with L-alpha-dimyristoyl phosphatidylcholine.

Human or bovine A-I apolipoproteins in solution form complexes with sonicated L-alpha-dimirystoyl phosphatidylcholine at 23 and 37 degrees, but not at 8 degrees, suggesting a strong dependence of the interaction on the physical state of the lipid (phase transition temperature 23 degrees). Complexes were isolated by gel filtration on a Sepharose 4B column and were subsequently analyzed for protein and lipid content, molecular weight, and physical state of the lipid portion. The average stoichiometry of all complexes, regardless of the initial concentrations or ratios of protein and lipid, was constant: 90 +/- 20 mol of phospholipid/mol of protein monomer, suggesting a highly cooperative interaction. Sedimentation equilibrium experiments indicated homogeneous macromolecular preparations and gave molecular weights around 235,000 (+/- 15%) for the complexes, with the human and bovine apo-A-I proteins contributing 77,000 (+/- 10%), i.e. about three protein subunits per complex. The lipid portion of the complexes retained some characteristics of a bilayer: it had a broad phase transition with a midpoint at 25.5 degrees as reported by the fluorescence polarization of the lipophilic probe diphenylhexatriene. Above the phase transition temperature the mobility of the phospholipids in the complexes with both apo-A-I proteins was considerably decreased relative to the pure L-alpha-dimyristoyl phosphatidylcholine dispersion; below the phase transition temperature the opposite was true, i.e. the protein fluidized the lipids. The results indicate that apol-A-I proteins interact stoichiometrically with L-alpha-dimyristoyl phosphatidylcholine vesicles above the gel to liquid-crystalline transition temperature of the lipid, promoting the destruction of vesicles and the formation of well defined particles of the general size of high density serum lipoproteins.     

Studies on the mechanism of membrane fusion: evidence for an intermembrane Ca2+-phospholipid complex, synergism with Mg2+, and inhibition by spectrin.

The interaction of Ca2+ and Mg2+ with phosphatidylserine (PS) vesicles in 0.1 M NaCl aqueous solution was studied by equilibrium dialysis binding, X-ray diffraction, batch microcalorimetry, kinetics of cation-induced vesicle aggregation, release of vesicle contents, and fusion. Addition of either cation causes aggregation of PS vesicles and produces complexes with similar stoichiometry (1:2 cation/PS) at saturating concentrations, although the details of the interactions and the resulting complexes are quite different. Addition of Ca2+ to PS vesicles at T greater than or equal to 25 degrees C induces the formation of an "anhydrous" complex of closely apposed membranes with highly ordered crystalline acyl chains and a very high transition temperature (Tc greater than 100 degrees C). The formation of this complex is accompanied by a release of heat (5.5 kcal/mol), rapid release of vesicle contents, and fusion of the vesicles into larger membranous structures. By contrast, addition of Mg2+ produces a complex with PS which is much more hydrated, has no crystallization of the acyl chains at T greater than or equal to 20 degrees C, and has comparatively little fusion. Studies with both Ca2+ and Mg2+ added simultaneously indicate that there is a synergistic effect between the two cations, which results in an enhancement of the ability of Ca2+ to form its specific complex with PS at lower concentrations. The presence of the erythrocyte protein "spectrin" inhibits this synergism and interferes with the formation of the specific PS/Ca complex. It also inhibits the fusion of PS vesicles. It is proposed that the unique PS/Ca complex, which involves close apposition of vesicle membranes, is an intermembrane "trans" complex. We further propose that such a complex is a key step for the resultant phase transition and fusion of PS vesicles. By contrast, the PS/Mg complex is proposed to be a "cis" complex with respect to each membrane. The results are discussed in terms of the mechanism of membrane fusion.     

Studies on the effect of the lipid phase transition on the interaction of glucagon with dimyristoyl glycerophosphocholine

Glucagon is found to interact with dimyristoyl glycerophosphocholine both above and below the phase transition temperature of the lipid. Above the phase transition temperature the interaction is manifested by an increase in the rate of vesicle aggregation and by an increased permeability of unilamellar vesicles to Eu³⁺ and to Fe(CN)₆³⁻. However, no stable lipoprotein complex can be detected by gel filtration. Below the phase transition glucagon can form stable complexes with dimyristoyl glycerophosphocholine vesicles but cannot rapidly rearrange these vesicles to disk-shaped particles until the phase transition temperature is approached. The energy of activation for the dissociation of glucagon from the disk-shaped lipoprotein particle is 29 kcal/mol at temperatures above 36°C but increases markedly at lower temperatures, as the region of the lipid phase transition is approached. This increase in energy of activation at lower temperatures is most probably due to the larger amount of energy required to rearrange gel-state lipid in the transition state and provides an explanation for the unusual kinetic stability of the glucagon-dimyristoyl glycerophosphocholine lipoprotein complex only at temperatures below the phase transition of the lipid.     

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the enthalpy state of vesicles of different size by their interaction with alpha-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with alpha-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with alpha-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of alpha-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, delta H, of the interaction of the three vesicle types with alpha-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24 degrees C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic delta H values around 24 degrees C for large vesicles approximates the transition enthalpy of the pure phospholipid.     

Interaction of rat lecithin-cholesterol acyltransferase with rat apolipoprotein A-I and with lecithin-cholesterol vesicles.

The interaction of rat plasma lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles and with rat apo-A-I was studied in comparison with that of human plasma lecithin-cholesterol acyltransferase to clarify the reaction mechanism of rat plasma lecithin-cholesterol acyltransferase. The interaction of both human and rat lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles was investigated by gel permeation chromatography on Superose 12. Both enzymes had almost the same affinity to the vesicles. The affinity of rat enzyme to rat apo-A-I was stronger than that of human enzyme to human apo-A-I when estimated on the apo-A-I-Sepharose 4B column. When human apo-A-I was added to the human enzyme/vesicle mixture which contained the enzyme-vesicle complex, the enzyme was effectively dissociated from the complex. But when rat apo-A-I was added to the rat enzyme/vesicle mixture, apo-A-I-enzyme-vesicle complex was still recognized by its elution pattern on gel permeation chromatography. This suggests that the mixture of rat enzyme, rat apo-A-I, and vesicles, which are the major components in the rat lecithin-cholesterol acyltransferase reaction, forms a stronger complex than do the components of the human reaction.     

Fusion and lipid exchange in vesicles containing lipophilic spin labels

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.     

Exchange and aggregation in dispersions of dimyristoyl phosphatidylcholine vesicles containing myristic acid

Processes occurring in dispersions of dimyristoyl phosphatidylcholine containing myristic acid have been studied by light scattering of dilute dispersions (concn. ≤ 1 mg/ml) at temperatures above and below the phase transition temperatures of these dispersions. The transition temperatures increase with increasing mol fraction of myristic acid. Above these temperatures, vesicles with different mol fractions of myristic acid exchange lipid molecules. The exchange process leads to vesicles having phase transition temperatures and radii, which are both intermediate between the initial transitions and radii, respectively. In contrast with the observations above the phase transitions, it was found that when dimyristoyl phosphatidylcholine/myristic acid vesicles were cooled to a few degrees below the phase transition, larger particles were formed. These observations are consistent with a mechanism consisting of vesicle aggregation followed by fusion of the aggregated vesicles. The aggregation process is of second order in the vesicle concentration, and its rate increases with increasing mol fraction of myristic acid.     

Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with α-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with α-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of α-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, $\text{ΔH}$, of the interaction of the three vesicle types with α-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24°C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic $\text{ΔH}$ values around 24°C for large vesicles approximates the transition enthalpy of the pure phospholipid     

Membrane fusion and the lamellar-to-inverted-hexagonal phase transition in cardiolipin vesicle systems induced by divalent cations.

The polymorphic phase behavior of bovine heart cardiolipin (CL) in the presence of different divalent cations and the kinetics of CL vesicle fusion induced by these cations have been investigated. (31)P-NMR measurements of equilibrium cation-CL complexes showed the lamellar-to-hexagonal (L(alpha)-H(II)) transition temperature (T(H)) to be 20-25 degrees C for the Sr(2+) and Ba(2+) complexes, whereas in the presence of Ca(2+) or Mg(2+) the T(H) was below 0 degrees C. In the presence of Sr(2+) or Ba(2+), CL large unilamellar vesicles (LUVs) (0.1 microm diameter) showed kinetics of destabilization, as assessed by determination of the release of an aqueous fluorescent dye, which strongly correlated with the L(alpha)-H(II) transition of the final complex: at temperatures above the T(H), fast and extensive leakage, mediated by vesicle-vesicle contact, was observed. On the other hand, mixing of vesicle contents was limited and of a highly transient nature. A different behavior was observed with Ca(2+) or Mg(2+): in the temperature range of 0-50 degrees C, where the H(II) configuration is the thermodynamically favored phase, relatively nonleaky fusion of the vesicles occurred. Furthermore, with increasing temperature the rate and extent of leakage decreased, with a concomitant increase in fusion. Fluorescence measurements, involving incorporation of N-NBD-phosphatidylethanolamine in the vesicle bilayer, demonstrated a relative delay in the L(alpha)-H(II) phase transition of the CL vesicle system in the presence of Ca(2+). Freeze-fracture electron microscopy of CL LUV interaction products revealed the exclusive formation of H(II) tubes in the case of Sr(2+), whereas with Ca(2+) large fused vesicles next to H(II) tubes were seen. The extent of binding of Ca(2+) to CL in the lamellar phase, saturating at a binding ratio of 0.35 Ca(2+) per CL, was close to that observed for Sr(2+) and Ba(2+). It is concluded that CL LUVs in the presence of Ca(2+) undergo a transition that favors nonleaky fusion of the vesicles over rapid collapse into H(II) structures, despite the fact that the equilibrium Ca(2+)-CL complex is in the H(II) phase. On the other hand, in the presence of Sr(2+) or Ba(2+) at temperatures above the T(H) of the respective cation-CL complexes, CL LUVs rapidly convert to H(II) structures with a concomitant loss of vesicular integrity. This suggests that the nature of the final cation-lipid complex does not primarily determine whether CL vesicles exposed to the cation will initially undergo a nonleaky fusion event or collapse into nonvesicular structures.     

Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles

We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers

Large unilamellar vesicles containing phosphatidylinositol (PI), neutral phospholipids, and cholesterol are induced to fuse by the catalytic activity of phosphatidylinositol-specific phospholipase C (PI-PLC). PI cleavage by PI-PLC is followed by vesicle aggregation, intervesicular lipid mixing, and mixing of vesicular aqueous contents. An average of 2-3 vesicles merge into a large one in the fusion process. Vesicle fusion is accompanied by leakage of vesicular contents. A novel method has been developed to monitor mixing of lipids located in the inner monolayers of the vesicles involved in fusion. Using this method, the mixing of inner monolayer lipids and that of vesicular aqueous contents are seen to occur simultaneously, thus giving rise to the fusion pore. Kinetic studies show, for fusing vesicles, second-order dependence of lipid mixing on diacylglycerol concentration in the bilayer. Varying proportions of PI in the liposomal formulation lead to different physical effects of PI-PLC. Specifically, 30-40 mol % PI lead to vesicle fusion, while with 5-10 mol % PI only hemifusion is detected, i.e., mixing of outer monolayer lipids without mixing of aqueous contents. However, when diacylglycerol is included in the bilayers containing 5 mol % PI, PI-PLC activity leads to complete fusion.     

Thermotropic behavior of dipalmitoylphosphatidylcholine vesicles reconstituted with the glycoprotein of vesicular stomatitis virus

The vesicular stomatitis virus glycoprotein reconstituted into dipalmitoylphosphatidylcholine (DPPC) vesicles exerts a profound effect upon the DPPC gel to liquid-crystalline phase transition. The glycoprotein was reconstituted into DPPC vesicles by octyl glucoside dialysis. The gel to liquid-crystalline phase transition of these vesicles was monitored by differential scanning calorimetry. Vesicles formed in the absence of glycoprotein (600--2100-A diameter) underwent the phase transition at 41.0 degrees C and had an associated enthalpy change of 8.0 +/- 1.6 kcal/mol. Increasing the mole ratio of glycoprotein to DPPC in the vesicles to 0.15 mol % reduced both the transition temperature and the transition enthalpy change. The enthalpy change as a function of the mole percent glycoprotein could be fit to a straight line by a least-squares procedure. Extrapolation of the results to the glycoprotein concentration where the enthalpy change was zero indicated one glycoprotein molecule bound 270 +/- 150 molecules of DPPC.     

Lipid mobility in the assembly and expression of the activity of the prothrombinase complex

A phospholipid or membrane surface is a required component of the prothrombinase complex, yet little is known about the influence of the lipid on the assembly and expression of this complex. Vesicles composed of synthetic phospholipids were used to investigate the effects of membrane "fluidity" on the prothrombinase complex. All vesicle types studied were capable of supporting the prothrombinase reaction which in each case was characterized by a similar apparent Km. The binding constants for the interaction of Factor Va and prothrombin with synthetic phospholipid vesicles were not significantly affected by temperature. The rate of thrombin production, however, increased with increasing temperature. The fluidity of the vesicles was assessed by measuring the fluorescence lifetimes, steady state anisotropies, and differential phase fluorometry of diphenylhexatriene embedded in the vesicles. No correlation was observed between the fluidity of the vesicles and the steady-state rate of thrombin production, even when the enzymatic activity was monitored below and above the phase transition temperature of the lipid vesicles. A distinct correlation, however, was found between the fluidity of the vesicle and the time required to reach the maximum rate of thrombin production (pre-steady-state interval). We believe that this "lag" time corresponds to the time required for the assembly of the prothrombinase complex. Thus, although lipid fluidity does affect the assembly of the prothrombinase complex, after the complex is assembled, this property has little effect on the catalytic process itself.     

Pulmonary surfactant secretion is regulated by the physical state of extracellular phosphatidylcholine.

Pulmonary alveolar type II cells synthesize, secrete, and recycle the components of pulmonary surfactant. In this report we present evidence that dipalmitoylphosphatidylcholine is a potent inhibitor of surfactant lipid secretion by type II cells. Monoenoic and dienoic phosphatidylcholines with fatty acids of 16 or 18 carbons are ineffective as inhibitors of surfactant lipid secretion. In contrast, disaturated phosphatidylcholines, with either symmetric or asymmetric pairs of fatty acids of 14, 16, or 18 carbons, exhibit inhibition of surfactant secretion that correlates extremely well with the phase transition temperature (Tc) of the phospholipid. The inhibitory activity of dipalmitoylphosphatidylcholine is not dependent upon lipid stereochemistry. N-Methylated derivatives of dipalmitoylphosphatidylethanolamine are significantly less effective than phosphatidylcholine as inhibitors. Phosphatidylcholines below their phase transition temperature are inhibitors of surfactant secretion, whereas those above their phase transition temperature are either ineffective or weakly inhibitory. The phase transition dependence of inhibition is observed when type II cells are incubated at 37 degrees C with different species of phosphatidylcholine. In addition, if type II cells are stimulated to secrete at different temperatures the efficacy of a given phospholipid as an inhibitor is dependent on its relationship to Tc (i.e. dipalmitoylphosphatidylcholine with a Tc of 41 degrees C significantly inhibits secretion at 37 degrees C but not at 42 degrees C). Inhibition of surfactant secretion by dipalmitoylphosphatidylcholine is abrogated when it is incorporated into the same liposome with dioleoylphosphatidylcholine as a 50:50 mixture. In contrast, the simultaneous addition of two separate populations of liposomes, one composed of dipalmitoylphosphatidylcholine and the other composed of dioleoylphosphatidylcholine, does not significantly alter the inhibitory activity found with dipalmitoylphosphatidylcholine alone. These data provide compelling evidence that the physical state of phosphatidylcholine can regulate surfactant secretion from alveolar type II cells and suggest a unique mechanism for regulating exocytosis in the alveolus of the lung.     

Permeability of vesicular phospholipid bilayer membranes to thallium and its facilitation

The rate of release of TI⁺ from phospholipid vesicles of different composition was measured by pulse polarography as a function of temperature or in the presence of valinomycin, tetraphenylboron (TPB⁻) or dipicrylamine (DPA⁻) as transport facilitators. The release from pure dipalmitoylphosphatidylcholine (DPPC) vesicles increased abruptly around the pretransition temperature. The release from lipid mixtures with broad transition temperature region increased continuously with temperature. The steepness of the increase decreased with the width of the transition peak. Valinomycin, TPB⁻ (tetraphenylboron) and DPA⁻ (dipicrylamine) facilitate release of TI⁺ from unilamellar vesicles above their phase transition temperature with a first-order release rate constant. They do not facilitate release below the phase transition. Bursts of release were observed upon their addition to the vesicles but after annealing, which was completed within less than a minute, the vesicles were resealed. No facilitated release from multilamellar vesicles could be discerned. The entrapped volume into the multilamellar vesicles is determined from the difference between the maximal facilitated release and the total release after lysis of the liposomes by Triton X-100. The volume entrapped in the multilamellar vesicles determined this way amounted to 10–20% of the total entrapped volume.     

Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study

A number of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are characterized by the intracellular deposition of fibrillar aggregates that contain a high proportion of alpha-synuclein (alphaS). The interaction with the membrane-water interface strongly modulates folding and aggregation of the protein. The present study investigates the lipid binding and the coil-helix transition of alphaS, using titration calorimetry, differential scanning calorimetry, and circular dichroism spectroscopy. Titration of the protein with small unilamellar vesicles composed of zwitterionic phospholipids below the chain melting temperature of the lipids yielded exceptionally large exothermic heat values. The sigmoidal titration curves were evaluated in terms of a simple model that assumes saturable binding sites at the vesicle surface. The cumulative heat release and the ellipticity were linearly correlated as a result of simultaneous binding and helix folding. There was no heat release and folding of alphaS in the presence of large unilamellar vesicles, indicating that a small radius of curvature is necessary for the alphaS-membrane interaction. The heat release and the negative heat capacity of the protein-vesicle interaction could not be attributed to the coil-helix transition of the protein alone. We speculate that binding and helix folding of alphaS depends on the presence of defect structures in the membrane-water interface, which in turn results in lipid ordering in the highly curved vesicular membranes. This will be discussed with regard to a possible role of the protein for the stabilization of synaptic vesicle membranes.     

Proton currents and protein motion in membranes

A specific interaction between purified liver transglutaminase and small unilamellar phospholipid vesicles at the lipid phase transition have been revealed. The enzyme-induced perturbation of the bilayer is sufficient for phase transition release of encapsulated carboxyfluorescein from the vesicles. The size of the enzyme-phospholipid recombinants depends upon the protein-phospholipid ratio as shown on Sepharose 4B elution profile. The activity of transglutaminase inserted into the bilayer is greatly reduced. The interaction does not occur when the phospholipid vesicle are in the solid or liquid phase and it requires the structural integrity of the enzyme.     

Depolarized light-scattering studies of bilayer structures in phospholipid vesicles

Depolarized light scattering has been used to investigate the hydrocarbon chain packing of phospholipids in vesicles below the phase transition and ordering of their chains above the phase transition. The chain packing and ordering have been demonstrated for vesicles of l-α-dipalmitoylphosphatidylethanolamine and some phosphatidylcholines of different hydrocarbon chain lenghts. Anisotropy ratios for phospholipid vesicles could be determined by measuring depolarization ratios for several vesicle sizes at low concentrations of the lipids. The following results were obtained. Hydrocarbon chains of l-α-dimyristoyl and distearoylphosphatidylcholines below their phase transitions pack at tilting angles in good agreement with X-ray diffraction data. On the other hand, hydrocarbon chains of dipalmitoylphosphatidylethanolamine pack perpendicular to the bilayer surface. Values of the averaged order parameter for dimyristoyl, dipalmitoyl and distearoylphosphatidylcholines at 2.5°C above their phase transition are all the same and the value for dipalmitoylphosphatidylcholine is in agreement with results from ²H-NMR experiments. The value of the order parameter for dipalmitoylphosphatidylethanolamine is slightly larger than that for dipalmitoylphosphatidylcholine.     

Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles.

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.