Effect of the fungicides tributyltin acetate and tributyltin chloride on multilamellar liposomes: fluorescence studies.

The influence of tri-n-butyltin acetate (TBTA) and tri-n-butyltin chloride (TBTC) on the physico-chemical state of charged and neutral phospholipids was investigated using multilamellar liposomes. The thermal dependence of steady state fluorescence polarization of DPH and its charged derivative TMA-DPH was recorded. The two fungicides lowered DPPC phase transition temperature and broadened the temperature range of the transition in different ways. The effects were concentration-dependent. The results show that TBTC interacts more effectively with DPPC model membranes rather than TBTA. Moreover, TBTC broadens and shifts the main phase transition (Tm) more effectively in DPPC rather than in DMPC liposomes. Below Tm, TBTC decreases fluorescence polarization (P) in all phospholipids used. Above Tm P is almost constant in phospholipids with saturated acyl chains, except for DMPG. In fact, an increase of P is detectable in this lipid as in PLs with unsaturated acyl chains. It is suggested that the effects of TBT on liposomal membranes are dependent on the anion moiety and phospholipids characteristics.     

A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol

The interaction of aqueous phospholipid dispersions of negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG) with the divalent cations Mg(2+), Ca(2+) and Sr(2+) at equimolar ratios in 100 mM NaCl at pH 7 was investigated by Fourier transform infrared spectroscopy. The binding of the three cations induces a crystalline-like gel phase with highly ordered and rigid all-trans acyl chains. These features are observed after storage below room temperature for 24 h. When the gel phase is heated after prolonged incubation at low temperature phase transitions into the liquid crystalline phase are observed at 58 degrees C for the DMPG:Sr(2+), 65 degrees C for the DMPG:Mg(2+), and 80 degrees C for the DMPG:Ca(2+) complex. By subsequent cooling from temperatures above T(m) these complexes retain the features of a liquid crystalline phase with disordered acyl chains until a metastable gel phase is formed at temperatures between 38 and 32 degrees C. This phase is characterized by predominantly all-trans acyl chains, arranged in a loosely packed hexagonal or distorted hexagonal subcell lattice. Reheating the DMPG:Sr(2+) samples after a storage time of 2 h at 4 degrees C results in the transition of the metastable gel to the liquid crystalline phase at 35 degrees C. This phase transition into the liquid crystalline state at 35 degrees C is also observed for the Mg(2+) complex. However, for DMPG:Mg(2+) at higher temperatures, a partial recrystallization of the acyl chains occurs and the high temperature phase transition at 65 degrees C is also detected. In contrast, DMPG:Ca(2+) exhibits only the phase transition at 80 degrees C from the crystalline gel into the fluid state upon reheating. Below 20 degrees C, the rate of conversion from the metastable gel to a thermodynamically stable, crystalline-like gel phase decreases in the order Ca(2+)&z. Gt;Mg(2+)>Sr(2+). This conversion into the crystalline gel phase is accompanied by a complete dehydration of the phosphate groups in DMPG:Mg(2+) and by a reorientation of the polar lipid head groups in DMPG:Ca(2+) and in DMPG:Sr(2+). The primary binding sites of the cations are the PO(2)(-) groups of the phosphodiester moiety. Our infrared spectroscopic results suggest a deep penetration of the divalent cations into the polar head group region of DMPG bilayers, whereby the ester carbonyl groups, located in the interfacial region of the bilayers, are indirectly affected by strong hydrogen bonding of immobilized water molecules. In the liquid crystalline phase, the interaction of all three cations with DMPG is weak, but still observable in the infrared spectra of the DMPG:Ca(2+) complex by a slight ordering effect induced in the acyl chains, when compared to pure DMPG liposomes.     

A comparison of the interaction of glucagon, human parathyroid hormone-(1-34)-peptide and calcitonin with dimyristoylphosphatidylglycerol and with dimyristoylphosphatidylcholine

The interaction of glucagon, human parathyroid hormone-(1-34)-peptide and salmon calcitonin with dimyristoylphosphatidylglycerol (DMPG) and with dimyristoylphosphatidylcholine (DMPC) was studied as a function of pH and temperature. The effect of lipid on the secondary structure of the peptide was assessed by circular dichroism and the effect of the peptide on the phase transition properties of the lipid was studied using differential scanning calorimetry. Some peptides interact more strongly with anionic than with zwitterionic phospholipids. This does not require an overall positive charge on the peptide. Increased thermal stability is observed in complexes formed between cationic peptides and anionic lipids. Particularly marked effects of glucagon and human parathyroid hormone-(1-34)-peptide on the phase transition properties of DMPG at pH 5 have been observed. The transition temperature is raised over 10 degrees C at a lipid/peptide molar ratio of less than 30:1 and the transition enthalpy is increased over 2-fold. These effects do not occur with any basic peptide and were not observed with metorphinamide, molluscan cardioexcitatory neuropeptide or myelin basic protein. The results demonstrate that certain peptides can affect the phase transition properties of lipids in a manner similar to divalent cations. The overall hydrophobicities of these peptides can be evaluated by their partitioning between aqueous and organic solvents. None of the above three peptide hormones partition into the organic phase. However, a closely related peptide, human calcitonin, does exhibit substantial partitioning into the organic phase. Nevertheless, human calcitonin has a weaker interaction with both DMPC and DMPG than does salmon calcitonin. The effects of human calcitonin on the phase transition of DMPC are qualitatively different from those of salmon calcitonin in that the human form more readily eliminates the pretransition but causes less change in the main transition. Like overall charge, overall hydrophobicity is not an overwhelming factor in determining the ability of peptides to interact with phospholipids but rather more specific interactions are required for strong complexes to form.     

Formation of a new stable phase of phosphatidylglycerols.

Dilauroyl and dimyristoylphosphatidylglycerol (DMPG) form a more stable gel state when aqueous suspensions are incubated several days at low temperature (0-2 degrees C), pH 7.4 with 0.15 M NaCl. This gel state is characterized by a higher transition temperature and a higher transition enthalpy. The geometry of this gel state is distinguishable from the metastable gel state that forms rapidly upon hydration on the basis of its x-ray diffraction pattern. Infrared spectra in the CH2 scissoring region indicate that the stable gel phase of DMPG is also characterized by reduced reorientational fluctuations of acyl chains and increased interchain interactions. Analysis of vibrational bands due to ester carbonyl groups of DMPG suggests that the transition to a new gel phase is initiated by changes in the interfacial and/or headgroup region of the bilayer, most likely via formation of interlipid hydrogen bonds. The melting of the stable gel phase of DMPG is accompanied by a gross morphological change resulting in vesiculation.     

Mechanism of membrane binding by the bovine seminal plasma protein,PDC-109: a surface plasmon resonance study

PDC-109, the major protein of bovine seminal plasma, binds to sperm plasma membranes upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. The binding process is mediated primarily by the specific interaction of PDC-109 with choline-containing phospholipids. In the present study the kinetics and mechanism of the interaction of PDC-109 with phospholipid membranes were investigated by the surface plasmon resonance technique. Binding of PDC-109 to different phospholipid membranes containing 20% cholesterol (wt/wt) indicated that binding occurs by a single-step mechanism. The association rate constant (k(1)) for the binding of PDC-109 to dimyristoylphosphatidylcholine (DMPC) membranes containing cholesterol was estimated to be 5.7 x 10(5) M(-1) s(-1) at 20 degrees C, while the values of k(1) estimated at the same temperature for the binding to membranes of negatively charged phospholipids such as dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA) containing 20% cholesterol (wt/wt) were at least three orders of magnitude lower. The dissociation rate constant (k(-1)) for the DMPC/PDC-109 system was found to be 2.7 x 10(-2) s(-1) whereas the k(-1) values obtained with DMPG and DMPA was about three to four times higher. From the kinetic data, the association constant for the binding of PDC-109 to DMPC was estimated as 2.1 x 10(7) M(-1). The association constants for different phospholipids investigated decrease in the order: DMPC > DMPG > DMPA > DMPE. Thus the higher affinity of PDC-109 for choline phospholipids is reflected in a faster association rate constant and a slower dissociation rate constant for DMPC as compared to the other phospholipids. Binding of PDC-109 to dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine, which are also zwitterionic, was found to be very weak, clearly indicating that the charge on the lipid headgroup is not the determining factor for the binding. Analysis of the activation parameters indicates that the interaction of PDC-109 with DMPC membranes is favored by a strong entropic contribution, whereas negative entropic contribution is primarily responsible for the rather weak interaction of this protein with DMPA and DMPG.     

Raman spectroscopic studies of dimyristoylphosphatidic acid and its interactions with ferricytochrome c in cationic binary and ternary lipid-protein complexes.

The vibrational Raman spectra of both pure 1-alpha-dimyristoylphosphatidic acid (DMPA) liposomes and DMPA multilayers reconstituted with ferricytochrome c at pH 7 and pH 4, with either sodium or calcium as the cation, are reported as a function of temperature. Multilayers composed of a 1:1 mol ratio DMPA and dimyristoylphosphatidylcholine with perdeuterated acyl chains (DMPC-d54) have also been reconstituted with approximately 10(-4) M ferricytochrome c for Raman spectroscopic observation. Total integrated band intensities and relative peak height intensity ratios, two spectral Raman scattering parameters used to characterize bilayer properties, are sensitive to the presence of both ferricytochrome c and the cation in the reconstituted liposomes. Temperature profiles, derived from the various Raman intensity parameters for the 3,100-2,800 cm-1 lipid acyl chain C-H stretching mode region specifically reflect bilayer perturbations due to the interactions of ferricytochrome c. At pH 4 the calcium DMPA multilamellar gel to liquid crystalline phase transition temperatures Tm, defined by either the C-H stretching mode I2850/I2880 and I2935/I2880 peak height intensity ratios, are 58.5 +/- 0.5 degrees C and 60.0 +/- 0.3 degrees C, respectively. This difference in Tm's resolves the phase transition process into first an expansion of the lipid lattice and then a melting of the lipid acyl chains. At pH 7 the calcium DMPA liposomes show no distinct phase transition characteristics below 75 degrees C. For sodium DMPA liposomes reconstituted with ferricytochrome c at either pH 4.0 or pH 7.0, spontaneous Raman spectra show altered lipid structures at temperatures above 40 degrees C. Resonance Raman spectra indicate that ferricytochrome c reconstituted in either calcium or sodium DMPA liposomes changes irreversibly above Tm. For either the binary lipid or ternary lipid-protein systems reconstituted with DMPC-d54, linewidth parameters of the DMPC-d54 acyl chain CD2 symmetric stretching modes at 2,103 cm-1 provide a sensitive measure of the conformational and dynamic properties of the perdeuterated lipid component, while the 3,000 cm-1 C-H spectral region reflects the bilayer characteristics of the DMPA species in the complex. Although calcium clearly induces a lateral phase separation in the DMPA/DMPC-d54 system at pH 7.5 (Kouaouci, R., J.R. Silvius, I. Grah, and M. Pezolet. 1985. Biochemistry. 24:7132-7140), no distinct lateral segregation of the lipid components is observed in the mixed DMPA/DMPC-d54 lipid system in the presence of either ferricytochrome c or the sodium and calcium cations at pH 4.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

DMPG gel-fluid thermal transition monitored by a phospholipid spin labeled at the acyl chain end

Low ionic strength aqueous dispersion of dimyristoyl phosphatidylglycerol (DMPG) presents a rather peculiar gel-fluid thermal transition behavior. The lipid main phase transition occurs over a large temperature interval (ca. 17 degrees C), along which several calorimetric peaks are observed. Using lipids spin labeled at the acyl chain end, a two-peak electron spin resonance (ESR) spectrum is observed along that temperature transition region (named intermediate phase), at three different microwave frequencies: L-, X- and Q-bands. The intermediate phase ESR spectra are analyzed, and shown to be most likely due to spin labels probing two distinct types of lipid organization in the DMPG bilayer. Based on the ESR spectra parameters, a model for the DMPG intermediate phase is proposed, where rather fluid and hydrated domains, possibly high curvature regions, coexist with patches that are more rigid and hydrophobic.     

Characterization by infrared spectroscopy of the interaction of a cardiotoxin with phosphatidic acid and with binary mixtures of phosphatidic acid and phosphatidylcholine.

The effect of cardiotoxin IIa from Naja mossambica mossambica, a small basic protein extracted from snake venom, on dimyristoylphosphatidic acid (DMPA) and on equimolar mixtures of DMPA and dimyristoylphosphatidylcholine (DMPC) has been studied by Fourier transform infrared spectroscopy. The interaction of cardiotoxin with DMPA dispersions decreases both the cooperativity of the phase transition of the lipid and the molecular order of the lipid acyl chains in the gel phase. This effect increases with the proportion of the toxin in the complexes and leads to the total abolition of the phase transition of DMPA at a lipid-to-protein molar ratio of 5. Small-angle X-ray results demonstrate that the structure of the lipid-protein complexes is poorly ordered and gives rise to broad diffusion peaks rather than to well-resolved diffraction patterns. Infrared spectra of oriented cardiotoxin-DMPA films show that the protein is not homogeneously oriented with respect to the bilayer surface. The destabilization of the gel-phase structure of DMPA by cardiotoxin also results in a deeper water penetration in the interfacial region of the lipid since more carbonyl ester groups appear to be hydrogen bonded in the presence of the toxin. The infrared results on the phosphate group vibrations also indicate clearly that the basic residues of cardiotoxin interact strongly with the phosphate group of DMPA that becomes partly ionized at a pH as low as 6.5. The results obtained on the interaction of cardiotoxin with an equimolar mixture of DMPA and DMPC clearly demonstrate the ability of this toxin to induce lateral phase separation in this mixture with one phase containing DMPA-rich domains perturbed by cardiotoxin while the second phase is composed of regions enriched in DMPC. Comparison of the results of the current study with those obtained on other basic proteins and polypeptides suggests that charge-induced phase separation occurs only when the charge density on certain regions of the protein structure is high enough to lead to efficient electrostatic interactions with anionic phospholipids. This condition occurs only when the conformation of the protein or polypeptide is well-ordered at the lipid interface.     

New approaches to the simulation of heat-capacity curves and phase diagrams of pseudobinary phospholipid mixtures.

A simulation program using least-squares minimization was developed to calculate and fit heat capacity (cp) curves to experimental thermograms of dilute aqueous dispersions of phospholipid mixtures determined by high-sensitivity differential scanning calorimetry. We analyzed cp curves and phase diagrams of the pseudobinary aqueous lipid systems 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol/ 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DMPG/DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid/1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DMPA/DPPC) at pH 7. The simulation of the cp curves is based on regular solution theory using two nonideality parameters rho g and rho l for symmetric nonideal mixing in the gel and the liquid-crystalline phases. The broadening of the cp curves owing to limited cooperativity is incorporated into the simulation by convolution of the cp curves calculated for infinite cooperativity with a broadening function derived from a simple two-state transition model with the cooperative unit size n = delta HVH/delta Hcal as an adjustable parameter. The nonideality parameters and the cooperative unit size turn out to be functions of composition. In a second step, phase diagrams were calculated and fitted to the experimental data by use of regular solution theory with four different model assumptions. The best fits were obtained with a four-parameter model based on nonsymmetric, nonideal mixing in both phases. The simulations of the phase diagrams show that the absolute values of the nonideality parameters can be changed in a certain range without large effects on the shape of the phase diagram as long as the difference of the nonideality parameters for rho g for the gel and rho l for the liquid-crystalline phase remains constant. The miscibility in DMPG/DPPC and DMPA/DPPC mixtures differs remarkably because, for DMPG/DPPC, delta rho = rho l -rho g is negative, whereas for DMPA/DPPC this difference is positive. For DMPA/DPPC, this difference is interpreted as being caused by a negative rho g value, indicating complex formation of unlike molecules in the gel phase.     

Apocytochrome c interaction with phospholipid membranes studied by Fourier-transform infrared spectroscopy.

Apocytochrome c, the heme-free precursor of cytochrome c, has been used extensively as a model to study molecular aspects of posttranslational translocation of proteins across membranes. In this report, we have used Fourier-transform infrared spectroscopy to gain further insight into the mechanism of apocytochrome c interaction with membrane phospholipids. Association of apocytochrome c with model membranes containing the acidic lipid dimyristoylphosphatidylglycerol (DMPG) as a single component results in a drastic perturbation of phospholipid structure, at the level of both the acyl chains and the interfacial carbonyl groups. However, in a binary mixture of DMPG with acyl chain perdeuterated dimyristoylphosphatidylcholine (DMPC-d54), the perturbing effect of the protein on the acidic phospholipid is greatly attenuated. In such a membrane with mixed lipids, the physical properties of the DMPG and DMPC components are affected in a similar fashion, indicating that apocytochrome c does not induce any significant segregation or lateral-phase separation of acidic and zwitterionic lipids. Analysis of the apocytochrome c spectrum in the amide I region reveals that binding to phospholipids causes considerable changes in the secondary structure of the protein, the final conformation of which depends on the lipid to protein ratio. In the presence of a large excess of DMPG, apocytochrome c undergoes a transition from an essentially unordered conformation in solution to an alpha-helical structure. However, in complexes of lower lipid to protein ratios (less than or equal to approximately 40:1), infrared spectra are indicative of an extended, intermolecularly hydrogen-bonded beta-sheet structure. The latter is suggestive of an extensive aggregation of the membrane-associated protein.     

The structure of anhydrous and hydrated dimyristoylphosphatidyl glycerol: a pressure tuning infrared spectroscopic study.

The addition of water to anhydrous phospholipids has profound effects on the intermolecular interactions and packing order of phospholipid molecules. These changes, as well as alterations induced by hydration in the orientation of the acyl chains, can be qualitatively assessed using high pressure infrared spectroscopy. Application of this technique to dimyristoylphosphatidyl glycerol (DMPG) showed that hydration causes major changes in (i) the orientation of the carboxyl groups in relation to the glycerol backbone; (ii) the degree of orientational disorder of the acyl chains and (iii) the proximity of neighbouring DMPG molecules. In addition, the frequency of the carbonyl group is shown to be strongly affected not only by changes in the hydrogen bond network, but also by subtle environmental perturbations induced by the conformation of nearby structural units.     

Effect of membrane fluidity and fatty acid composition on the prothrombin-converting activity of phospholipid vesicles.

Vesicles composed of phospholipids with different fatty acyl side chains have been utilized to examine the importance of the nonpolar membrane region for the prothrombin-converting activity of procoagulant phospholipid vesicles. Membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with unsaturated fatty acyl side chains were more active in prothrombin activation than membranes composed of phospholipids with saturated fatty acyl chains. This phenomenon was observed above the phase transition temperature, i.e., on membranes in the liquid-crystalline state. The prothrombin-converting activity of saturated phospholipids approached the activity of unsaturated phospholipids at high factor Va concentrations, which is indicative for a less favorable equilibrium constant for prothrombinase assembly on membrane surfaces composed of saturated phospholipids. The difference between saturated and unsaturated phospholipids was annulled on membranes with high mole percentages of PS. This may result from a compensating contribution of electrostatic forces to the binding equilibria involved in prothrombinase assembly. Additional effects on the prothrombin-converting activity were observed when membranes containing saturated phospholipids were studied below their phase transition temperature. In agreement with Higgins et al. [(1985) J. Biol. Chem. 260, 3604-3612], we found that the time required for the assembly of prothrombinase from membrane-bound factors Xa and Va is considerably prolonged on solid membranes. However, we also observed an effect of membrane fluidity on the steady-state rate of prothrombin activation. Kinetic experiments at saturating factor Va concentrations showed that the transition from the liquid-crystalline to the gel state caused a more than 9-fold decrease of the kcat of prothrombin activation without affecting the Km for prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of lipid structure on peptide-lipid interactions. Complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon calcitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin- DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol . There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

NMR study of the interactions of polymyxin B, gramicidin S, and valinomycin with dimyristoyllecithin bilayers

The interactions of three polypeptide antibiotics (polymyxin B, gramicidin S, and valinomycin) with artificial lecithin membranes were studied by nuclear magnetic resonance (NMR). Combination of 31P and 2H NMR allowed observation of perturbations of the bilayer membrane structure induced by each of the antibiotics in the regions of the polar headgroups and acyl side chains of the phospholipids. The comparative study of the effects of these membrane-active antibiotics and the lipid bilayer structure demonstrated distinct types of antibiotic-membrane interactions in each case. Thus, the results showed the absence of interaction of polymyxin B with the dimyristoyllecithin membranes. In contrast, gramicidin S exhibited strong interaction with the lipid above the gel to liquid-crystalline phase transition temperature: disordering of the acyl side chains was evident. Increasing the concentration of gramicidin S led to disintegration of the bilayer membrane structure. At a molar ratio of 1:16 of gramicidin S to lecithin, the results are consistent with coexistence of gel and liquid-crystalline phases of the phospholipids near the phase transition temperature. Valinomycin decreased the phase transition temperature of the lipids and increased the order parameters of the lipid side chains. Such behavior is consistent with penetration of the valinomycin molecule into the interior of the lipid bilayers.     

The influence of Ca2+ on the turbidity of DPPC-DMPA vesicles within the temperature range of the phase transition

The influence of the addition of Ca2+ on the phase behaviour of vesicles, composed of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidic acid (DMPA) in a ratio of 4 to 1, has been investigated by means of turbidity measurements. As expected one single phase transition for the mixed phospholipids was observed in the absence of Ca2+. Passing through the temperature range of this transition after the addition of Ca2+, conditions appeared to favor fusion of the vesicles. A possible reason for this is that during the transition Ca2+ may permeate through the vesicle membranes and gain access to the inside DMPA binding sites. Therefore it is not unambiguously possible to determine phase transition temperatures from the turbidity changes that occur under these conditions. However, when within the temperature range of the phase transition of the mixed phospholipids the influence of Ca2+ addition to the vesicles was recorded isothermally, at each temperature separately, the final plot of turbidity versus temperature turned out to be far less confused by fusion events and adopted the form of two separate phase transitions. The temperatures at which these two transitions occur closely resemble the phase transition temperatures that may be observed in the absence of Ca2+ for DMPA and DPPC alone, 39 degrees C and 43 degrees C respectively. The results of this study suggest that when Ca2+ has only access to the outside of the vesicle membranes it may segregate the neutral and the acidic phospholipids into separate domains, both domains adopting their proper phase condition at the actual temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular Dynamics Simulations of Mixed Acidic/Zwitterionic Phospholipid Bilayers

Anionic lipids are key components in the cell membranes. Many cell-regulatory and signaling mechanisms depend upon a complicated interplay between them and membrane-bound proteins. Phospholipid bilayers are commonly used as model systems in experimental or theoretical studies to gain insight into the structure and dynamics of biological membranes. We report here 200-ns-long MD simulations of pure (DMPC and DMPG) and mixed equimolar (DMPC/DMPG, DMPC/DMPS, and DMPC/DMPA) bilayers that each contain 256 lipids. The intra- and intermolecular interaction patterns in pure and mixed bilayers are analyzed and compared. The effect of monovalent ions (Na⁺) on the formation of salt-bridges is investigated. In particular, the number of Na⁺-mediated clusters in the presence of DMPS is higher than with DMPG and DMPA. We observe a preferential clustering of DMPS (and to some extent DMPA) lipids together rather than with DMPC molecules, which can explain the phase separation observed experimentally for DMPC/DMPS and DMPC/DMPA bilayers.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing dl-methyl anteisobranched fatty acids. 1. Differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of aqueous dispersions of nine dl-methyl branched anteisoacylphosphatidylcholines was studied by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy. The calorimetric studies demonstrate that these compounds all exhibit a complex phase behavior, consisting of at least two minor, low-enthalpy, gel-state transitions which occur at temperatures just prior to the onset of the gel/liquid-crystalline phase transition. In addition, at still lower temperatures, anteisobranched phosphatidylcholines containing fatty acyl chains with an odd number of carbon atoms show a major, higher enthalpy, gel-state transition, which was assigned to a conversion from a condensed to a more loosely packed gel phase. No such transition was observed for the even-numbered compounds in aqueous dispersion, but when dispersed in aqueous ethylene glycol, a major gel-state transition is clearly discernible for two of the even-numbered phospholipids. The major gel-state transition exhibits heating and cooling hysteresis and is fairly sensitive to the composition of the bulk aqueous phase. 31P NMR spectroscopic studies indicate that the major gel-state transition is accompanied by a considerable change in the mobility of the phosphate head group and that, at temperatures just prior to the onset of the gel/liquid-crystalline phase transition, the mobility of the phosphate head group is comparable to that normally exhibited by the liquid-crystalline state of most other phospholipids. The temperatures at which the gel/liquid-crystalline phase transition occurs and the enthalpy change associated with this process are considerably lower than those of the saturated n-acyl-PC's of comparable acyl chain length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-induced lateral phase separations in phosphatidylcholine-phosphatidic acid mixtures. A Raman spectroscopic study

The effects of calcium ions on mixed membranes of dimyristoylphosphatidic acid (DMPA) and dimyristoylphosphatidylcholine (DMPC) with either the PA or the PC component deuterated have been studied by Raman spectroscopy. The spectra of the pure components show that the acyl chains of hydrated DMPA bilayers are less tightly packed and have more trans bonds than those of DMPC. This behavior appears to be due to the particular arrangement of the polar head groups of DMPA for which the glycerol chain is oriented parallel to the bilayer surface. In agreement with the calorimetrically determined phase diagram [Graham, I., Gagné, J., & Silvius, J. R. (1985) Biochemistry (preceding paper in this issue)], the Raman results show that, in the absence of calcium, DMPA and DMPC are completely miscible at an equimolar ratio but undergo extensive phase separation in the presence of excess calcium. DMPC in phase-separated DMPC-DMPA (Ca2+) mixtures has a conformation that is very similar to that of pure DMPC bilayers, but it is packed more tightly since, depending on the temperature, it is at least partly incorporated into either a solid solution in DMPA or a DMPA-Ca2+-rich "cochleate" phase. This latter shows the same characteristics as the cochleate phase of pure DMPA-Ca2+ which is highly ordered and does not give rise to a thermotropic transition between 5 and 100 degrees C. However, the cochleate phase in DMPA (Ca2+)-DMPC mixtures contains some 20 mol % of DMPC trapped in small domains. These clusters do not melt cooperatively but become as fluid as pure DMPC at 50 degrees C.     

The oxysterol 3β-hydroxy-5-oxo-5,6-secocholestan-6-al changes the phase behavior and structure of phosphatidylethanolamine–phosphatidylcholine mixtures

An oxidized form of cholesterol, atheronal, is a form found in vivo that has been associated with human pathologies. We have studied mixtures of this oxidized sterol with the phospholipids phosphatidylethanolamine and phosphatidylcholine. We used phospholipids either with palmitoyl and oleoyl acyl chains on the C1 and C2 carbon atoms of glycerol or with both acyl chains being palmitoleoyl. We also compared the effects of atheronal on the curvature properties of these lipids with the action of cholesterol. We studied the bilayer to hexagonal phase transition temperature of mixtures of these lipids using differential scanning calorimetry as well as the dimensions of the hexagonal phase cylinders using X-ray diffraction. Disordering of the lamellar phase was also qualitatively assessed by the loss of sharp diffraction peaks. Our results demonstrate that the modulation of membrane curvature in these systems depends not only on the nature of the sterol, but also on the acyl chain composition of the phospholipids used. In addition, some of the effects of atheronal could be ascribed to reaction of the aldehyde and ketone groups of this oxidized sterol with the amino group of phosphatidylethanolamine.     

Interaction of alpha-melanocyte stimulating hormone with binary phospholipid membranes: structural changes and relevance of phase behavior

The interaction of alpha-melanocyte stimulating hormone (alpha-MSH) with negatively charged binary membrane systems composed of either 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], (DMPC/DMPG) or DMPC/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA), both at a 3:1 ratio, was studied using complementary techniques (differential scanning calorimetry, infrared and ultraviolet absorption spectroscopy, and steady-state and time-resolved fluorescence). The peptide structure in buffer, at medium to high concentrations, is a mixture of aggregated beta-strands and random coil, and upon increasing the temperature the random coil configuration becomes predominant. At low concentrations (micromolar) there are essentially no aggregates. When in interaction with the lipidic systems this transition is prevented and the peptide is stabilized in a specific conformation different from the one in solution. The incorporation of alpha-MSH into phosphatidic acid-containing systems produced a significant alteration of the calorimetric data. Lateral heterogeneity can be induced by the peptide in the DMPA-containing mixture, at variance with the one of DMPG. In addition, the lipid/water partition coefficient for the peptide in the presence of DMPC/DMPA is greater in the gel phase as compared to the fluid phase. From the high values of limiting anisotropies it can be concluded that the peptide presents a very reduced rotational dynamics when in interaction with the lipids, pointing out to a strong interaction. Overall, these results show that the structure and stability of alpha-MSH in a negatively charged membrane environment are substantially different from those of the peptide in solution, being stabilized in a specific conformation that could be important to eliciting its biological activity.