Surface distribution of the fatty acid side chains of phosphatidylethanolamine in mixed phospholipid vesicles

A method has been developed for the selective determination of the fatty acid side chain distribution associated with the amino containing phospholipids located in the inner and outer surfaces of membranes. Using sonicated phosphatidylethanolamine/phosphatidylcholine vesicles as a model, the analysis consists of selective labeling of the outer surface amino groups with the membrane impermeable reagent 2,4,6-trinitrobenzenesulfonic acid. Outer and inner surface phosphatidylethanolamine fractions are separated by thin-layer chromatography. Analysis of methyl esters derived from these two fractions, by gas-liquid chromatography, yields the fatty acid side chain distribution. Our results show that there is no mol fraction dependence of the incorporation of any specific fatty acid side chains of egg yolk phosphatidylethanolamine into the vesicle or any preferential distribution of these side chains in the inner or outer vesicle surface. The surface distribution of the egg yolk phosphatidylethanolamine molecules in these vesicles appears to be determined by the head group packing requirements and not the fatty acid side chain composition.     

Magnetic resonance study of the distribution of 2,2,6,6-tetramethylpiperidine-N-oxyl in phosphatidylcholine bilayers.

With the aid of paramagentic praseodymium ions the resonances at 60 MHz of the inward and outward facing choline methyl protons of sonicated egg yolk phosphatidylcholine vesicles were resolved. The subsequent addition of 2,2,6,6,-tetramethylpiperidine-N-oxyl (TEMPO) to the vesicle suspension broadened the inner and outer resonances equally. TEMPO easily penetrates the egg yolk phosphatidylcholine vesicles and has free access to the entire lipid volume above the gel to liquid crystalline transition temperature. The electron spin resonance (ESR) spectrum of TEMPO in the egg yolk phosphatidylcholine suspension exhibits features indicating that TEMPO dissolves principally in the hydrocarbon portion of the egg yolk phosphatidylcholine bilayer. The egg yolk phosphatidylcholine methylene chain proton resonances are also broadened by TEMPO notably to a greater extent than the choline methyl resonances. These data indicate that TEMPO should be more sensitive to the melting behavior of the fatty acyl chains of phospholipid suspensions than to the polar head groups.     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus.

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the identity of the fatty acyl chains can uniquely specify or determine which side of the membrane individual phosphatidylethanolamine molecules come to occupy. Although fatty acyl chain asymmetry and phosphatidylethanolamine asymmetry are correlated in VSV, no simple rules can be discerned which uniquely relate the two paramaters.     

Transmembrane asymmetry of vesicle lipids.

The role of fatty acyl chain unsaturation in promoting asymmetry in phospholipid vesicle bilayers was investigated in mixed lipid systems with differing acyl chains and a constant phosphatidylcholine headgroup. Ratios of outside to inside components were determined by nuclear magnetic resonance spectroscopy of 13C-enriched egg phosphatidylcholine. An asymmetry or disproportionation ratio is defined and used to express quantitatively how a mixture of two lipids distributes in the outer and inner vesicle surfaces. In mixed systems with 13C-enriched egg phosphatidylcholine as one component, increasing fatty acyl unsaturation in the other component results in an increasing preference of the unsaturated chains for the outer surface.     

Acyl chain unsaturation modulates distribution of lecithin molecular species between mixed micelles and vesicles in model bile. Implications for particle structure and metastable cholesterol solubilities.

We determined the distribution of lecithin molecular species between vesicles and mixed micelles in cholesterol super-saturated model biles (molar taurocholate-lecithin-cholesterol ratio 67:23:10, 3 g/dl, 0.15 M NaCl, pH approximately 6-7) that contained equimolar synthetic lecithin mixtures or egg yolk or soybean lecithins. After apparent equilibration (48 h), biles were fractionated by Superose 6 gel filtration chromatography at 20 degrees C, and lecithin molecular species in the vesicle and mixed micellar fractions were quantified as benzoyl diacylglycerides by high performance liquid chromatography. With binary lecithin mixtures, vesicles were enriched with lecithins containing the most saturated sn-1 or sn-2 chains by as much as 2.4-fold whereas mixed micelles were enriched in the more unsaturated lecithins. Vesicles isolated from model biles composed of egg yolk (primarily sn-1 16:0 and 18:0 acyl chains) or soy bean (mixed saturated and unsaturated sn-1 acyl chains) lecithins were selectively enriched (6.5-76%) in lecithins with saturated sn-1 acyl chains whereas mixed micelles were enriched with lecithins composed of either sn-1 18:1, 18:2, and 18:3 unsaturated or sn-2 20:4, 22:4, and 22:6 polyunsaturated chains. Gel filtration, lipid analysis, and quasielastic light scattering revealed that apparent micellar cholesterol solubilities and metastable vesicle cholesterol/lecithin molar ratios were as much as 60% and 100% higher, respectively, in biles composed of unsaturated lecithins. Acyl chain packing constraints imposed by distinctly different particle geometries most likely explain the asymmetric distribution of lecithin molecular species between vesicles and mixed micelles in model bile as well as the variations in apparent micellar cholesterol solubilities and vesicle cholesterol/lecithin molar ratios.(ABSTRACT TRUNCATED AT 250 WORDS)     

Asymmetric distribution of phosphatidylethanolamine fatty acyl chains in the membrane of vesicular stomatitis virus

The membrane of vesicular stomatitis virus (VSV) contains two distinct pools of phosphatidylethanolamine molecules which reside in the inner and outer phospholipid monolayers, respectively. 36% of the total membrane phosphatidylethanolamine is found in the outer monolayer while 64% is found in the inner. The two pools of VSV phosphatidylethanolamine can be distinguished operationally by the fact that only outer phosphatidylethanolamine is reactive in intact virions with the membrane-impermeable reagent trinitrobenzenesulfonate (TNBS). We have made use of this property to separate inner from outer VSV phosphatidylethanolamine and to determine the fatty acyl chain compositions of the two phosphatidylethanolamine pools separately. The results show that compared to outer phosphatidylethanolamine, inner phosphatidylethanolamine molecules contain a significantly higher proportion of unsaturated fatty acyl chains. Furthermore, whereas the proportion of unsaturated fatty acyl chains was found to be quite similar at the 1 and 2 glycerol carbon atoms in inner phosphatidylethanolamine, a marked dissimilarity was observed in outer phosphatidylethanolamine; outer phosphatidylethanolamine was enriched in saturated fatty acyl chains at the 1 position and in unsaturated fatty acyl chains at the 2 position. The differential fatty acyl chain composition of inner compared to outer phosphatidylethanolamine indicates that rapid, random transmembrane migration (flip-flop) of phosphatidylethanolamine does not occur in the VSV membrane. The nature of the fatty acyl chain asymmetry observed in VSV phosphatidylethanolamine does not support the view that the     

A large-scale purification of phosphatidylethanolamine, lysophosphatidylethanolamine, and phosphatidylethanolamine, and phosphatidylcholine by high performance liquid chromatography: a partial resolution of molecular species

Egg yolk phospholipids, on a 10 g scale, were resolved by high-performance liquid chromatography on an 8-m silica column with elution by a stepwise chloroformmethanol gradient into homogenous phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and lysophosphatidylethanolamine fractions. Within these fractions, partial resolution on the basis of fatty acyl side chain composition was achieved.     

Egg yolk triglycerides during development of the domestic chicken

The triglycerides isolated from egg yolk lipids of eggs at various stages of incubation were fractionated according to the degree of unsaturation by argentation chromatography, and individual fractions were analyzed for their fatty acids by gas liquid chromatography. The proportions of the various fractions were constant during development. Fatty acid composition of the fractions were constant also. Fractions with one saturated fatty acid and two monoenoic fatty acids (SM₂) constituted over 40% of the total triglyceride. Palmitic acid constituted over 30%, and oleic acid over 45% of the fatty acid of total triglycerides. It is suggested that during development of thick embryo there is no selective utilization of the egg yolk triglycerides.     

Rapid transbilayer movement of phospholipids induced by an asymmetrical perturbation of the bilayer

Phospholipase D is used to convert egg phosphatidylcholine to phosphatidic acid in unilamellar vesicles. The transbilayer distribution of both lipids is determined by 31P NMR using paramagnetic ions. Phosphatidic acid formed in the outer monolayer is translocated to the inner monolayer with a halftime of 30-40 min or less. This is accompanied by an equally fast movement of part of the phosphatidylcholine from the inner to the outer monolayer. During these fast transbilayer movements the barrier properties of the vesicle bilayer are maintained.     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidyl-choline (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates. 1/T_1.5L, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.     

Saponins can cause the agglutination of phospholipid vesicles

The interaction of saponins with phospholipid vesicles was investigated by means of liposomal agglutination or a precipitation assay. Ginsenoside-Rc, which has an α-l-arabinofuranose residue at the non-reducing terminus, exhibited remarkable agglutinability toward egg yolk phosphatidylcholine vesicles, while other saponins lacking this characteristic sugar residue showed less or no agglutinability. The molar ratio of ginsenoside-Rc to egg phosphatidylcholine in the aggregates was estimated to be 0.4–0.5 by a precipitation assay using ¹⁴C-labeled egg phosphatidylcholine vesicles. The agglutination was inhibited by p-nitrophenyl α-l-arabinofuranoside but not by p-nitrophenyl β-d-glucopyranoside or arabinogalactan. The results indicated that the α-l-arabinofuranose residue in ginsenoside-Rc should be important for the expression of the agglutinability. The agglutinability of ginsenoside-Rc toward lipid vesicles depended on both the polar head groups and fatty acyl chains of phospholipids. Egg yolk phosphatidylcholine vesicles were strongly agglutinated by ginsenoside-Rc, although sphingomyelin, phosphatidylethanolamine, phosphatidic acid and phosphatidylserine were less agglutinated. The agglutinability of ginsenoside-Rc was effective for phosphatidylcholines with short or unsaturated fatty acyl chains. The results suggested that the interaction of ginsenoside-Rc with phospholipid membranes should be affected not only by the chemical structure of the phospholipid but also by the membrane fluidity.     

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.     

Effects of lipid headgroup and packing stress on poly(ethylene glycol)-induced phospholipid vesicle aggregation and fusion.

The effect of lipid headgroup and curvature-related acyl packing stress on PEG-induced phospholipid vesicle aggregation and fusion were studied by measuring vesicle and aggregate sizes using the quasi-elastic light scattering and fluorescence energy transfer techniques. The effect of the lipid headgroup was monitored by varying the relative phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents in the vesicles, and the influence of hydrocarbon chain packing stress was controlled either by the relative amount of PE and PC content in the vesicles, or by the degree of unsaturation of the acyl chains of a series of PEs, e.g., dilinoleoylphosphatidylethanolamine (dilin-PE), lysophosphatidylethanolamine (lyso-PE), and transacylated egg phosphatidylethanolamine (TPE). The PEG threshold for aggregation depends only weakly on the headgroup composition of vesicles. However, in addition to the lipid headgroup, the curvature stress of the monolayer that forms the vesicle walls plays a very important role in fusion. Highly stressed vesicles, i.e., vesicles containing PE with highly unsaturated chains, need less PEG to induce fusion. This finding applies to the fusion of both small unilamellar vesicles and large unilamellar vesicles. The effect of electrostatic charge on vesicle aggregation and fusion were studied by changing the pH of the vesicle suspension media. At pH 9, when PE headgroups are weakly charged, increasing electrostatic repulsion between headgroups on the same bilayer surface reduces curvature stress, whereas increasing electrostatic repulsion between apposing bilayer headgroups hinders intervesicle approach, both of which inhibit aggregation and fusion, as expected.     

Movement of cholesterol between vesicles prepared with different phospholipids or sizes

The rates of exchange of [4-14C]cholesterol between lipid vesicles prepared with different phospholipids and with different sizes have been measured. The first-order rate constants were higher using vesicles prepared from phosphatidylcholines with highly branched or polyunsaturated fatty acyl chains than with saturated diacyl or di-O-alkyl chains. The rate measurements indicate that the affinity of cholesterol for phospholipid does not vary significantly on change of the type of linkage (ether or ester) in phosphatidylcholine (PC) or of the positions of the fatty acyl chains in 1,2-diacyl-PC bearing one saturated and one unsaturated chain; furthermore, egg phosphatidylglycerol and egg phosphatidylethanolamine appear to have comparable affinities for cholesterol. However, the molecular packing in the bilayer and nearest-neighbor interactions involving cholesterol appear tightened more by N-palmitoylsphingomyelin than by dipalmitoyl-PC; on incorporation of 44 mol % of these phospholipids (which have the same fatty acyl chain composition) into either small or large unilamellar vesicles prepared with egg phosphatidylglycerol, the exchange rates were strikingly slower when the donor species contained sphingomyelin compared with PC. The rate of cholesterol exchange was 100% faster with small unilamellar vesicles than with large unilamellar vesicles as donors, suggesting that the looser packing in the highly curved small vesicles facilitates cholesterol desorption. The cholesterol exchange rate did not vary with the size of the acceptor vesicles, which indicates that desorption is the rate-limiting step in the exchange process in the presence of excess acceptors.     

Factors contributing to the distribution of free fatty acids among phospholipid vesicles

The distribution of free fatty acids at equilibrium after incubation of small sonicated unilamellar vesicles (SUV) with large unilamellar vesicles (LUV) of different lipid composition has been determined. Stearic acid (SA) and oleic acid (OA) showed similar preferences for SUV and LUV of egg yolk phosphatidylcholine (EYPC). Both ionized and protonated forms of the free fatty acids (FFAs) behaved similarly with respect to the equilibrium distribution between EYPC of different size. The charge of the vesicles was found, however, to be important, since both FFAs in their ionized form preferentially associated to vesicles of phosphatidylcholine (PC) as compared with vesicles of phosphatidylglycerol (PC). While SA preferred membranes in the gel state, OA showed preference for the membrane in fluid state. The insertion of both OA and SA in phosphatidylethanolamine (PE)/phosphatidylcholine vesicles is less favourable than in vesicles of pure PC. All these data suggest that membrane lipid content may play a role in determining the distribution of free fatty acids among the membranes of a cell.     

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.     

The asymmetric transmembrane distribution of phosphatidylethanolamine,phosphatidylserine, and fatty acids of the bovine retinal rod outer segment disk membrane

Summary The transmembrane distribution of the major aminophospholipids in the bovine retinal rod outer segment disk membrane, phosphatidylethanolamine and phosphatidylserine, was determined using a novel pair of permeable and impermeable covalent modification reagents. The values for the percentages of phosphatidylethanolamine and phosphatidylserine in the outer monolayer were calculated from a simple expression which takes into account the leakage of impermeable reagent into the disk lumen as monitored by the extent of labeling of lysine entrapped in the lumen. We infer from our results that at least 73 to 87% of the disk phosphatidylethanolamine and 77 to 88% of the disk phosphatidylserine are in the outer disk membrane monolayer. The fatty acid composition of the inner aminophospholipids is slightly more saturated than the outer aminophospholipids. Calculations using the lateral surface areas occupied by the disk membrane lipids suggest that 65 to 100% of the disk phosphatidylcholine is on the inner membrane surface. Since the disk phosphatidylcholine is also somewhat more saturated than the phosphatidylethanolamine and phosphatidylserine of the outer monolayer, the total inner membrane monolayer fatty acid composition is more saturated than that of the outer monolayer fatty acid composition.     

Cytochrome b5 induced flip-flop of phospholipids in sonicated vesicles

Cytochrome b5 induced flip-flop of phosphatidylethanolamine (PE) in sonicated vesicles prepared from a 9:1 mixture of phosphatidylcholine (PC) to phosphatidylethanolamine was determined as follows. First, vesicles having a nonequilibrium distribution of PE across the bilayer were prepared by amidinating the external amino groups with isethionyl acetimidate. Amidinated cytochrome b5 was then added, and after the protein was completely bound, the rate of appearance of fresh PE on the outer surface was determined by removing aliquots at timed intervals and titrating the external amino groups with trinitrobenzenesulfonic acid. The results show an initial rapid phase of flip-flop (especially in the presence of salt) followed by a very slow phase, at 25 degrees C. Similar results were obtained when cytochrome b5 was introduced into the amidinated vesicles by spontaneous transfer from PC donor vesicles. These results indicate that the accumulation of the transferable ("loose") form of cytochrome b5 on the outer surface of a vesicle causes a transient, global destabilization of the bilayer that is relieved by lipid flip-flop. We speculate that this mechanism may be a significant driving force for the transfer of amphipathic molecules across membranes.     

Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients

The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366]