Phosphatidylserine Dispersions Research Articles

Phase separation of cholesterol from phosphatidylserine–cholesterol mixtures in the presence of the local anesthetic tetracaine

Addition of the local anesthetic tetracaine (TTC) to multilamellar dispersions of natural phosphatidylserine (PS) causes changes in the thermotropic properties of the membrane, which can be detected by differential scanning calorimetry, and in the structure of the membrane as detected by X-ray diffraction. At molar ratio [PS]/[TTC]∼8.5, the melting temperature of the phospholipid shifts downwards by approximately 2.5 °C. The melting endotherm is broadened; however, there is little change in the enthalpy of melting. In ternary mixtures (PS–TTC–cholesterol), the thermotropic changes are enhanced. At [PS]/[TTC]∼13, the onset of phase separation of cholesterol crystals from PS in the liquid crystalline state occurs at molar fraction cholesterol ( X chol)∼0.28, marginally smaller than that found in the absence of the anesthetic.

Phase separation of cholesterol from phosphatidylserine?cholesterol mixtures in the presence of the local anesthetic tetracaine

Addition of the local anesthetic tetracaine (TTC) to multilamellar dispersions of natural phosphatidylserine (PS) causes changes in the thermotropic properties of the membrane, which can be detected by differential scanning calorimetry, and in the structure of the membrane as detected by X-ray diffraction. At molar ratio [PS]/[TTC]∼8.5, the melting temperature of the phospholipid shifts downwards by approximately 2.5 °C. The melting endotherm is broadened; however, there is little change in the enthalpy of melting. In ternary mixtures (PS–TTC–cholesterol), the thermotropic changes are enhanced. At [PS]/[TTC]∼13, the onset of phase separation of cholesterol crystals from PS in the liquid crystalline state occurs at molar fraction cholesterol ( X chol )∼0.28, marginally smaller than that found in the absence of the anesthetic.

The mitochondrial precursor protein apocytochrome c strongly influences the order of the headgroup and acyl chains of phosphatidylserine dispersions. A deuterium and phosphorus-31 NMR study

Deuterium and phosphorus nuclear magnetic resonance techniques were used to study the interaction of the mitochondrial precursor protein apocytochrome c with headgroup-deuterated (dioleoylphosphatidyl-L-[2-2H1]serine) and acyl chain deuterated (1,2-[11,11-2H2]dioleoylphosphatidylserine) dispersions. Binding of the protein to dioleoylphosphatidylserine liposomes results in phosphorus nuclear magnetic resonance spectra typical of phospholipids undergoing fast axial rotation in extended liquid-crystalline bilayers with a reduced residual chemical shift anisotropy and an increased line width. 2H NMR spectra on headgroup-deuterated dioleoylphosphatidylserine dispersions showed a decrease in quadrupolar splitting and a broadening of the signal on interaction with apocytochrome c. Addition of increasing amounts of apocytochrome c to the acyl chain deuterated dioleoylphosphatidylserine dispersions results in the gradual appearance of a second component in the spectra with a 44% reduced quadrupolar splitting. Such large reduction of the quadrupolar splitting has never been observed for any protein studied yet. The lipid structures corresponding to these two components could be separated by sucrose gradient centrifugation, demonstrating the existence of two macroscopic phases. In mixtures of phosphatidylserine and phosphatidylcholine similar effects are observed. The induction of a new spectral component with a well-defined reduced quadrupolar splitting seems to be confined to the N-terminus since addition of a small hydrophilic amino-terminal peptide (residues 1-38) also induces a second component with a strongly reduced quadrupolar splitting. A chemically synthesized peptide corresponding to amino acid residues 2-17 of the presequence of the mitochondrial protein cytochrome oxidase subunit IV also has a large perturbing effect on the order of the acyl chains, indicating that the observed effects may be a property shared by many mitochondrial precursor proteins. In contrast, binding of the mature protein, cytochrome c, to acyl chain deuterated phosphatidylserine dispersions has no effect on the deuterium and phosphorus nuclear magnetic resonance spectra, thereby demonstrating precursor-specific perturbation of the phospholipid order. The inability of holocytochrome c to perturb the phospholipid order is due to folding of this protein, since unfolding of cytochrome c by heat or urea treatment results in similar effects on dioleoylphosphatidylserine bilayers, as observed for the unfolded precursor. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.

Specificity of the interaction of amino- and carboxy-terminal fragments of the mitochondrial precursor protein apocytochrome c with negatively charged phospholipids. A spin-label electron spin resonance study

The contribution of the various regions of the mitochondrial precursor protein apocytochrome c to the interaction of the protein with phosphatidylserine dispersions has been studied with chemically and enzymatically prepared fragments of horse heart apocytochrome c and phospholipids spin-labeled at different positions of the sn-2 chain. Three amino-terminal heme-less peptides, two heme-containing amino-terminal fragments, one central fragment, and three carboxy-terminal fragments were studied. The electron spin resonance spectra of phospholipids spin-labeled at the C5 position of the fatty acid chain indicate that both amino-terminal and carboxy-terminal fragments of the apocytochrome c molecule cause a restriction of motion of the lipids, whereas the heme-containing peptides and protein have less effect. In addition, a second motionally more restricted lipid component, which is observed for apocytochrome c interacting with phosphatidylserine dispersions containing lipids spin-labeled at the C12 or C14 position [Görrissen, H., Marsh, D., Rietveld, A., & de Kruijff, B. (1986) Biochemistry 25, 2904-2910], was observed both on binding the carboxy-terminal fragments and on binding of the amino-terminal fragments of the precursor protein. Interestingly, even a small water-soluble peptide consisting of the 24 carboxy-terminal residues gave rise to a two-component spectrum, with an outer hyperfine splitting of the restricted lipid component of 59 G, indicating a considerable restriction of the chain motion. This suggests that both the carboxy- and amino-terminal parts of the protein penetrate into the center of the bilayer and cause a strong perturbation of the fatty acyl chain motion. The implications of these findings for the mechanism of apocytochrome c translocation across membranes are discussed.

Monolayer characteristics and thermal behavior of natural and synthetic phosphatidylserines.

The monolayer properties and thermal behavior of different phosphatidylserines are presented. At neutral pH and 22 degrees C, saturated phosphatidylserines form condensed monolayers while unsaturated phosphatidylserines form liquid-expanded films. Under similar conditions, dimyristoylphosphatidylserine undergoes a transition from the liquid-expanded to the condensed state. At pH 4 and 22 degrees C, the surface pressure-area isotherms are shifted to smaller areas relative to the monolayers recorded at neutral pH. The condensation observed at pH 4 is close to that produced at pH 7.4 by the addition of 10 mM CaCl2. As regards the molecular packing in monolayers and the thermal behavior, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) and its ether analogue are similar, albeit not identical. Below 30 mN/m, monolayers of the ether analogue are even more condensed than those of DPPS. The order-disorder transition of the ether analogue occurs usually at higher temperatures than that of the diacyl compound. Sonicated phosphatidylserine dispersions consisting of small unilamellar vesicles show anomalous thermal properties compared to sonicated phosphatidylcholine dispersions. They exhibit sharp order-disorder transitions at similar or even slightly elevated temperatures compared to unsonicated phosphatidylserine dispersions. This anomaly is explained in terms of a pH gradient across the bilayer membrane of the small unilamellar phosphatidylserine vesicle. The internal surface pH is more acidic than the external pH, leading to some protonation of phosphatidylserine molecules. This in turn leads to a condensation of phosphatidylserine molecules on the inner bilayer surface. Such a gradient is proposed to be responsible for the thermodynamic stability of highly curved negatively charged bilayer vesicles.

A calorimetric examination of the effect of myotoxin a on the thermotropic phase behavior of model lipid membranes

The effect of myotoxin a on the thermotropic phase behavior of aqueous dispersions of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) was examined using differential scanning calorimetry (DSC). Myotoxin a significantly altered the normal phase behvior of DMPC in a concentration dependent fashion. This effect is perturbed by Ca e+ and is sensitive to ionic strength and pH. High concentrations of toxin eliminate the characteristic pretransition associated with the polar head group of DMPC. They also increase the temperature of the main gel-to-liquid crystal transition from 23°C to 32–235°C. At low concentrations of toxin, the first visible effect is upon the pretransition which is split into two components that diminish with time. The main transition is less affected at low toxin concentrations, although the magnitude of the transition is reduced while it is simultaneously shifted to higher temperatures. The main transition is also split into multiple components. The toxin also had pH specific effects on the phase behavior of DMPS. Above physiological pH (8.5) the normal transition of DMPS at 36–38°C was split in the presence of myotoxin a and new components appeared centered at 31°C and 35°C. These observations are consistent with reports that the skeletal muscle membrane system is the major site of the myonecrotic effect of myotoxin a.

Apocytochrome c binding to negatively charged lipid dispersions studied by spin-label electron spin resonance

The interaction of apocytochrome c with aqueous dispersions of phosphatidylserine from bovine spinal cord and with other negatively charged phospholipids has been studied as a function of pH and salt concentration by using spin-label electron spin resonance (ESR) spectroscopy and chemical binding assays. The ESR spectra of phospholipids spin-labeled at different positions on the sn-2 chain indicate a generalized decrease in mobility of the lipids, while the characteristic flexibility gradient toward the terminal methyl end of the chain is maintained, on binding of apocytochrome c to phosphatidylserine dispersions. This perturbation of the bulk lipid mobility or ordering is considerably greater than that observed on binding of cytochrome c. In addition, a second, more motionally restricted, lipid component is observed with lipids labeled close to the terminal methyl ends of the chains. This second component is not observed on binding of cytochrome c and can be taken as direct evidence for penetration of apocytochrome c into the lipid bilayer. It is less strongly motionally restricted than similar spectral components observed with integral membrane proteins and displays a steep flexibility gradient. The proportion of this second component increases with increasing protein-to-lipid ratio, but the stoichiometry per protein bound decreases from 4.5 lipids per 12 000-dalton protein at low protein contents to 2 lipids per protein at saturating amounts of protein. Apocytochrome c binding to phosphatidylserine dispersions decreases with increasing salt concentration from a saturation value corresponding to approximately 5 lipids per protein in the absence of salt to practically zero at 0.4 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

A 2H-NMR study on the interactions of the local anesthetic tetracaine with membranes containing phosphatidylserine

The interaction of the local anesthetic tetracaine with phosphatidylserine-containing model membranes has been studied by 2H-NMR. Charged tetracaine exhibited an unusually large partition coefficient into multilamellar dispersions of phosphatidylserine. The 2H-NMR spectra consisted of a Pake doublet and a narrow line, with the former corresponding to tetracaine in the bilayer and the latter to tetracaine free in solution. A strong pH dependence of the quadrupole splittings indicated different membrane locations for charged and uncharged tetracaine. In equimolar mixtures of phosphatidylserine and phosphatidylcholine the partition coefficients and 2H-NMR spectra were much more like those observed in neat phosphatidylcholine than in neat phosphatidylserine. Dilution studies at pH 5.5 indicated that in phosphatidylserine/phosphatidylcholine mixtures tetracaine experiences a three-site exchange similar to that found earlier for tetracaine in phosphatidylcholine. Tetracaine is in fast exchange between sites weakly bound to membrane and free in solution, and in slow exchange with a strongly bound site in the membrane.

Activation of histamine secretion from rat mast cells by aqueous dispersions of phosphatidylserine.

Activation of histamine secretion from rat mast cells by aqueous dispersions of phosphatidylserine.

Differences in the interaction of inorganic and organic (hydrophobic) cations with phosphatidylserine membranes

The interaction of phosphatidylserine dispersions with “hydrophobic”, organic cations (acetylcholine, tetraethylammonium ion) is compared with that of simple inorganic cations (Na +, Ca 2+); differences in the hydration properties of the two classes of ions exist in the bulk phase as evident from spin-lattice relaxation time T 1, measurements. It is shown that the reaction products (cation-phospholipid) differ markedly in their physicochemical behaviour. With increasing concentration both classes of ions reduce the ζ-potential of phosphatidylserine surfaces, the monovalent inorganic cations being only slightly more effective than the hydrophobic cations. Inorganic cations cause precipitation of the lipid once the surface charge of the bilayer is reduced to a certain threshold value. This is not the case with the organic cations. The difference is probably associated with the different hydration properties of the resulting complexes. Thus binding of Ca 2+ causes displacement of water of hydration and formation of an anhydrous, hydrophobic calcium-phosphatidylserine complex which is insoluble in water, whereas the product of binding of the organic cations is hydrated, hydrophilic and water soluble. The above findings are consistent with NMR results which show that the phosphodiester group is involved in the binding of both classes of cations as well as being the site of the primary hydration shell. Besides affecting interbilayer membrane interactions such as those involved in cell adhesion and membrane fusion, the binding of both classes of cation can affect the molecular packing within a bilayer.

Structure and morphology of phosphatidylserine dispersions

1. 1. In contrast to egg phosphatidylcholine, aqueous dispersions of phosphatidylserine exist as a single lamellar liquid crystalline phase up to a water content of approx. 75% with all the water intercalated between the bilayers. In the absence of salt the lamellae of the large multilamelar particles present in dispersions ( c = (g lipid)/(g lipid + g water) > 0.2) are corrugated, particularly the outer lamellae. In the absence of salt planar phosphatidylserine bilayers are less stable than those of neutral phosphatidylcholine. 2. 2. At low lipid concentrations ( c < 0.05) the structural organisation is not yet clearly defined but theoretical X-ray scattering curves calculated for model particles suggest that no extensive multilamellar packing remains. 3. 3. Sonication of phosphatidylserine dispersions produces vesicles varying in size and surrounded by a closed, single bilayer in which the structural organisation of the fundamental lipid bilayer is maintained. In the absence of salt the average vesicle diameter is 16.5 mm (165 Å), about 7–10 nm (70–100 Å) smaller than the average diameter of phosphatidylcholine vesicles. In the presence of salt (0.1 M NaCl, 0.01 M Tris, pH 8.1) the average vesicle diameter is 25 nm (250 Å) identical with that of phosphatidylcholine vesicles. 4. 4. Reducing the double layer repulsion by adding NaCl to unsonicated dispersions ( c > 0.05) leads to the extrusion of water from the interbilayer space and a reduction in the lamellar repeat distance. The multilamellar particles shrink and their lamellae become smooth, indicative of an ordering in the packing of the lamellae. In dilute unsonicated dispersions ( c > 0.05) increasing the ionic strength increases the order and extent of the multilamellar structures. Increasing the ionic strength in sonicated dispersions containing single shelled vesicles leads to aggregation and/or coalescence producing larger single bilayer vesicles as well as multilamellar structures. 5. 5. Time-dependent structural changes in unsonicated phosphatidylserine dispersions in salt-free water are observed which are due to the reorganization of small multilamellar regions, randomly oriented with respect to each other, into more extensive and more ordered multilamellar regions. Aggregation and/or coalescence of single bilayer vesicles occurs in sonicated phosphatidylserine dispersions. This process is temperature dependent and increases with increasing temperature. The structures present in aqueous dispersions after preparation are not in thermodynamic equilibrium. 6. 6. Structural details derived from X-ray diffraction and electron micrographs of free-etched preparations of phosphatidylserine dispersions are consistent with each other. The negative-staining procedure, however, produces artifacts with dispersions of acidic phospholipids.

Phospholipid hydration studied by deuteron magnetic resonance spectroscopy

Deuteron magnetic resonance spectra were obtained from 2H 2O in mixtures with egg lecithin, egg phosphatidylethanolamine, and ox brain sodium phosphatidylserine. The acid form of phosphatidylserine does not hydrate. Details of the different hydration “shells” were obtained by studying the spectral splittings as a function of 2H 2O concentration. Several different types of water are present, including bulk water (exchanging only slowly with water associated with the lipid), “trapped” water (not present with phosphatidylethanolamine), and up to three types of bound water. The spectral splittings characteristic of each water environment yielded information about the water binding energies and degrees of anisotropy of motion of the phospholipid polar groups; lecithin polar groups have least motional restriction and sodium phosphatidylserine most, while phosphatidylethanolamine binds water most tightly. Spectra of some lecithin and phosphatidylserine dispersions varied with time, due to a slow reorganization of randomly oriented multilamellar regions into longer, more ordered systems, with a length of about 1 μm. At −20°C the timescales of the change were of the order of a week and a month for lecithin and phosphatidylserine respectively. Complex changes in the spectra were observed as the temperature was raised; these are interpreted in terms of changes in the motions of the phospholipid molecules.

Structures of Aqueous Dispersions of Phosphatidylserine

Abstract Dilute (g10% w/v) unsonicated phosphatidylserine dispersions in H2O contain spherical particles of a wide size distribution (0.1 to 15 µm). The majority have diameters between 0.1 to 2 µm and lack the multilamellar arrangement of bilayers typical of unsonicated lecithin dispersions. Sonication produces small thermodynamically unstable vesicles surrounded by a single closed bilayer with an average vesicle diameter of 160 A (range 125 to 250 A). The vesicles are more homogeneous with respect to size and their curvature is significantly greater than that of vesicles prepared from lecithins of similar chain length. While phosphatidylserine forms inverted micelles in organic solvent, no micelles were detected in sonicated, aqueous dispersions. Increasing the ionic strength leads to an increase in particle size and to the formation of multilamellar structures both with sonicated and unsonicated dispersions and eventually flocculation of a metal-phosphatidylserine complex occurs. Unsonicated phosphatidylserine dispersions give no high resolution spectrum. Proton nuclear magnetic resonance measurements on sonicated dispersions in 2H2O show that the molecular motion in the polar group of the molecule is sufficient for all protons, including those of the glycerol group, to contribute to the high resolution spectrum. Increasing the ionic strength leads to a closer packing of the polar groups and of the hydrocarbon chain CH2 groups close to the glycerol group without significantly affecting the correlation times of motion of most of the protons along the hydrocarbon chains. However, the anisotropy of the hydrocarbon chain motion and thus the degree of order within the bilayer increases significantly.

NMR Spectroscopy: Antibiotic Interactions with Phospholipids

<h2>Abstract:</h2> Sonicated dispersions of phosphatidylcholine, lysophosphatidylcholine, phosphatidylinositol, and phosphatidylserine in deuterium oxide were studied by NMR, and the changes in spectra produced on addition of penicillin G and ampicillin to the dispersions were observed. The antibiotics appeared to reduce the configurational freedom and kinetic motion of the phospholipid hydrocarbon chains due to hydrophobic interaction between the hydrocarbon chains and the phenyl portion of the antibiotic side chain. Penicillin G interacted with the phospholipid systems to a greater degree than ampicillin, while the phospholipids interacted in the order phosphatidylserine > phosphatidylcholine > phosphatidylinositol > lysophosphatidylcholine.

23Na NMR studies of sodium ion interactions: Phosphatidyl serine, phosphoserine, and phosphoethanolamine

23Na nuclear magnetic resonance (NMR) relaxation time measurements of sodium ion solutions were studied as a function of pH. Results were obtained for aqueous solutions containing phosphate, phosphoethanolamine, and phosphoserine, and an aqueous dispersion of phosphatidyl serine. Changes in the relaxation rate ( T 1 −1) going from low pH to high pH exhibit the predominantly ionic nature of the associations. The change in relaxation rate correlates directly with the charge-to-radius ratio of the phosphate, phosphoserine, and phosphoethanolamine anions in that order. The results obtained with phosphatidyl serine dispersions suggest that there is only a weak association of sodium ion with phosphatidyl serine at physiological pH.

Nuclear magnetic resonance studies of the polypeptide alamethicin and its interaction with phospholipids

High resolution nuclear magnetic resonance spectroscopy is used to study alamethicin (a cyclic polypeptide antibiotic which induces interesting ion transport properties in both natural and artificial membranes) and the interaction of alamethicin with phospholipids. The aggregation of alamethicin in aqueous solution at neutral pH is largely unaffected by Na +, K + or Ca 2+ ions, and the nuclear magnetic resonance spectrum of the polypeptide is not changed upon formation of complexes with these ions either in deuterium oxide or in organic solvent. Large changes are induced in the nuclear magnetic resonance spectra of sonicated aqueous dispersions of phosphatidylcholine and phosphatidylserine on the addition of small quantities of alamethicin. The peaks from the lipid protons are broadened, indicating a loss of freedom of motion of the lipid molecules. The effect on phosphatidylserine dispersions is the larger, and it is estimated that each alamethicin molecule induces a transition of about 600 lipid molecules from the normal multilamellar state to some new form of aggregate. Particle size measurements, using analytical column chromatography and ultracentrifugation, show that the reduction in motion of the lipid molecules is not due to a decrease in tumbling rate, but is probably primarily due to hydrophobic interactions. This is confirmed by electron spin resonance spin label experiments. X-ray diffraction and electron microscopy experiments reveal that the phospholipid multilamellar structure is broken down by the alamethicin. Electrophoresis shows that, in the new structure, the alamethicin molecules are oriented with their polar side-chains at the lipid-water interface. These results are significant for an understanding of the general field of lipid-peptide interactions, and may help to explain the antibiotic effect of alamethicin and similar molecules and its effects on the ion transport properties of membranes.

Ultrasonic attenuation measurements in phospholipid dispersions

Ultrasonic attenuation measurements have been used to show that relaxation processes occur in the time range 1 · 10 −7 1 · 10 −8 sec in dispersions of phosphatidylserine and, to a lesser extent, in phosphatidylcholine dispersions. The ultrasonic attenuation is modified by changes in phospholipid concentration, pH, cholesterol and divalent metal ion content. A quantitative analysis of the data is not possible, but the most probable mechanisms for the relaxation processes are intraliposome conformational changes and/or solvation equilibria.

The involvement of phosphatidylserine in adenosine triphosphatase activity of the sodium pump.

1. A study has been made to see whether the ATPase of the Na pump is activated by phospholipids.2. Solubilization of a membrane-containing (microsomal) fraction from ox brain cortex with deoxycholate removed most or all of the ouabain-inhibited ATPase and p-nitrophenylphosphatase (pNPPase) activities. Addition of sonicated dispersions of crude commercial samples of phosphatidic acid, phosphatidylinositol or phosphatidylserine stimulated both enzymic activities, but neither phosphatidylcholine nor phosphatidylethanolamine affected them. After partial purification by chromatography, however, definite activation was demonstrated only with that component of crude samples which migrated like phosphatidylserine.3. The ATPase activity dependent on phosphatidylserine was eliminated by removal of Na and K or by addition of ouabain and was substantially inhibited by oligomycin. The corresponding component of pNPPase activity was partially inhibited by ouabain or by removal of most of the K, but was little affected by oligomycin. The phosphatidylserine-dependent enzyme(s) therefore exhibited properties characteristic of the ATPase of the Na pump.4. Phosphatidylserine dispersions similarly activated both the ATPase and the pNPPase of the particulate enzyme preparations, untreated with deoxycholate.5. The results suggest that the system for active transport of Na and K involves a complex unit of phosphatidylserine and the protein of the ATPase.

REACTION OF LOCAL ANESTHETICS WITH PHOSPHOLIPIDS. A POSSIBLE CHEMICAL BASIS FOR ANESTHESIA.

Local anesthetics (LA) have been found to interact with phospholipids and lipids extracted from nerve and muscle. This reaction is demonstrated by: (a) Inhibition by LA of phospholipid (and tissue lipid) facilitated transport of calcium from a methanol: water phase into chloroform. This action is dependent upon the cationic form of the LA. (b) LA increase the electrical resistance of "membranes" prepared by impregnating Millipore filters with cephalin:cholesterol or tissue lipid extracts and bathed with NaCl or KCl solutions. (c) LA coagulate aqueous dispersions of cephalin, phosphatidyl serine, phosphatidyl ethanolamine, and inositide, an action shared by calcium. The order of potency in coagulating cephalin sols is tetracaine > calcium > butacaine > procaine. Na(+) and K(+) do not coagulate phospholipid dispersions at 0.1 M concentration and antagonize the effect of Ca(2+). (d) LA produce a marked fall in the pH of cephalin sols equivalent to that produced by calcium, (e) Ca(2+) and LA form 1:2 molar complexes with phospholipids probably by ion-ion and ion-induced polar type of binding at the phosphate groups of the lipid. It is suggested that such reactions with cell membrane phospholipids may underlie inhibitory effects of LA on cellular ion fluxes and provide a chemical basis for anesthetic action.