Calcium-induced lipid phase separations and interactions of phosphatidylcholine/anionic phospholipid vesicles. Fluorescence studies using carbazole-labeled and brominated phospholipids

A novel method that uses a carbazole-labeled fluorescent phosphatidylcholine, which partitions preferentially into liquid-crystalline lipid domains, to monitor the kinetics and the extents of thermotropic and ionotropic lateral phase separations in vesicles combining brominated and nonbrominated phosphatidylcholines (PCs), phosphatidic acids (PAs), and phosphatidylserines (PSs) is described. The calcium-induced segregation of several nonbrominated PA species in liquid-crystalline brominated PC bilayers behaves as a well-defined lateral phase separation; the residual solubility of the PA component in the PC-rich phase in the presence of calcium can vary severalfold depending on the PA acyl chain composition. PC/PS mixtures show a pronounced tendency to form metastable solutions in the presence of calcium, particularly when they contain less than equimolar proportions of PS. This metastability is not readily relaxed by repeated freeze-thawing of vesicles in the presence of calcium, by avidin-mediated contacts between PC/PS vesicles containing biotinylated lipids, or by calcium-induced lateral segregation of PA in the same vesicles. Different PS species exhibit different apparent residual solubilities in liquid-crystalline PC bilayers, ranging from less than 10 mol % for dimyristoyl-PS to ca. 45 mol% for dioleoyl-PS, after prolonged incubations of PC/PS multilamellar vesicles with excess calcium. Results are presented, obtained by using the above lipid-segregation assay and parallel assays of intervesicle lipid mixing, that raise questions concerning the relevance of the equilibrium behavior of calcium-treated PS/PC mixtures to the relatively rapid interactions (fusion and lipid mixing) of PC/PS vesicles that follow initial exposure to calcium.     

Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles

The interactions of unilamellar vesicles containing phosphatidylcholine (PC) and phosphatidic acid (PA) in the presence of calcium and magnesium were examined by fluorometric assays of vesicle lipid mixing, contents mixing, and contents leakage and by spray-freezing freeze-fracture electron microscopy. These results were correlated with calorimetric and fluorometric measurements of divalent cation induced lateral segregation of lipids in these vesicles under comparable conditions. PA-PC vesicles in the presence of calcium show a rapid but limited intermixing of vesicle lipids and contents, the extent of which increases as the vesicle size decreases or the PA content increases. Calcium produces massive aggregation and efficient mixing of the contents of vesicles containing high proportions of dioleoyl-PA or egg PA, but vesicle coalescence in the latter case is followed rapidly by vesicle collapse and massive leakage of contents. The effects of magnesium are similar for vesicles of very high PA content. However, in the presence of magnesium, vesicles containing lower amounts of PA exhibit "hemifusion", a mode of interaction in which vesicles aggregate and mix approximately 50% of their lipids, apparently representing the lipids of the outer monolayer of each vesicle, without significant mixing of vesicle contents or collapse of the vesicles. Fluorometric measurements of lipid lateral segregation demonstrate that lateral redistribution of lipids in PA-PC vesicles begins at submillimolar concentrations of divalent cations and shows no abrupt change at the "threshold" divalent cation concentration, above which coalescence of vesicles is observed. By correlating calorimetric and fluorometric measurements of lipid lateral segregation and mixing of vesicle components, we can demonstrate that lipid segregation is at least strongly correlated with calcium-promoted coalescence of PA-PC vesicles and is essential to the magnesium-promoted interactions of vesicles of low PA contents.     

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.     

Ca<Superscript>2+</Superscript>-Induced Phase Separation in the Membrane of Palmitate-Containing Liposomes and Its Possible Relation to Membrane Permeabilization

A Ca(2+)-induced phase separation of palmitic acid (PA) in the membrane of azolectin unilamellar liposomes has been demonstrated with the fluorescent membrane probe nonyl acridine orange (NAO). It has been shown that NAO, whose fluorescence in liposomal membranes is quenched in a concentration-dependent way, can be used to monitor changes in the volume of lipid phase. The incorporation of PA into NAO-labeled liposomes increased fluorescence corresponding to the expansion of membrane. After subsequent addition of Ca(2+), fluorescence decreased, which indicated separation of PA/Ca(2+) complexes into distinct membrane domains. The Ca(2+)-induced phase separation of PA was further studied in relation to membrane permeabilization caused by Ca(2+) in the PA-containing liposomes. A supposition was made that the mechanism of PA/Ca(2+)-induced membrane permeabilization relates to the initial stage of Ca(2+)-induced phase separation of PA and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer.     

Structural changes in dipalmitoylphosphatidylcholine bilayer promoted by Ca2+ ions: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60mM CaCl2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 degrees C, the lipid bilayer thickness, dL, goes through a maximum as a function of CaCl2 concentration (dL=54.4A at approximately 2.5mM of CaCl2). Simultaneously, both the area per DPPC molecule AL, and the number of water molecules nW located in the polar head group region decrease (DeltaAL=AL(DPPC))-AL(DPPC+Ca)=2.3A2 and Deltan=n(W(DPPC))-n(W(DPPC+Ca))=0.8mol/mol at approximately 2.5mM of CaCl2). In the fluid phase, at 60 degrees C, the structural parameters d(L), A(L), and n(W) show evident changes with increasing Ca2+ up to a concentration C(Ca)(2+) < or = 10mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60mM CaCl2 (at 20 and 60 degrees C) are nearly the same as those for unilamellar vesicles without Ca2+.     

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.     

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.     

Structural effects of neutral lipids on divalent cation-induced interactions of phosphatidylserine-containing bilayers.

Ca2+ is known to induce the adhesion and collapse of phosphatidylserine (PS) bilayers into dehydrated multilamellar structures. The aim of this study was to examine how that interaction and the resultant structures might be modified by neutral lipid species. A combination of rapid mixing, x-ray diffraction, thin-layer chromatography, density gradient centrifugation, and freeze-fracture electron microscopy was used in conjunction with osmotic stress techniques to characterize the structures formed by the Ca(2+)-induced interaction of multilamellar liposomes and of large unilamellar vesicles. The results showed that dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine at concentrations of up to approximately 30 mol % are accommodated in a single dehydrated multilamellar structure. Similar results were obtained using mixed PS species isolated from bovine brain. Principally, the data indicate that neutral lipid is both dehydrated during the rapid collapse process of Ca(PS)2 formation and accommodated within this dehydrated structure. The large energies available on formation of the Ca(PS)2 bilayers contribute to the dehydration of neighboring neutral lipids that likely form continuous bilayers with them. Higher concentrations of these neutral lipids modify Ca(2+)-induced bilayer interactions, leading to progressively weaker interactions, larger bilayer separations, and in some cases separation into two structures; phosphatidylethanolamine species favoring nonbilayer structures tended to promote such separation at lower concentrations than bilayer lipids.     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: a small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca(2+) and Mg(2+) cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA=1:1 mol/base and in the range of concentration of the cation(2+) 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L(x) phase with repeat distance d(Lx) approximately 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L(DOPC) phase with repeat distance d(DOPC) approximately 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L(DOPC) phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC+DNA+Ca(2+) aggregates was investigated in the range 20-80 degrees C. The transversal thermal expansivities of both phases were evaluated.     

Visualization of domain formation in the inner and outer leaflets of a phospholipid bilayer

Large vesicles (5-10-micron in diameter) were formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera and digital image processor. When such vesicles contained a fluorescent phosphatidic acid (PA) and were exposed to 2 mM CaCl2 or 0.5 mM PrCl3, it was possible to visualize PA-enriched domains within the vesicles. Calcium-induced domain formation was reversible in the presence of 4 mM EGTA. Vesicles were formed containing fluorescent PA on either the inner or outer leaflet of the bilayer and the patching and dissolution of patching were studied under conditions where calcium was present on the outside of the vesicle and where calcium was distributed across the bilayer. In addition, vesicles were formed with two different fluorescent PA's, one on the inner leaflet and a different one on the outer leaflet of the bilayer. The results of the experiments show that in vesicles formed primarily with naturally occurring phospholipids such as egg phosphatidylcholine or brain phosphatidylethanolamine, there was no coordinate action of the two leaflets of the bilayer. An exception to this was found, however, if the vesicles were formed in the presence of primarily dioleoyl phospholipids (greater than 95 mol %). In these vesicles there was a coordinate or coupled response to calcium by the two leaflets of the bilayer. In most cases, however, the two leaflets of the bilayer showed independent or uncoupled domain formation.     

Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex.

The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.     

Polyamines as modulators of membrane fusion: aggregation and fusion of liposomes

We have studied the effect of the polyamines (spermine, spermidine, and putrescine) on the aggregation and fusion of large (approximately 100 nm in diameter) unilamellar liposomes in the presence of 100 mM NaCl, pH 7.4. Liposome fusion was monitored by the Tb/dipicolinic acid fluorescence assay for the intermixing of internal aqueous contents, and the release of contents was followed by carboxyfluorescein fluorescence. Spermine and spermidine at physiological concentrations aggregated liposomes composed of pure phosphatidylserine (PS) or phosphatidate (PA) and mixtures of PA with phosphatidylcholine (PC) but did not induce any fusion. However, liposomes composed of mixtures of acidic phospholipids, cholesterol, and a high mole fraction of phosphatidylethanolamine could be induced to fuse by spermine and spermidine in the absence of divalent cations. Putrescine alone in the physiological concentration range was ineffective for both aggregation and fusion of these liposomes. Liposomes made of pure PC did not aggregate in the presence of polyamines. Addition of aggregating concentrations of spermine caused a drastic increase in the rate of Ca(2+)-induced fusion of PA liposomes and a large decrease in the threshold Ca(2+) concentration required for fusion. This effect was less pronounced in the case of PS or PA/PC vesicles. Preincubation of PA vesicles with spermine before the addition of Ca(2+) resulted in a 30-fold increase in the initial rate of fusion. We propose that polyamines may be involved in the regulation of membrane fusion phenomena accompanying cell growth, cell division, exocytosis, and fertilization.     

Effects of diacylglycerols on the structure of phosphatidylcholine bilayers: a 2H and 31P NMR study

The interaction of four diacylglycerols (DAGs) with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts was investigated by a combination of 31P and 2H NMR spectrometry. We found that saturated and unsaturated long-chain DAGs induce different types of perturbations into the bilayer structure. The saturated DAGs dipalmitin and distearin induce lateral phase separation of the lipids into (i) DAG-enriched gellike domains and (ii) relatively DAG-free regions in the liquid-crystalline phase. In the latter regions, the order parameters along the fatty acyl chains of DPPC are practically identical with the control. This phase separation effect was observed in both model systems studied, and its extent is dependent upon DAG concentration and temperature. Only bilayer phases were present upon addition of dipalmitin or distearin at all concentrations and temperatures studied. The unsaturated DAGs diolein and DAG derived from egg PC (egg-DAG) affect PC bilayers in the following two ways: (i) by increasing the order parameters of the side chains, as observed for both DPPC and BL-PC model systems; (ii) by inducing nonbilayer lipid phases, as observed for BL-PC, but not DPPC. At a concentration of 25 mol % of an unsaturated DAG in mixed PC bilayers, a peak corresponding to isotropic lipid conformation appeared and increased in intensity with increase in temperature, while at 32 mol % hexagonal and bilayer phases coexisted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length

The kinetics and thermodynamics of the transmembrane movement (flip-flop) of fluorescent analogs of phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were investigated to determine the contributions of headgroup composition and acyl chain length to phospholipid flip-flop. The phospholipid derivatives containing n-octanoic, n-decanoic or n-dodecanoic acid in the sn-1 position and 9-(1-pyrenyl)nonanoic acid in the sn-2 position were incorporated at 3 mol% into sonicated single-bilayer vesicles of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC). The kinetics of diffusion of the pyrene-labeled phospholipids from the outer and inner monolayers of the host vesicles to a large pool of POPC acceptor vesicles were monitored by the time-dependent decrease of pyrene excimer fluorescence. The observed kinetics of transfer were biexponential, with a fast component due to the spontaneous transfer of pyrenyl phospholipids in the outer monolayer of labeled vesicles and a slower component due to diffusion of pyrenyl phospholipid from the inner monolayer of the same vesicles. Intervesicular transfer rates decreased approx. 8-fold for every two carbons added to the first acyl chain. Correspondingly, the free energy of activation for transfer increased approx. 1.3 kcal/mol. With the exception of PE, the intervesicular transfer rates for the different headgroups within a homologous series were nearly the same, with the PC derivative being the fastest. Transfer rates for the PE derivatives were 5-to 7-fold slower than the rates observed for PC. Phospholipid flip-flop, in contrast, was strongly dependent on headgroup composition with a smaller dependence on acyl chain length. At pH 7.4, flip-flop rates increased in the order PC less than PG less than PA less than PE, where the rates for PE were at least 10-times greater than those of the homologous PC derivative. Activation energies for flip-flop were large, and ranged from 38 kcal/mol for the longest acyl chain derivative of PC to 25 kcal/mol for the PE derivatives. Titration of the PA headgroup at pH 4.0 produced an approx. 500-fold increase in the flip-flop rate of PA, while the activation energy decreased 10 kcal/mol. Increasing acyl chain length reduced phospholipid flip-flop rates, with the greatest change observed for the PC analogs, which exhibited an approx. 2-fold decrease in flip-flop rate for every two methylene carbons added to the acyl chain at the sn-1 position.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR study of the interaction of retinoids with phospholipid bilayers

The interaction of three vitamin A derivatives or retinoids: all-trans-retinoic acid, 13-cis-retinoic acid and retinol with multilamellar phospholipid bilayers was studied using a combination of 2H- and 31P-NMR measurements. The following model membrane systems were used: (1) dipalmitoylphosphatidylcholine (DPPC) bilayers; (2) bilayers composed of a mixture of DPPC and bovine heart phosphatidylcholine (PC); (3) mixed PC/phosphatidylethanolamine (PE) bilayers. Only a weak interaction was observed between 13-cis-retinoic acid and DPPC membranes. Addition of all-trans-retinoic acid at a molar ratio of 1:2 to the lipid causes a small decrease (5 C degrees) in the gel to liquid crystalline phase-transition temperature of DPPC, a small increase in the order parameters of the lipid side-chains of single component bilayers and no measurable effect in the other lipid systems studied. Considerably larger perturbation in the lipid bilayer structure is introduced by addition of retinol which, at a molar ratio of 1:2 to the lipid, lowered the gel to liquid crystalline phase-transition temperature of DPPC by 21 C degrees and caused a decrease of order parameters of the lipid side-chains in all three lipid bilayer systems. These effects are consistent with intercalation of retinol molecules into the bilayer interior. The results for the mixed PC/PE bilayers indicate that the presence of retinol caused lateral separation of PE- and retinol-enriched regions.     

Importance of phosphatidylethanolamine for the interaction of apocytochrome c with model membranes containing phosphatidylserine

The effect of phosphatidylethanolamine (PE) on the binding of apocytochrome c to model membranes was examined. When 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of the standard vesicles composed of 80% of this lipid and 20% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) was gradually replaced with upward of 50% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), the binding increased appreciably. Ca(2+), causing the phase separation of PS, also brought about increased binding of apocytochrome c in the PC/PS system, underlining the importance of PS properties in membranes for the protein binding. The resonance energy transfer between Trp-59 in apocytochrome c and pyrene-PS incorporated into bilayers showed that the replacement of PC with PE increased the extent of apocytochrome c penetration into membranes by a PE concentration-dependent manner. However, in the absence of PS, PE had no apparent effect on these functions of apocytochrome c, suggesting that PE-induced change(s) of acidic membrane properties is important to the association of apocytochrome c with vesicles. From the observations that the excimer to monomer fluorescence ratio of pyrene-PS increased and the fluorescence of NBD-PS was quenched with increasing concentration of PE, it was deduced that PE caused PS-enriched domains in PC/PE/PS membranes. The colocalization of pyrene-PS with BODIPY-PS by PE further supported the possibility. We suggest that PE-induced formation of PS-enriched domains acts as binding sites for apocytochrome c in membranes.     

Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped

A novel liposomal method permits studies of Ca movements across the bilayers of multilamellar vesicles (MLV) which had entrapped the Ca-dependent, fluorescent indicator dye Fura 2. Ionomycin-mediated Ca translocation across MLV of phosphatidylcholine (PC)/dicetyl phosphate (DCP), 9:1, obeyed simple first-order kinetics since log-log plots of initial rates versus ionomycin or Ca concentration yielded slopes of approximately 1. Since Ca is translocated in a Ca-dependent fashion in the course of stimulus-response coupling of cells which form diacylglycerol (DAG) and phosphatidate (PA) from polyphosphoinositides, we compared effects of PA with those of DAG. PA and DAG were preincorporated in PC/DCP vesicles, in which trace amounts of ionomycin provided transmembrane potential (due to Ca2+/H+ exchange). Significant increases in Ca movements were observed in the presence of egg lecithin PA, dioleoyl-PA, and dipalmitoyl-PA when compared with DCP- or DAG-containing MLV. DAGs such as 1-oleoyl-2-acetoylglycerol or 1,2-dioleoylglycerol in liposomes decreased rates of Ca translocation. Ca influx into PA-containing MLV was dependent on the mole percent of the PA in bilayers; the complex kinetics of Ca influx were compatible with the formation of nonbilayer states. Incorporation of cholesterol into the liposomes inhibited initial rates of Ca uptake by MLV presumably by condensing the bilayers. Ca influx increased with increasing pH of the external medium from 6.9 to 7.9 in liposomes with an internal pH of 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)