Calcium-independent activation of prothrombin on membranes with positively charged lipids

The activation of prothrombin by factor Xa is strongly accelerated by negatively charged phospholipids plus calcium ions. In this paper we report that positively charged membranes can also stimulate prothrombin activation provided that the activation reaction is carried out in the absence of calcium ions. Membranes composed of a mixture of phosphatidylcholine (PC) and positively charged lipids like stearylamine, sphingosine, or hexadecyltrimethylammonium bromide caused a more than 1000-fold increase of the rate of prothrombin activation. Prothrombin activation by the factor Xa-factor Va complex was also considerably stimulated by such membranes. Stimulation of prothrombin activation by positively charged membranes was suppressed at high ionic strength. This suggests that electrostatic attraction of negatively charged proteins by positively charged membranes is the major driving force in the association of prothrombin and factor Xa with the lipid surface. Calcium ions strongly inhibited prothrombin activation on vesicles composed of PC and stearylamine (80/20 M/M), which indicates that the regions of prothrombin and/or factor Xa containing gamma-carboxyglutamic acid (gla) are important for the interaction of these proteins with positively charged membranes. The importance of the gla domain was confirmed by the observation that PC/stearylamine vesicles had much less effect on the reactions between proteins that lack gla residues [gla-domainless (des-1-45) prothrombin, prethrombin 1, prethrombin 2, or gla-domainless (des-1-44) factor Xa]. The efficiency of prothrombin and prothrombin derivatives to act as substrate decreased in the order prothrombin greater than des-1-45-prothrombin = prethrombin 1 greater than prethrombin 2, while prothrombin activation by gla-domainless (des-1-44) factor Xa was hardly stimulated by positively charged membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The assembly factor P17 from bacteriophage PRD1 interacts with positively charged lipid membranes.

The interactions of the assembly factor P17 of bacteriophage PRD1 with liposomes were investigated by static light scattering, fluorescence spectroscopy, and differential scanning calorimetry. Our data show that P17 binds to positively charged large unilamellar vesicles composed of the zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine and sphingosine, whereas only a weak interaction is evident for 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles. P17 does not bind to negatively charged membranes composed of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and 1-palmitoyl-2-oleoyl-phosphatidylcholine. Our differential scanning calorimetry results reveal that P17 slightly perturbs the phase behaviour of neutral phosphatidylcholine and negatively charged multilamellar vesicles. In contrast, the phase transition temperature of positively charged dimyristoylphosphatidylcholine/sphingosine multilamellar vesicles (molar ratio 9 : 1, respectively) is increased by approximately 2.4 degrees C and the half width of the enthalpy peak broadened from 1.9 to 5.6 degrees C in the presence of P17 (protein : lipid molar ratio 1 : 47). Moreover, the enthalpy peak is asymmetrical, suggesting that lipid phase separation is induced by P17. Based on the far-UV CD spectra, the alpha-helicity of P17 increases upon binding to positively charged micelles composed of Triton X-100 and sphingosine. We propose that P17 can interact with positively charged lipid membranes and that this binding induces a structural change on P17 to a more tightly packed and ordered structure.     

The rippled structure in bilayer membranes of phosphatidylcholine and binary mixtures of phosphatidylcholine and cholesterol

Freeze-fracture electron microscopy is used to study the rippled texture in pure dimyristoyl and dipalmitoyl phosphatidylcholine membranes and in mixtures of dimyristoyl phosphatidylcholine and cholesterol. Evidence is presented that the apparent phase transition properties of multilamellar liposomes may be dependent on the manner in which liposomes are prepared. Under certain conditions the ripple structures as visualized by freeze-fracture electron microscopy for the pure phosphatidylcholines are observed to be temperature dependent in the vicinity of the pretransition. Thus the transition can sometimes appear to be a gradual transition rather than a sharp, first-order phase transition. In mixtures of dimyristoyl phosphatidylcholine and cholesterol, the ripple repeat distance is found to increase as the cholesterol concentration is increased between 0 and 20 mol%. Above 20 mol%, no rippling is observed. A simple theory is presented for the dependence of ripple repeat spacing on cholesterol concentration in the range 0–20 mol%. This theory accounts for the otherwise inexplicable abrupt increase in the lateral diffusion coefficients of fluorescent lipids in binary mixtures of phosphatidylcholine and cholesterol when the cholesterol concentration is increased above 20 mol%.     

Effects of very small amounts of cholesterol on gel-phase phosphatidylcholine membranes

The mobility of 5-doxyl stearic acid spin label (5-SASL) in the gel phase of dipalmitoylphosphatidylcholine membranes between the main transition and subtransition temperatures was studied as a function of cholesterol content. Very small amounts of cholesterol (0.01-1 mol%) cause a dramatic increase in the mobility of 5-SASL. Temperature-drop experiments from 38 degrees C to 28 degrees C were made across the pretransition temperature and the rate of approach to equilibrium was measured. Cholesterol at low concentrations also affects this rate. The membrane reached equilibrium after 10 h in the absence of cholesterol, 3 h at 0.01 mol% cholesterol, and less than 10 min at 0.03 mol% cholesterol.     

EPR investigation of clomipramine interaction with phosphatidylcholine membranes in presence and absence of cholesterol

The effects of tricylic antidepressant clomipramine (CLO) on the membrane properties of saturated dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as well as on unsaturated egg yolk phosphatidylcholine liposomes were investigated by the electron paramagnetic resonance spin-labeling technique, in combination with the simulation of the spectra, taking into account that the membrane is heterogeneous and composed of the regions with different fluidity characteristics. Different spin labels, monitoring membrane properties in the upper and inner parts of the membrane, were used. In general, two spectral components, having different motional characteristics, were detected in all liposomes investigated. In liposomes with saturated chains, CLO decreased the phase-transition temperature, disordered the membrane, and increased polarity in the upper part of the membrane. However, less impact was observed in liposomes with unsaturated chains. In dipalmitoyl phosphatidylcholine liposomes, it also induced molecular rearrangements near the pretransition temperature. The presence of 30 mol% cholesterol increased the fluidizing effect of CLO and modified the lateral diffusion of nitroxide in the inner part of the membrane. A unique anomalous increase in diffusion of nitroxide, dependent on CLO concentration, was detected in the temperature region where the phosphatidylcholine membrane without cholesterol experiences the phase transitions. Since the changes in the central part of the membrane were even more pronounced than in the upper part of the membrane, it could be concluded that CLO incorporates into the membrane with its hydrophobic ring parallel to the phospholipid chains.     

Organization and interaction of cholesterol and phosphatidylcholine in model bilayer membranes

The molecular organization of sterols in liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at 37 degrees C is examined by utilizing the fluorescent analogue of cholesterol cholesta-5,7,9-trien-3 beta-ol (cholestatrienol). (1) Cholestatrienol is shown to be indistinguishable from native cholesterol in terms of its ability to condense POPC, as determined by (i) pressure/area studies of mixed-lipid monolayers and (ii) its ability to increase the order of POPC bilayers (determined by electron spin resonance studies) whether on its own or admixed with cholesterol at various ratios. (2) By analysis of the perturbation of the absorption spectra, cholestatrienol was found to be freely miscible in aggregates of cholesterol in buffer. In contrast, a lack of any detectable direct interaction of the sterol molecules in POPC bilayers was detected. (3) Fluorescence intensity and lifetime measurements of POPC/sterol (1:1 mol/mol) at various cholesterol/cholestratrienol molar ratios (0.5:1 up to 1:1 cholestatrienol/POPC) confirmed that sterol molecules in the membrane matrix were not associated to any great degree. (4) A quantitative estimate of how close sterol molecules approach each other in the membrane matrix was evaluated from the concentration dependence of the steady-state depolarization of fluorescence and was found to be 10.6 A. From geometrical considerations, the sterol/phospholipid phase at 1:1 mol/mol is depicted as each sterol having four POPC molecules as nearest neighbors. We term this arrangement of the lipid matrix an "ordered bimolecular mesomorphic lattice". (5) The concentration dependence of depolarization of fluorescence of cholestatrienol in POPC liposomes in the absence of cholesterol yielded results that were consistent with the cholestatrienol molecules being homogeneously dispersed throughout the phospholipid phase at sterol/POPC ratios of less than 1:1. (6) From qualitative calculations of the van der Walls' hydrophobic interactions of the lipid species, the phospholipid condensing effect of cholesterol is postulated to arise from increased interpenetration of the flexible methylene segments of the acyl chains, as a direct result of their greater mutual attraction compared to their attraction for neighboring sterol molecules. (7) The interdependence of the ordered bimolecular mesomorphic lattice and the acyl chain condensation is discussed in an effort to understand the ability of cholesterol to modulate the physical and mechanical properties of biological membranes.     

Lipolysis in model membranes in the presence of positively charged soluble proteins]

Phospholipase A2 hydrolysis of neutral and negatively charged lipid membranes modified by positively charged proteins has been studied using liposomes composed of either dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) alone or their equimolar mixture in the presence of cytochrome c, histone H1, cytochrome b5, and polylysine. Twenty minutes after the reaction had been initiated, DOPC hydrolysis was 58%, while that in the equimolar mixture with DOPG was 35%. DOPG hydrolysis was more complete in binary mixtures of liposomes. The same was observed for liposomes in the presence of cytochrome c. Hydrolysis of phospholipids in binary liposomes in the presence of histone H1 was 3 times faster than that in protein-free liposomes. In the presence of polylysine the rate of DOPG hydrolysis was decreased. The results obtained are suggestive of electrostatic interactions between hydrophilic proteins and negatively charged phospholipids, the phospholipase A2 catalytic activity being affected by these interactions.     

Translocation of positively charged copoly(Lys/Tyr) across phospholipid membranes

Much attention has recently been paid to the study of positively charged polypeptides as a possible carrier for therapeutic protein or DNA delivery to cells. In this study, we have investigated the translocation of positively charged copoly(Lys/Tyr) (MW=72000, DP=385) across lipid membranes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), as well as soybean phospholipids (SBPL) using zeta potential method, circular dichroism spectroscopy (CD), electrophysiology technique, fluorescence spectroscopy, and confocal laser scanning microscopy. Results of zeta potentials show that copoly(Lys/Tyr) associate with lipid membranes and become gradually saturated on the membranes either hydrophobically or electrostatically or both. CD studies demonstrate that the copoly(Lys/Tyr) takes and remains beta-sheet conformation during its interaction with liposome membranes, indicating that the translocation process should be carpet-mode like. Data from the electrophysiology technique reveal that positively charged copoly(Lys/Tyr) can cause transmembrane currents under an applied voltage, confirming its transfer across lipid membranes. Fluorescence spectroscopy results display a three-step mechanism of translocation across membrane: adsorption, transportation, and desorption, which has been verified by results from confocal laser scanning microscopy. We provided the first direct observation that the positively charged polypeptides, copoly(Lys/Tyr), can translocate through SBPL and EPC/DOPE lipid bilayer membranes. In addition, we found that the translocation efficiency of copoly(Lys/Tyr) was higher on the EPC/DOPE lipid membrane than on the SBPL lipid membrane.     

Controlled release of ionic drugs from complex micelles with charged channels

Oral administration of ionic drugs generally encounters with significant fluctuation in plasma concentration due to the large variation of pH value in the gastrointestinal tract and the pH-dependent solubility of ionic drugs. Polymeric complex micelles with charged channels on the surface provided us with an effective way to reduce the difference in the drug release rate upon change in pH value. The complex micelles were prepared by self-assembly of PCL-b-PAsp and PCL-b-PNIPAM in water at room temperature with PCL as the core and PAsp/PNIPAM as the mixed shell. With an increase in temperature, PNIPAM collapsed and enclosed the PCL core, while PAsp penetrated through the PNIPAM shell, leading to the formation of negatively charged PAsp channels on the micelle surface. Release behavior of ionic drugs from the complex micelles was remarkably different from that of usual core-shell micelles where diffusion and solubility of drugs played a key role. Specifically, it was mainly dependent on the conformation of the PAsp chains and the electrostatic interaction between PAsp and drugs, which could partially counteract the influence of pH-dependent diffusion and solubility of drugs. As a result, the variation of drug release rate with pH value was suppressed, which was favorable for acquiring relatively steady plasma drug concentration.     

Complexation of phosphatidylcholine lipids with cholesterol

It is postulated that the specific interactions between cholesterol and lipids in biological membranes are crucial in the formation of complexes leading subsequently to membrane domains (so-called rafts). These interactions are studied in molecular dynamics simulations performed on a dipalmitoylphosphatidylcholine (DPPC)-cholesterol bilayer mixture and a dilauroylphosphatidylcholine (DLPC)-cholesterol bilayer mixture, both having a cholesterol concentration of 40 mol %. Complexation of the simulated phospholipids with cholesterol is observed and visualized, exhibiting 2:1 and 1:1 stoichiometries. The most popular complex is found to be 1:1 in the case of DLPC, whereas the DPPC system carries a larger population of 2:1 complexes. This difference in the observed populations of complexes is shown to be a result of differences in packing geometry and phospholipid conformation due to the differing tail length of the two phosphatidylcholine lipids. Furthermore, aggregation of these complexes appears to form hydrogen-bonded networks in the system containing a mixture of cholesterol and DPPC. The CH...O hydrogen bond plays a crucial role in the formation of these complexes as well as the hydrogen bonded aggregates. The aggregation and extension of such a network implies a possible means by which phospholipid:cholesterol domains form.     

Synthesis of positively charged galactosurfactants

An approach to synthesis of cationic carbohydrate surfactants with potential antimicrobial or transfected activities is described.     

Influence of cholesterol on gramicidin-induced HII phase formation in phosphatidylcholine model membranes

The influence of cholesterol incorporation on gramicidin-induced hexagonal HII phase formation in different phosphatidylcholine model systems was investigated by 31P- and 2H-NMR, small-angle X-ray diffraction and differential scanning calorimetry. In liquid-crystalline distearoylphosphatidylcholine systems cholesterol inhibits gramicidin-induced HII phase formation. In dioleoylphosphatidylcholine the opposite effect is observed. Cholesterol appears to preferentially interact with gramicidin under liquid-crystalline conditions in both systems. Two phenomena that had been reported for gramicidin-treated erythrocyte membranes and derived liposomes (Tournois, H., Leunissen-Bijvelt, J., Haest, C.W.M., De Gier, J. and De Kruijff, B. (1987) Biochemistry, 26, 6613-6621) could also be observed in more simple dioleoylphosphatidylcholine-gramicidin-cholesterol systems. These are (i) an increase in tube diameter in the gramicidin-induced HII phase with increasing temperature, which is ascribed to the presence of cholesterol in this phase, and (ii) the loss of the hexagonal HII phase related 31P-NMR line shape at lower temperatures despite the presence of this phase as demonstrated with X-ray diffraction. This latter phenomenon appears to be due to restrictions in the rate of lateral diffusion of the phospholipids around the HII tubes due to the presence of gramicidin.     

Growth of linear charged micelles

The electrostatics of micellar growth is reviewed and extended for solutions containing excess salt. In dilute solution the expansion of a linear micelle with increasing salt concentration is explained for a wide range of ionic strength. When the micellar charge density is very high, counterions condense nonuniformly onto the micellar rod. In that case the micelle may contract upon the addition of salt. In semidilute solutions the excluded-volume effect is an additional factor complicating the ionic strength dependence of micellar growth.     

Synthesis of positively charged galactose-bearing surfactants

An approach to the synthesis of cationic carbohydrate surfactants with potential antimicrobial and transfecting activities is proposed.     

Interactions of pyrethroids with phosphatidylcholine liposomal membranes

Interactions of several pyrethroids with membrane lipids in the form of dipalmitoylphosphatidylcholine (DPPC) liposomes have been studied using fluorescent membrane probes. Fluorescence anisotropy values and lifetimes (determined by phase-shift and demodulation techniques) of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, were decreased in gel phase liposomes by pyrethroids at concentrations on the order of 10 microM. The pyrethroids containing a cyano substituent were also observed to cause collisional quenching of diphenylhexatriene fluorescence. Pyrethroids differed in their effectiveness at lowering the phase transition temperature of DPPC, and in their ability to broaden the temperature range of this transition. The fluorescence intensity of DPPC-incorporated chlorophyll a was used to monitor the pretransition of DPPC and the lateral diffusion of a membrane component located in the polar headgroup region. Permethrin did not affect chlorophyll a fluorescence intensity at any temperature. It may be concluded from these results that pyrethroids are preferentially located in the interior hydrophobic regions of the lipid bilayer, and that these compounds can disorder hydrocarbon packing in the bilayer core. However, polar headgroups were not disordered, and diffusion of membrane components in the polar headgroup region was not altered.     

Lipid raft components cholesterol and sphingomyelin increase H+/OH- permeability of phosphatidylcholine membranes

H+/OH- permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH- and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH- permeability from a baseline of 2.4x10(-2) cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH- permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH- permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484-1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH- conductivity, (iii) H+/OH- fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH- permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results.     

Detailed molecular dynamics simulations of model biological membranes containing cholesterol

Detailed molecular dynamics simulations performed to study the nature of lipid raft domains that appear in model membranes are reviewed in this paper. The described simulations were performed on hydrated bilayers containing binary mixtures of cholesterol with phospholipids and also on ternary mixtures containing cholesterol, a phospholipid with a high main transition temperature T_m, and a phospholipid with a low transition temperature T_m. These simulations provide qualitative and semi-quantitative information about cholesterol-lipid interactions and also a testing ground for major assumptions made to explain the nature of lipid rafts in model membranes.