The effect of cholesterol on measured interaction and compressibility of dipalmitoylphosphatidylcholine bilayers

We have examined the phase diagram of dipalmitoylphosphatidylcholine (DPPC)--cholesterol-water mixtures at low cholesterol content, and report phase separation between 3 and 10 mol% cholesterol. The two lamellar phases at equilibrium in this region appear to be pure DPPC and 11 mol% cholesterol in DPPC. For these two lamellar phases, which are made up of alternating layers of water and bimolecular lipid leaflets, we have measured the forces of interaction between leaflets and the lateral pressure and compressibility of the leaflets. Both bilayers experience a strong repulsive force when forced together only a few ?ngstr?ms (1 A = 0.1 nm) closer than their maximum separation in excess water. However, the presence of 11 mol% cholesterol causes the bilayers to move apart of 35-A separation from the 19-A characteristic of pure DPPC in excess water. This swelling may result from a decrease in van der Waals attraction between bilayers or from an increase in bilayer repulsion. Differences in bilayer interaction can be a cause for phase separation. More importantly these differences can cause changes in the composition of regions of membranes approaching contact. At 11 mol%, cholesterol substantially increases the lateral compressibility of DPPC bilayers leading to higher lateral density fluctuations and potentially higher bilayer permeability.     

Effect of divalent cations on the structure of dipalmitoylphosphatidylcholine and phosphatidylcholine/phosphatidylglycerol bilayers: an 2H-NMR study

The interactions of CaCl2 or MgCl2 with multilamellar phospholipid bilayers were studied by 2H-NMR. Two model membrane systems were used: (1) dipalmitoylphosphatidylcholine (DPPC) bilayers and (2) bilayers composed of a mixture of phosphatidylcholine and phosphatidylglycerol at a molar ratio of 5:1. Addition of 0.25 M CaCl2 to DPPC bilayers resulted in significant uniform increase of the order parameters of the lipid side chains; the effect of 0.25 M MgCl2 was insignificant. Both phosphatidylcholine and phosphatidylglycerol components of the mixed bilayers were affected by the presence of 0.25 M CaCl2 and, to a much smaller degree, by MgCl2. The addition of Ca2+ induced significantly larger increase of the order parameters of the phosphatidylcholine component. The results are consistent with the long-range effects of Ca2+ binding on the packing of the lipid membranes.     

Effect of glycosaminoglycans and divalent cations on the thermal properties of dipalmitoylphosphatidylcholine

The effect of glycosaminoglycans (GAG) and divalent cations on the thermal properties of dipalmitoyl-phosphatidylcholine (DPPC)-water systems was examined in order to model some interactions taking place on low density lipoprotein (LDL) surfaces. The thermal properties of these systems were measured by differential scanning calorimetry (DSC). According to the results, all three glycosaminoglycans used (chondroitin-4-sulfate, chondroitin-6-sulfate and heparin) were effective but to a different extent. Calcium ions enhance the interaction more than magnesium ions, probably because divalent cations form bridges between the negatively charged groups of GAGs and the headgroups of lipids. It is conceivable that similar processes might occur in the case of LDL.     

Interactions of divalent cations with phosphatidylserine bilayer membranes

The interaction of divalent cations with a homologous series of diacylphosphatidylserines (diacyl-PS) has been studied by differential scanning calorimetry and X-ray diffraction. Hydrated di-C14-PS (DMPS) exhibits a gel leads to liquid-crystal bilayer transition at 39 degrees C (delta H = 7.2 kcal/mol of DMPS). With increasing MgCl2 concentration, progressive conversion to a phase exhibiting a high melting (98 degrees C), high enthalpy (delta H congruent to 11.0 kcal/mol of DMPS) transition is observed. Similar behavior is observed for DMPS with increasing CaCl2 concentration. In this case, the high-temperature transition of the Ca2+-DMPS complex occurs at approximately 155 degrees C and is immediately followed by an exothermic transition probably associated with PS decomposition. For di-C12-, di-C14-, di-C16- (DPPS), and di-C18-PS, the transition temperatures of the Ca2+-PS complexes are in the range 151-155 degrees C; only di-C10-PS exhibits a significantly lower value, 142 degrees C. A different pattern of behavior is exhibited by DPPS in the presence of Sr2+ or Ba2+, with transitions in the range 70-80 degrees C being observed. X-ray diffraction of the Ca2+-PS complexes at 20 degrees C provides evidence of structural homology. All Ca2+-PS complexes exhibit bilayer structures, the bilayer periodicity increasing linearly from 35.0 A for di-C10-PS to 52.5 A for di-C18-PS. Wide-angle X-ray diffraction data indicate that hydrocarbon chain "crystallization" occurs on Ca2+-PS complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The adsorption of divalent cations to phosphatidylcholine bilayer membranes

Electrophoretic mobility and ³¹P NMR measurements were combined to test whether the combination of the Henry, Boltzmann and Grahame equations is capable of describing the adsorption of divalent cations to phosphatidylcholine membranes. Cobalt was chosen for this study because, of all the common divalent cations, its effects on the ³¹P NMR spectrum of phosphatidylcholine membranes are easiest to interpret. Both the ³¹P NMR data on the adsorption of cobalt and the zeta potential data calculated from the electrophoretic mobility in the presence of cobalt are well described by the combination of these three equations. Electrophoretic mobility measurements were also performed with a number of other divalent cations and the zeta potentials were, in all cases, well described by the combination of these three equations. The binding deduced from such measurements decreases in the sequence: Mn²⁺, Mg²⁺, Ca²⁺, Co²⁺, Ni²⁺, Sr²⁺, Ba²⁺. If we assume that a lipid molecule occupies an area of 60 Ų and that there is a 1: 1 stoichiometry for the binding of the divalent ions to phosphatidylcholine, the dissociation constants are, respectively: 0.3, 1.0, 1.0, 1.2, 1.2, 2.8, 3.6 M.     

The adsorption of divalent cations to phosphatidylglycerol bilayer membranes

The ability of the Stern equation to describe the adsorption of divalent cations to phosphatidylglycerol membranes was tested by combining ³¹P-NMR and electrophoretic mobility measurements. In 0.1 M sodium chloride both the ³¹P-NMR and the zeta potential data are well described by the Stern equation. ³¹P-NMR and ¹³C-NMR results indicate that cobalt forms inner-sphere complexes only with the phosphate group of phosphatidylglycerol molecules and that a substantial fraction of the adsorbed cobalt ions form outer-sphere complexes. Evidence is presented that suggests the alkaline earth cations also bind to phospholipids mainly by forming outer sphere complexes. Electrophoretic mobility measurements were performed with several different divalent cations. In all cases the zeta potentials in 0.1 M sodium chloride were well described by the Stern equation. The intrinsic 1 : 1 association constants (M^?1) for the phosphatidylglycerol complexes decreased in the sequence: Mn²⁺, 11.5; Ca²⁺, 8.5; Ni²⁺, 7.5; Co²⁺, 6.5; Mg²⁺, 6.0; Ba²⁺, 5.5 and Sr²⁺, 5.0.     

Adsorption of divalent cations to bilayer membranes containing phosphatidylserine

The Stern equation, a combination of the Langmuir adsorption isotherm, the Boltzmann relation, and the Grahame equation from the theory of the diffuse double layer, provides a simple theoretical framework for describing the adsorption of charged molecules to surfaces. The ability of this equation to describe the adsorption of divalent cations to membranes containing brain phosphatidylserine (PS) was tested in the following manner. Charge reversal measurements were first made to determine the intrinsic 1:1 association constants of the divalent cations with the anionic PS molecules: when the net charge of a PS vesicle is zero one-half of the available sites are occupied by divalent cations. The intrinsic association constant, therefore, is equal to the reciprocal of the divalent cation concentration at which the mobility of a PS vesicle reverses sign. The Stern equation with this association constant is capable of accurately describing both the zeta potential data obtained with PS vesicles at other concentrations of the divalent cations and the data obtained with with vesicles formed from mixtures of PS and zwitterionic phospholipids. Independent measurements of the number of ions adsorbed to sonicated PS vesicles were made with a calcium-sensitive electrode. The results agreed with the zeta potential results obtained with multilamellar vesicles. When membranes are formed at 20 degrees C in 0.1 M NaCl, the intrinsic 1:1 association constants of Ni, Co, Mn, Ba, Sr, Ca, and Mg with PS are 40, 28, 25, 20, 14, 12, and 8 M-1, respectively.     

Images of divalent cations in unstained symmetric and asymmetric lipid bilayers

Divalent cations have been microscopically visualized in association with simple lipid bilayers. Symmetric and asymmetric oriented bilayers were constructed from fatty acid monolayers and were cut in thin transverse sections for examination by bright field electron microscopy in the absence of stains, fixatives or embedding materials. It has been found that bilayers formed of lipid molecules having alkaline earth head groups exhibit natural electron contrast. The intrinsic image has been linked to local variations in the bilayer absolute electron density profile determined by X-ray diffraction analysis of the same specimens (McIntosh, T.J., Waldbillig, R. C. and Robertson J. D. (1976) Biochim. Biophys. Acta 448, 15–33). By combining the microscopic, chemical and X-ray evidence it has been estimated that local increments of about 1 g/cm³ can produce detectable electron contrast in 500 Å transverse sections of bilayers.     

Lipopolysaccharide bilayer structure: effect of chemotype, core mutations, divalent cations, and temperature.

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a protective and permeability barrier. X-ray diffraction analysis of osmotically stressed LPS multilayers was used to determine the structure and interactive properties of LPSs from strains containing the minimum number of sugars necessary for bacterial survival (Re chemotype) to the maximum number of sugars found in rough bacteria (Ra chemotype). At 20 degrees C in the absence of divalent cations, LPS suspensions gave a sharp wide-angle reflection at 4.23 A and a broad low-angle band centered at 50-68 A depending on the chemotype, indicating the presence of gel phase bilayers separated by large fluid spaces. As osmotic pressure was applied, the apposing bilayers were squeezed together and lamellar diffraction at 6 A resolution was obtained. At low applied pressures (<10(6) dyn/cm2), the total repulsive pressure between bilayers could be explained by electrostatic double layer theory. At higher applied pressures, there was a sharp upward break in each pressure-distance relation, indicating the presence of a hydrophilic steric barrier whose range depended strongly on the LPS chemotype. The positions of these upward breaks, along with electron density profiles, showed that the sugar core width systematically increased from 10 A for the Re chemotype to 27 A for the Ra chemotype. In excess buffer, the addition of divalent cations brought the bilayers into steric contact. Electron density profiles were used to determine the locations of cation binding sites and polar substituents on the LPS oligosaccharide core. The area per hydrocarbon chain was approximately 26 A2 in liquid-crystalline LPS bilayers, an indication of an acyl chain packing that is much tighter than that found in bilayers composed of typical membrane lipids. This unusually tight packing could be a critical factor in the permeability barrier provided by LPS.     

Effect of the mitochondrial ionophore for divalent cations on bilayer phospholipid membranes]

It has been shown that ionophore of bivalent cations (IBC) isolated from fatless, subjected to partial triptic hydrolysis cattle heart or liver mitochondria decreases BPM resistance inducing Ca2+ conductivity. Ions of lanthane in micromolar concentrations decrease calcium conductivity induced with IBC. When ten-fold gradient in Ca2+ was created on BPM the intitiation of the membrane potential fo 9-11 mV was observed. The role fo IBC and water soluble factors binding Ca2+ with high affinity, in the mitochrondial mechanism of Ca2+ translocation is discussed.     

Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.     

Adsorption of uni and divalent cations to planar bilayer membranes containing sulphatides or phosphatidylserine

It has been postulated that sulphatides may be the K+ binding site of the sodium pump. In order to test this hypothesis we studied the binding of K+ to bilayer membranes containing sulphatides or phosphatidylserine. The adsorption constants of Na+, K+ and Ca2+ to planar bilayers containing these acidic lipids were determined from changes in the electrostatic potential at the membrane surface. Our results indicate that univalent cations adsorb weakly to both lipids and Ca2+ binds more strongly. The sequence of ion binding was Ca2+ greater than Na+ greater than K+. These results indicate that K+ does not bind specifically to sulphatides or phosphatidylserine and rule out the proposal that sulphatides by themselves provide the K+ binding site of the sodium pump.     

The effect of ergosterol on dipalmitoylphosphatidylcholine bilayers: a deuterium NMR and calorimetric study

We have studied the effect of ergosterol, an important component of fungal plasma membranes, on the physical properties of dipalmitoylphosphatidylcholine (DPPC) multibilayers using deuterium nuclear magnetic resonance ((2)H NMR) and differential scanning calorimetry (DSC). For the (2)H NMR experiments the sn-1 chain of DPPC was perdeuterated and NMR spectra were taken as a function of temperature and ergosterol concentration. The phase diagram, constructed from the NMR spectra and the DSC thermograms, exhibits both solid-ordered (so) + liquid-ordered (lo) and liquid-disordered (ld) + lo phase coexistence regions with a clear three-phase line. This is the first demonstration that lo domains exist in liquid crystalline membranes containing ergosterol. The domain sizes in the ld+lo phase coexistence region were estimated by analyzing the exchange of labeled DPPC between the two regions, and depend on ergosterol concentration. The DPPC-ergosterol phase diagram is similar to that of the DPPC-cholesterol multibilayer system except that the so+lo and ld+lo phase coexistence regions are considerably broader.     

Calorimetric,volumetric and structural studies of the interaction between chlorogenic acid and dipalmitoylphosphatidylcholine bilayers

Chlorogenic acid (CGA) is the main component of coffee and an antioxidant. CGA has been reported to bear various good health effects. At the same time, it has been found that the addition of CGA induces an undesirable deformation of red blood cells. This fact suggests that CGA may bind to the proteins or/and membrane lipids of red blood cells. This study aimed to examine how CGA binds the bilayers of phosphatidylcholine (PC), one of red blood cells' primary lipids. To this end, we investigated the effect of CGA on the phase behavior and the structure of dipalmitoyl-PC (DPPC) bilayers in the form of multi-lamellar vesicles. Calorimetry and dilatometry measurements showed that the DPPC chain melting transition cooperativity decreases as increasing CGA concentrations. In addition, X-ray diffraction results showed that the lamellar repeat periodicity becomes disordered, and the periodicity disappears completely at high CGA concentrations. Together with these findings, it can be inferred that the CGA molecules do not penetrate inside the DPPC bilayers but bind to their surface in a negatively charged form.     

Effect of divalent cations on the assembly of neutral and charged phospholipid bilayers in patch-recording pipettes.

Monolayers of the negatively charged phospholipid phosphatidylserine (PS) and of the amphoteric phospholipid dioleoylphosphatidylethanolamine (DOPE) were used to assemble bilayers at the tip of patch-recording pipettes. PS bilayers, with seal resistances in the range of gigaohmns (gigaseals), could only be generated when millimolar concentration of divalent cations, Ca++, Mg++, or Ba++ were present in the pipette and bath solutions. In contrast, gigaseals of DOPE were independent of divalent ion concentration in the pH range where DOPE is predominantly neutral (pH 6.5) or positively charged (pH 1.5). At pH 10.0, when most DOPE molecules bear a net negative charge, gigaseals became divalent cation dependent, in a manner quantitatively similar to that of PS at neutral pH. The results indicate that divalent cations play an important role in stabilizing gigaseals of negatively charged lipid but are of no consequence in neutral or positively charged seals.     

Effects of butanol isomers on dipalmitoylphosphatidylcholine bilayer membranes

Differential scanning calorimetry and (31)P-NMR were used to study the effects of butanol isomers on the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers. The threshold concentration for the onset of interdigitation for each isomer was determined by the disappearance of the pretransition and the onset of a large hysteresis between the heating and cooling scans of the gel-to-liquid main transition. The threshold concentration was found to correlate with increased solubility of the isomers in the aqueous phase, led by tert-butanol. However, as the solution concentration of tert-butanol increased, there was an abrupt shrinking of the hysteresis, initially with well-resolved shoulder peaks indicating mixed phases. The eventual disappearance of the shoulder peaks was correlated with a breakdown of the multilamellar structure identified using (31)P-NMR.     

Permeation and interaction of divalent cations in calcium channels of snail neurons

We have studied the current-carrying ability and blocking action of various divalent cations in the Ca channel of Lymnaea stagnalis neurons. Changing the concentration or species of the permeant divalent cation shifts the voltage dependence of activation of the Ca channel current in a manner that is consistent with the action of the divalent cation on an external surface potential. Increasing the concentration of the permeant cation from 1 to 30 mM produces a twofold increase in the maximum Ca current and a fourfold increase in the maximum Ba current; the maximum Ba current is twice the size of the maximum Ca current for 10 mM bulk concentration. Correcting for the changing surface potential seen by the gating mechanism, the current-concentration relation is almost linear for Ba2+, and shows only moderate saturation for Ca2+; also, Ca2+, Ba2+, and Sr2+ are found to pass through the channel almost equally well. These conclusions are obtained for either of two assumptions: that the mouth of the channel sees (a) all or (b) none of the surface potential seen by the gating mechanism. Cd2+ blocks Lymnaea and Helix Ca channels at concentrations 200 times smaller than those required for Co2+ or Ni2+. Ca2+ competes with Cd2+ for the blocking site; Ba2+ binds less strongly than Ca2+ to this site. Mixtures of Ca2+ and Ba2+ produce an anomalous mole fraction effect on the Ca channel current. After correction for the changing surface potential (using either assumption), the anomalous mole fraction effect is even more prominent, which suggests that Ba2+ blocks Ca current more than Ca2+ blocks Ba current.     

Fusogenic capacities of divalent cations and effect of liposome size

The initial kinetics of divalent cation (Ca2+, Ba2+, Sr2+) induced fusion of phosphatidylserine (PS) liposomes, LUV, is examined to obtain the fusion rate constant, f11, for two apposed liposomes as a function of bound divalent cation. The aggregation of dimers is rendered very rapid by having Mg2+ in the electrolyte, so that their subsequent fusion is rate limiting to the overall reaction. In this way the fusion kinetics are observed directly. The bound Mg2+, which by itself is unable to induce the PS LUV to fuse, is shown to affect only the aggregation kinetics when the other divalent cations are present. There is a threshold amount of bound divalent cation below which the fusion rate constant f11 is small and above which it rapidly increases with bound divalent cation. These threshold amounts increase in the sequence Ca2+ less than Ba2+ less than Sr2+, which is the same as found previously for sonicated PS liposomes, SUV. While Mg2+ cannot induce fusion of the LUV and much more bound Sr2+ is required to reach the fusion threshold, for Ca2+ and Ba2+ the threshold is the same for PS SUV and LUV. The fusion rate constant for PS liposomes clearly depends upon the amount and identity of bound divalent cation and the size of the liposomes. However, for Ca2+ and Ba2+, this size dependence manifests itself only in the rate of increase of f11 with bound divalent cation, rather than in any greater intrinsic instability of the PS SUV. The destabilization of PS LUV by Mn2+ and Ni2+ is shown to be qualitatively distinct from that induced by the alkaline earth metals.