Cholesterol effects on the interaction of cardiolipin with anti-cardiolipin antibody

(1) Human antibodies to cardiolipin, phosphatidic acid and phosphatidylserine were assessed by binding to nitrocellulose paper and subsequent reaction with an enzyme-linked or radioactively labelled second antibody to human IgG. (2) The addition of cholesterol to constant amounts of cardiolipin impregnated in the nitrocellulose paper resulted in a profound fall in antibody binding beginning at a 0.5 to 1 molar ratio of cholesterol to cardiolipin and stabilizing at about 15% of the original level. (3) Antibody binding to phosphatidic acid and phosphatidylserine also showed extensive cholesterol-induced inhibition beginning at a slightly lower molar ratio of cholesterol to phospholipid. (4) The structural array of neither the cardiolipin alone impregnated in nitrocellulose nor the phospholipid together with cholesterol is known. It is possible that the specific cardiolipin phase structure required for human antibody recognition was disrupted by cholesterol.     

Segregation of phosphatidic acid-rich domains in reconstituted acetylcholine receptor membranes

Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.     

Contents

The thermotropic behavior of mixtures of cholesterol with phosphatidylserine (PS) or galactocerebroside was investigated.In PS/cholesterol mixtures at low cholesterol concentrations only one peak in the thermograms is obtained. At about Xchol = 0.3 another peak at higher temperatures is also seen, this peak stems from phase transition of almost pure cholesterol phase. However, increase of Xchol results in further incorporation of cholesterol into the mixture.In galactocerebroside-cholesterol mixtures above Xchol 0.4, at least two domains differing in cholesterol content and thermotropic properties coexist.In the presence of cholesterol even at 1:1 molar ratios the phosphatidylserine or galactocerebroside are still undergoing melting.     

Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lipid peroxidation in hemoglobin-containing liposomes. Effects of membrane phospholipid composition and cholesterol content

The effects of phospholipid-oxidation state and vesicle composition on lipid peroxidation in hemolysate-containing liposomes (hemosomes) were studied by the thiobarbituric acid assay. Liposomes (hemosomes) were prepared from egg phosphatidylcholine (PC) with either low (PC0.08) or high (PC0.66) oxidation indices reflecting low and high conjugated diene/lipid hydroperoxy contents. Thiobarbituric acid reactivity was negligible over 6 h at 38 degrees C in buffer-containing (control) liposomes prepared from PC0.08, whereas it was slightly increased in those prepared from PC0.66. Encapsulated hemolysate had no effect in PC0.08 liposomes, but significantly increased thiobarbituric acid reactivity in those prepared from PC0.66. Inclusion of either phosphatidylethanolamine or phosphatidylinositol in the membrane further increased lipid peroxidation in hemosomes prepared from PC0.66, whereas phosphatidic acid and phosphatidylserine were inhibitory. Inclusion of cholesterol in the membrane had no effect in PC0.66 hemosomes, but significantly inhibited lipid peroxidation in the presence of phosphatidylethanolamine or phosphatidylinositol. The effects of phosphatidic acid and cholesterol were dose-dependent. Co-incorporation of cholesterol and phosphatidic acid or phosphatidylserine in the membrane resulted in almost complete elimination of hemoglobin (Hb)-induced lipid peroxidation. Lysophosphatidic acid had similar effect as phosphatidic acid, whereas lysophosphatidylserine exerted inhibition only in the presence of phosphatidylethanolamine. The rate of lipid peroxidation showed no correlation with the amount of encapsulated Hb, neither with the oxidation indices nor the polyunsaturated fatty acid contents of negatively charged phospholipids. The above findings suggest a possible role for the high cholesterol content and preferential localization of phosphatidylserine in the inner bilayer leaflet of erythrocyte membrane in protecting against Hb-induced lipid peroxidation in the membrane.     

Calcium-induced phase separation phenomena in multicomponent unsaturated lipid mixtures

The ability of calcium to induce phase separation in multicomponent lipid mixtures containing various unsaturated species of acidic and neutral phospholipids has been investigated by 31P NMR, 3H NMR, and small-angle X-ray diffraction techniques. It is shown that, in unsaturated (dioleoyl-) phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) (1:1) and phosphatidic acid (PA)/phosphatidylcholine (PC) (1:1) mixtures, calcium is unable to induce lateral phase separation of the acidic and neutral lipids and that all the lipids adopt a hexagonal (HII) phase in the presence of calcium. In multicomponent mixtures containing one or more acidic species the presence of cholesterol either facilitates calcium-induced lamellar to hexagonal (HII) transitions for all the lipid components or, in systems already in a hexagonal (HII) phase, mitigates against calcium-induced lateral phase separations. Further, cholesterol is shown to exhibit no preferential interaction on the NMR time scale with either PC, PE, or phosphatidylserine (PS) when the lipids are in the liquid-crystal state. The ability of cholesterol to directly induce HII phase formation in PC/PE mixtures is also shown to be common to various other sterols including ergosterol, stigmasterol, coprostanol, epicoprostanol, and androstanol.     

Volume enlargement and recovery of Na+-dependent amino acid transport in proteoliposomes derived from Ehrlich ascites cell membranes

Na+-dependent amino acid transport can be reconstituted from solubilized Ehrlich cell plasma membranes by addition of asolectin vesicles, gel filtration, and a freeze-thaw cycle. Removal of phosphatidic acid (approximately 10% of the total lipid) by Ba2+ precipitation reduces the efficiency of reconstitution of Na+-dependent amino acid transport by approximately 73% and decreases intravesicular volume of the proteoliposomes by approximately 43%. The loss of transport activity is not due to exclusion of specific proteins during reconstitution. The phosphatidic acid-free liposomes are less permeable and require more time to attain an equilibrium distribution of solute. Transport activity and intravesicular volume can be restored to Ba2+-precipitated asolectin proteoliposomes by addition of egg-phosphatidic acid during reconstitution. The extent of recovery of transport activity is proportional to the change in intravesicular volume and depends on the amount of phosphatidic acid present. Replacement of phosphatidic acid with 20% phosphatidylserine or phosphatidylglycerol leads to increases in intravesicular volume with little or no increase in amino acid transport. Generation of phosphatidic acid in situ by treatment of Ba2+-precipitated proteoliposomes with phospholipase D also restored transport. The observed increase in transport activity (9-fold) is accompanied by a 46% increase in intravesicular volume, presumably caused by vesicle fusion. Phosphatidic acid is also required for successful reconstitution of Na+-dependent amino acid transport from pure phosphatidylcholine:phosphatidylethanolamine (1:1) mixtures with only a small change (approximately 16%) in intravesicular volume. The results provide evidence for both indirect and direct effects of phosphatidic acid on reconstitution of Na+-dependent amino acid transport. The indirect effects occur through enlargement of intravesicular volume, large vesicles showing higher rates of transport. However, there is also evidence to indicate a specific effect of phosphatidic acid on the Na+-dependent amino acid transporter, since other acidic lipids may change intravesicular volume without a commensurate change in transport activity.     

Calcium-induced condensation-reorganization phenomena in multilamellar vesicles of phosphatidic acid. pH potentiometric and 31P-NMR, Raman and ESR spectroscopic studies

In biological membranes, the anionic characteristics of the polar headgroup of phosphatidic acids are responsible for structural changes induced by Ca2+ in many cellular processes. The very simple headgroup structure of dipalmitoylphosphatidic acid (DPPA) offers particular advantages as a model to study the interactions between Ca2+ and natural phosphatidic acids such as cardiolipin and phosphatidylserine. The effects of calcium ions on DPPA membranes have been studied as a function of temperature by potentiometry and by Raman, ESR and 31P-NMR spectroscopies. The protons in monosodic DPPA liposomes have been considered as a probe to detect pH variations resulting from introduction of Ca2+ inside the membrane. This method has also allowed us to determine the stoichiometry of this reaction: 2 DPPA(H) + Ca2+----Ca(DPPA)2 + 2H+. 31P-NMR spectroscopy has been used to detect reorganization-condensation phenomena in multilamellar vesicles of DPPA under the influence of calcium and temperature. Furthermore, the temperature profiles obtained from Raman spectra for Ca(DPPA)2 membranes provide conclusive evidence that Ca2+ induces major reorganization of the phosphatidic acid component into a highly ordered phase. Quantitative estimates of the degree of motional restriction of spin-labeled soaps embedded inside membranes composed of DPPA with or without Ca2+ have been made using ESR technique. These results are discussed and compared to those found previously for a natural phosphatidic acids such as phosphatidylserine.     

The Fusion of Liposomes to Rat Brain Microsomal Membranes Regulates Phosphatidylserine Synthesis

Rat brain microsomal membranes were fused to liposomes prepared with several pure lipids, namely, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and mixtures of phosphatidic acid and phosphatidylcholine or phosphatidylethanolamine. The fusion between liposomes and microsomes was measured by the octadecyl rhodamine B chloride method. The extent and other properties of fusion largely depend on the lipid used to prepare liposomes; phosphatidic acid and phosphatidylinositol fuse more extensively than other lipid classes. The activity of serine base exchange is affected by the fusion between rat brain microsomes and lipids. It is strongly inhibited by phosphatidylserine, but it is activated by phosphatidic acid. The inhibition produced by phosphatidylserine on its own synthesis is proposed as a mechanism for controlling the formation of phosphatidylserine in rat brain microsomes.     

A new high-temperature transition of crystalline cholesterol in mixtures with phosphatidylserine

Phosphatidylserine and cholesterol are two major components of the cytoplasmic leaflet of the plasma membrane. The arrangement of cholesterol is markedly affected by the presence of phosphatidylserine in model membranes. At relatively low mol fractions of cholesterol in phosphatidylserine, compared with other phospholipids, cholesterol crystallites are formed that exhibit both thermotropic phase transitions as well as diffraction of x-rays. In the present study we have observed and characterized a novel thermotropic transition occurring in mixtures of phosphatidylserine and cholesterol. This new transition is observed at 96 degrees C by differential scanning calorimetry (DSC), using a heating scan rate of 2 degrees C/min. Observation of the transition requires that the hydrated lipid mixture be incubated for several days, depending on the temperature of incubation. The rate of formation of the material exhibiting a transition at 96 degrees C is more rapid at higher incubation temperatures. At 37 degrees C the half-time of conversion is approximately 7 days. Concomitant with the appearance of the 96 degrees C peak the previously known transitions of cholesterol, occurring at approximately 38 degrees C and 75 degrees C on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrous cholesterol and to the dehydration of cholesterol monohydrate, respectively. The loss of the 75 degrees C peak takes a longer time than that of the 38 degrees C peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75 degrees C and subsequently is converted by additional time of incubation to an altered form of the monohydrate, showing a phase transition at 96 degrees C. After several weeks of incubation at 37 degrees C, only the form with a phase transition at 96 degrees C remains. If such a sample undergoes several successive heating and cooling cycles, the 96 degrees C peak disappears and the 38 degrees C transition reappears on heating. For samples of 1-palmitoyl-2-oleoyl phosphatidylserine or of 1-stearoyl-2-oleoyl phosphatidylserine having mol fractions of cholesterol between 0.4 and 0.7, the 38 degrees C transition that reappears after the melting of the 96 degrees C component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at least for these samples, the amount of anhydrous cholesterol crystallites formed is indeed a property of the lipid mixture. We have also examined variations in the method of preparation of the sample and find similar behavior in all cases, although there are quantitative differences. The 96 degrees C transition is partially reversible on cooling and reheating. This transition is also scan rate dependent, indicating that it is, at least in part, kinetically determined. The enthalpy of the 96 degrees C transition, after incubation of the sample for 3 weeks at 37 degrees C is dependent on the ratio of cholesterol to 1-palmitoyl-2-oleoyl phosphatidylserine or to 1-stearoyl-2-oleoyl phosphatidylserine, with the enthalpy per mole cholesterol increasing between cholesterol mol fractions of 0.2 and 0.5. Dimyristoyl phosphatidylserine at a 1:1 molar ratio with cholesterol, after incubation at 37 degrees C, exhibits a transition at 95 degrees C that reverses on cooling at 44 degrees C, instead of 60 degrees C, as observed with either 1-palmitoyl-2-oleoyl phosphatidylserine or 1-stearoyl-2-oleoyl phosphatidylserine. These findings along with the essential absence of the 96 degrees C transition in pure cholesterol or in cholesterol/phosphatidylcholine mixtures, indicates that the phospholipid affects the characteristics of the transition, and therefore the cholesterol crystallites must be in direct contact with the phospholipid and are not simply in the form of pure crystals of cholesterol. These observations are particularly important in view of recent observations of the presence of cholesterol crystals in biological systems.     

Comparative studies on the effects of pH and Ca2+ on bilayers of various negatively charged phospholipids and their mixtures with phosphatidylcholine.

(1) The thermotropic behaviour of dimyristoyl phosphatidylglycerol, phosphatidylserine, phosphatidic acid and phosphatidylcholine was investigated by differential scanning calorimetry and freeze-fracture electron microscopy as a function of pH and of Ca2+ concentration. (2) From the thermotropic behaviour as a function of pH, profiles could be constructed from which apparent pK values of the charged groups of the lipids could be determined. (3) Excess Ca2+ induced a shift of the total phase transition in 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-glycerophosphoglycerol mixtures. In 14 : 0/14 : 0-glycerophosphocholine bilayers containing 16 : 0/16 : 0-glycerophosphoglycerol lateral phase separation was induced by Ca2+. (4) Up to molar ratios of 1 : 2 of 14 : 0/14 : 0-glycerophosphoserine to 14 : 0/14: 0-glycerophosphocholine, excess Ca2+ induced lateral phase separation. Addition to mixtures of higher molar ratios caused segregation into different structures: the liposome organization and the stacked lamellae/cylindrical organization. (5) Addition of excess Ca2+ to mixtures of 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-phosphatidic acid caused, independent of the molar ratio, separation into two structural different organizations. (6) The nature of Ca2+-induced changes in bilayers containing negatively charged phospholipids is strongly dependent on the character of the polar headgroup of the negatively charged phospholipid involved.     

Associations of B- and C-Raf with cholesterol, phosphatidylserine, and lipid second messengers: preferential binding of Raf to artificial lipid rafts

The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B- and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K(D)) in the range of 0.5-50 nm. Raf association with liposomes was accompanied by displacement of 14-3-3 proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called rafts. We demonstrate here that purified Raf proteins have moderate binding affinity for cholesterol. However, under conditions of lipid raft formation, Raf association with cholesterol (or rafts) increased dramatically. Since ceramides also support formation of rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in raft regions via association with cholesterol and ceramides and in non-raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf protein that is already present at the membrane.     

Differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylserine bilayer membranes

We have examined the effects of cholesterol on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylserines by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. We find that the incorporation of increasing quantities of cholesterol progressively reduces the temperature, enthalpy, and cooperativity of the gel-to-liquid-crystalline phase transition of the host phosphatidylserine bilayer, such that a cooperative chain-melting phase transition is completely or almost completely abolished at 50 mol % cholesterol, in contrast to the results of previous studies. We are also unable to detect the presence of a separate anhydrous cholesterol or cholesterol monohydrate phase in our binary mixtures, again in contrast to previous reports. We further show that the magnitude of the reduction in the phase transition temperature induced by cholesterol addition is independent of the hydrocarbon chain length of the phosphatidylserine studied. This result contrasts with our previous results with phosphatidylcholine bilayers, where we found that cholesterol increases or decreases the phase transition temperature in a chain length-dependent manner (1993. Biochemistry, 32:516-522), but is in agreement with our previous results for phosphatidylethanolamine bilayers, where no hydrocarbon chain length-dependent effects were observed (1999. Biochim. Biophys. Acta, 1416:119-234). However, the reduction in the phase transition temperature by cholesterol is of greater magnitude in phosphatidylethanolamine as compared to phosphatidylserine bilayers. We also show that the addition of cholesterol facilitates the formation of the lamellar crystalline phase in phosphatidylserine bilayers, as it does in phosphatidylethanolamine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of cholesterol. We ascribe the limited miscibility of cholesterol in phosphatidylserine bilayers reported previously to a fractional crystallization of the cholesterol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. In general, the results of our studies to date indicate that the magnitude of the effect of cholesterol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipid dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.     

The effect of protons or calcium ions on the phase behavior of phosphatidylserine-cholesterol mixtures.

The influence of protons or calcium ions on the miscibility of cholesterol in phosphatidylserine has been examined using differential scanning calorimetry and X-ray diffraction. At pH 2.6, where the carboxyl group of the serine moiety is protonated, two endothermic transitions are observed in cholesterol-phosphatidylserine mixtures. The midpoint of the first is at 35 degrees C in the absence of cholesterol and decreases to approx. 15 degrees C for molar fraction of cholesterol 0.5. The second transition is centered at approx. 44 degrees C, almost independent of cholesterol content. The two lower temperature phases are lamellar and the high temperature phase has hexagonal symmetry. Cholesterol is more miscible in protonated phosphatidylserine than in the sodium form: cholesterol crystals are detected at a molar ratio of phosphatidylserine to cholesterol of about 1.7:1 as compared to about 2.3:1 at neutral pH. In the presence of calcium ions (1.3 Ca2+ per phosphatidylserine), a lamellar phase is observed with layer spacing 53 A which is independent of temperature (25 degrees C-65 degrees C) and of cholesterol content. Calcium ions cause reduced cholesterol solubility: crystallites are detected already at a molar ratio of 4:1.     

Lipid phase separation induced by a hydrophobic protein in phosphatidylserine--phosphatidylcholine vesicles

Differential scanning calorimetry (DSC) was used to detect phase separation induced by hydrophobic myelin protein, lipophilin, in a mixture of phosphatidylserine (PS) and dipalmitoylphosphatidylcholine (DPPC). Preferential binding of PS to the boundary layer of lipophilin causes a decrease in the PS content of the remaining lamellar phase with a resultant shift in the phase-transition temperature to a higher temperature. The phase diagram for this mixture in the presence and absence of lipophilin is presented. From the phase diagram, it can be estimated that for an equimolar mixture of PS and DPPC, the boundary layer contains only PS, although for higher DPPC contents, some DPPC can also be found in the boundary layer. In the case where partial phase separation in induced in this mixture by Ca2+ alone, lipophilin increases the phase separation indicating that it also binds PS preferentially in the presence of Ca2+. Preferential binding of two other acidic lipids, phosphatidic acid and phosphatidyl-glycerol, to the boundary layer was also found, including a mixture where the acidic lipid was the higher melting component in the mixture.     

Interrelationship between Ca2+-dependent phospholipid base-exchange reaction and phospholipase D-like activity in bovine retina

Endogenous substrates (phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine) for the Ca2+-dependent base-exchange reaction were investigated using bovine retinal microsomes. The amounts of the three bases, serine, ethanolamine and choline, released from the membranes and the amount of phosphatidic acid generated in the membranes were measured in the presence of Ca2+ with or without exogenous bases. When the membranes were incubated with Ca2+ alone, the three bases were liberated into the water-soluble fractions accompanied by accumulation of phosphatidic acid, suggesting the presence of Ca2+-dependent phospholipase D-like activity. When an exogenous base was added to the reaction mixture, the liberation of the other two bases increased slightly and the formation of phosphatidic acid decreased markedly. The exogenous base also stimulated the liberation of the same base from prelabeled phospholipids. Accompanying these changes, the exogenous base was incorporated into the membrane phospholipid. With respect to pH profile, time course and metal requirements, both the base incorporation and phospholipase D-like activity were quite similar. The amount of base incorporated generally agreed with both the decreased amount of phosphatidic acid formed and the increased amount of base released. These results suggest that, beside the base-exchange reaction, phospholipase D-like activity plays an important role in Ca2+-dependent base incorporation into bovine retinal membranes.     

Oleic- and docosahexaenoic acid-containing phosphatidylethanolamines differentially phase separate from sphingomyelin

A central tenet of the lipid raft model is the existence of non-raft domains. In support of this view, we have established in model membranes that a phosphatidylethanolamine (PE)-containing docosahexaenoic acid (DHA) forms organizationally distinct non-raft domains in the presence of sphingomyelin (SM) and cholesterol (Chol). We have shown that formation of DHA-rich domains is driven by unfavorable molecular interactions between the rigid Chol molecule and the highly flexible DHA acyl chain. However, the molecular interactions between SM and the DHA-containing PE, which could also contribute to the formation of DHA-rich non-raft domains, have not been sufficiently investigated. To address this issue, we use differential scanning calorimetry (DSC) to study the phase behavior of mixtures of SM with either 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE), an oleic acid (OA)-containing control, over a wide range of concentrations. Deconvolution of binary DSC scans shows that both 16:0-22:6PE and 16:0-18:1PE phase separate from SM. Analysis of transition temperatures and partial phase diagrams, constructed from the DSC scans for the first time, shows that 16:0-22:6PE displays greater non-ideal mixing with SM compared to 16:0-18:1PE. Our findings support a model in which DHA- and OA-containing PEs differentially phase separate from SM over a wide range of molar ratios to initiate the formation of non-raft domains, which is greatly enhanced by DHA, but not OA, in the presence of cholesterol.     

Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities

The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion.     

The phospholipid requirement for activity of the lactose carrier of Escherichia coli

The transport activity of the lactose carrier of Escherichia coli has been reconstituted in proteoliposomes composed of different phospholipids. The maximal activity was observed with the natural E. coli lipid as well as mixtures containing phosphatidylethanolamine or phosphatidylserine. Phosphatidylcholine or mixtures of phosphatidylcholine with phosphatidylglycerol, phosphatidic acid, or cardiolipin showed low activity. The lactose carrier reconstituted with amino phospholipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine, and dioleoylphosphatidylcholine) revealed a progressive decrease in both counterflow and proton motive force-driven lactose uptake activities. Trinitrophenylation of phosphatidylethanolamine in the E. coli proteoliposomes resulted in a marked reduction in lactose carrier activity. Partial restitution of transport activity was obtained by detergent extraction of the carrier from these inactive proteoliposomes and reconstitution of the carrier into proteoliposomes containing normal E. coli lipid. These results suggest that the amino group of the amino phospholipids (e.g. phosphatidylethanolamine and phosphatidylserine) is required for the full function of the lactose carrier from E. coli.     

Fourier transform infrared spectroscopy of aqueous dispersions of phosphatidylserine-cholesterol mixtures

The effect of cholesterol on vibrational spectra in the non polar and in the polar region of dimyristoyl phosphatidylserine (DMPS) and of phosphatidylserine from bovine spinal cord (PS) has been investigated. The small shifts in the methylene CH stretching frequencies after taking into account the contribution of the cholesterol spectrum were interpreted as a combined effect of cholesterol on the conformation of the chains and of the lesser contributions of the cholesterol methyl groups. Cholesterol also influences the ratio of the trans (1465 cm^–1) to the lower wavelength (1457 cm^–1) CH₂ bending bands. No significant direct effect of cholesterol on the vibration of the polar residues was discerned. The small shift of the carboxylate band observed below the phase transition is probably due to the change in the intermolecular zwitterions when the average distance between the neighboring polar groups increases due to incorporation of cholesterol molecules.Abbreviations PS phosphatidylserine natural - DMPS dimyristoyl phosphatidylserine - DPPC dipalmitoyl phosphatidylcholine - FTIR Fourier transform infrared spectroscopy - DSC differential scanning calorimetry - PE phosphatidylethanolamine Offprint requests to: D. Bach