Transbilayer exchange of phosphatidylethanolamine for phosphatidylcholine and N-acetimidoylphosphatidylethanolamine in single-walled bilayer vesicles.

A preparation of small single-walled liposome vesicles containing a 9:1 mole ratio of phosphatidylcholine to phosphatidylethanolamine was subjected to reaction with the membrane-impermeable reagent, isethionyl acetimidate hydrochloride. This reagent converted 90% of the external phosphatidylethanolamine groups to the amidine derivative, leaving the mole ratio of unreacted phosphatidylethanolamine to phosphatidylcholine on the outside surface of the vesicle much lower than that on the inside surface. Equilibration of phosphatidylethanolamine across the bilayer was then measured as a function of time by monitoring the appearance of phosphatidylethanolamine on the outside surface utilizing the reaction of the amino groups with 2, 4, 6-trinitrobenzenesulfonic acid. The results show that no new phosphatidylethanolamine appeared on the external surface of the vesicles over a period of 12 days at 22 degrees. A conservative estimate of the precision of the measurements is +/- 10%. On this basis, the estimated half-time for the equilibration of phosphatidylethanolamine across the bilayer of these vesicles must be at least 80 days at 22 degrees.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A(2). We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Transbilayer distribution of sterols in mycoplasma membranes: a review

The polyene antibiotic, filipin, binds to 3 beta-hydroxysterols. The initial rate of filipin-sterol association, monitored in a stopped-flow spectrophotometer, was first order in each reacting partner. The ratio of rate constants in intact mycoplasma cells relative to isolated, unsealed membranes provides an estimate of sterol distribution in the membrane bilayer. Cholesterol is distributed symmetrically in the bilayer of M. gallisepticum cells from the early exponential phase. However, in the M. capricolum membrane two-thirds of the unesterified cholesterol is localized in the outer leaflet; alkyl-sterols are distributed predominantly in the external monolayer. Cholesterol is translocated rapidly in the bilayer of M. capricolum cells. Exogenous phospholipids incorporated into the membrane had no effect on the cholesterol distribution in M. capricolum.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A₂. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

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.     

Transbilayer distribution of aminophospholipids and the oxidative stability of their component polyunsaturated fatty acids

We examined the relationship between the transbilayer distribution of aminophospholipids, such as phosphatidylethanolamine (PE), PE plasmalogen and phosphatidylserine, and the oxidative stability of polyunsaturated fatty acids (PUFAs) in the aminophospholipids. To modulate the transbilayer distribution of aminophospholipid in liposomes, we used phosphatidylcholine (PC) with two types of acyl chain region: dipalmitoyl (PC16:0) or dioleoyl (PC18:1). In the smaller-sized liposomes, the proportions of aminophospholipid in the liposomal external layer were significantly higher in liposomes containing PC18:1 than in those containing PC16:0. Additionally, aminophospholipids in the external layer of smaller-sized liposomes were able to protect their component PUFAs from 2,2'-azobis(2-amidinopropane)dihydrochloride-mediated lipid peroxidation.     

Transbilayer movement of cholesterol in phospholipid vesicles under equilibrium and non-equilibrium conditions

1. The exchange of [3H] cholesterol between phospholipid: cholesterol vesicles and an excess of red cell ghosts is examined. 2. Using a number of different phophatidylcholines, only the cholesterol thought to be associated with the outer half of the bilayer (about 70 percent) is available for exchange, suggesting that at least at equilibrium the transbilayer movement of cholesterol or "flip-flop", occurs very slowly, if it occurs at all. 3. The rate of exchange of cholesterol between the vesicles and the ghosts is dependent on the nature of the fatty acid chain of the phospholipids, being a function of both the fatty acid chain length and the degree of unsaturation. 4. Under non-equilibrium conditions, when cholesterol is being both exchanged and depleted from the lipid vesicles to red cell ghosts, the previously non-exchangeable vesicle cholesterol becomes available for exchange, suggesting that under these conditions "flip-flop" can occur.     

Transbilayer distribution and movement of lysophosphatidylcholine in liposomal membranes

Single bilayer vesicles were prepared by sonication of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine. Incubation with lysophospholipase results in a fast hydrolysis of 80–90% of lysophosphatidylcholine. The remaining lysophosphatidylcholine is only very slowly hydrolysed. There results are interpreted as lysophosphatidylcholine being asymmetrically distributed over the two halves of the bilayer. The slow phase of lysophosphatidylcholine hydrolysis sets an upper limit to the rate of transbilayer movement of lysophosphatidylcholine. The half time of this process at 37° C is estimated to be about 100 h. Incorporation of cholesterol in the vesicles reduces the distributional asymmetry of lysophosphatidylcholine to the extent of an outside-inside ratio of 60 : 40. [¹⁴C]Lysophosphatidylcholine introduced into the outer monolayer of such vesicles by intervesicular transfer of lysophosphatidylcholine remains virtually completely available for hydrolysis by lysophospholipases, corroborating the interpretation that transbilayer movement of lysophosphatidylcholine in these vesicles is an extremely slow process.In handshaken liposomes consisting of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine 15–20% of lysophosphatidylcholine is readily available for exogenous lysophospholipase. This pool may represent lysophosphatidylcholine in the outer monolayer of the liposomes.     

Transbilayer movement of fluorescent phospholipid analogues in the cytoplasmic membrane of Escherichia coli

We investigated the transmembrane movement of fluorescent labeled phospholipids in inverted inner membrane vesicles (IIMV) of Escherichia coli (E. coli) wild-type strain (MG1655), as well as in proteoliposomes reconstituted from detergent extracts of the IIMV. The transbilayer movement of 1-myristoyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoethanolamine (M-C6-NBD-PE) and -phosphocholine (M-C6-NBD-PC) was measured by a fluorescence stopped-flow back-exchange assay. Both analogues were rapidly translocated across the IIMV membrane, with half-times of <1 min (outward movement) and approximately 3 min (inward movement). No flip-flop was detected in protein-free liposomes, but in IIMV-derived proteoliposomes flip-flop of M-C6-NBD-PE occurred similarly to IIMV and could be largely eliminated by proteinase K treatment.     

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.     

NMR of molecules interacting with lipids in small unilamellar vesicles

Detailed characterization of protein, peptide or drug interactions with natural membrane is still a challenge. This review focuses on the use of nuclear magnetic resonance (NMR) for the analysis of interaction of molecules with small unilamellar vesicles (SUV). These phospholipid vesicles are often used as model membranes for fluorescence or circular dichroism experiments. The various NMR approaches for studying molecule-lipid association are reviewed. After a brief survey of the SUV characterization, the use of heteronuclear NMR (phosphorous, carbon, fluorine) is described. Applications of proton NMR through transferred nuclear Overhauser effect to perform structural determination of peptide are presented. Special care is finally given to the influence of the kinetic of the interactions for the proton NMR of bound molecules in SUV, which can constitute a good model for the study of dynamical processes at the membrane surface. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Transbilayer movement of 24Na in sonicated phosphatidylcholine vesicles exposed to frequency-modulated microwave radiation

Sonicated egg phosphatidylcholine vesicles loaded with ²⁴Na⁺ were exposed at 20mW to a frequency-modulated (3 Hz) microwave field in the range of 2350 to 2550 MHz, or at 80 mW to a 2450-MHz CW (continuous wave) field, in a waveguide. The vesicle suspension absorbed microwaves at about 1 mW/ml and 25 mW/ml (CW experiment). The average temperature change of the irradiated suspension was < 0.1 °C from ambient. Leakage of ²⁴Na⁺ from the vesicles for up to 19 hours was measured. No difference was noted in the movement of ²⁴Na⁺ from the vesicles in the irradiated and control dispersions.     

Interaction of trypsin with multilamellar vesicles of soybean lipids

The pH dependence of complex formation of trypsin with multilamellar vesicles (MLV) of soybean lipids has been investigated. The lipids were characterized by the same phospholipid composition, but the content of other lipids differed. Decrease of pH or introduction of negatively charged components into the lipid samples increased trypsin content in the protein-lipid complexes. This suggests electrostatic interaction between the protein and soybean lipids. The dependence of trypsin activity in the complexes with MLV on their concentration and on the presence of an ionic detergent was studied. Trypsin-MLV interaction did not result in complete inactivation of the protein molecules. Moreover, the effects of dilution and addition of ionic detergent on trypsin activity were additive. Using a fluorescence technique, complex formation with MLV was found to stabilize trypsin molecules, preventing their autolysis.     

Transbilayer distribution of alpha-tocopherol and lipid asymmetry in nerve tissue membranes

The alpha-tocopherol content and fatty acid composition of lipids in various types of nervous tissue membranes were studied. The transbilayer distribution of alpha-tocopherol and polyunsaturated fatty acids in liposomes and plasma membranes of synaptosomes was examined. It was shown that both phosphatidylethanolamine and phosphatidylserine are localized predominantly in the inner monolayer and they contain the bulk of polyenoic fatty acid residues. alpha-Tocopherol incorporated into liposomes from synaptosome plasma membrane lipids and present in synaptosome plasma membranes is also predominantly localized in the inner monolayers. No asymmetrical distribution of incorporated alpha-tocopherol was observed in liposomes prepared from a single phospholipid, e.g., dioleoylphosphatidylcholine.     

Movement of vesicles in cytoplasmic streaming in plasmodium

The moving velocities of vesicles in the cytoplasmic streaming of a slime mold were measured, in which all of the vesicles passing through a designated window were counted. Vesicles in the streaming are distributed in their moving velocities and the distribution itself varies with time. The mean velocity of vesicles and its standard deviation were found to exhibit a linear relationship, suggesting a possibility that vesicles in the cytoplasm would also be involved in force generation.     

Transbilayer distribution of phospholipids in photoreceptor membrane studied with trinitrobenzenesulfonate alone and in combination with phospholipase D

In a further study of the transbilayer distribution of phospholipids in rod disk membranes, the amino group reagent, trinitrobenzenesulfonate, and the phospholipid-hydrolyzing enzyme, phospholipase D, have been used alone and in combination.Under carefully defined conditions (1 mM trinitrobenzenesulfonate, pH 7.4, 20°C, darkness), trinitrobenzenesulfonate yields limited final levels of modification of phosphatidylethanolamine and phosphatidylserine, suggesting only minor reagent penetration and membrane disturbance under these conditions.Treatment of stacked disks with trinitrobenzenesulfonate under these conditions leads to a biphasic modification of the a aminophospholipids. Relatively fast (less than 1 h) modification of 50% phosphatidylethanolamine and 40% phosphatidylserine occurs, slowly rising (approx. 3 h) to 60 and 50%, respectively.Extensive treatment of stacked disks with phospholipase D leads to the hydrolysis of 55% phosphatidylcholine and 50% phosphatidylethanolamine, while phosphatidylserine is hardly attacked by this enzyme.Treatment of stacked disks with trinitrobenzenesulfonate after prior treatment with phospholipase D leads to no further modification than that maximally obtained with either reagent alone: about one-half of the three major phospholipid classes is accessible. Although both reagents differ greatly in molecular size, mode of action and other properties, they apparently see the same pool of phosphatidylethanolamine, their joint substrate. Considering that we start with the original right-side-out configuration, that all phospholipids can in principle be modified (no shielding) and that the membrane remains essentially intact, we conclude that the accessible lipid pool represents the outer face of the disk membranes.These results confirm our earlier conclusions from treatment with three phospholipases that the three major phospholipids are nearly symmetrically distributed over the two faces of the disk membrane.The divergence with the conclusions of other investigators is most likely explained by their use of disk membranes (disk vesicles) in which the original phospholipid distribution had not been maintained and/or of conditions under which trinitrobenzenesulfonate markedly penetrates the membrane.     

Transbilayer phosphatidylcholine distributions in small unilamellar sphingomyelin-phosphatidylcholine vesicles: effect of altered polar head group

The effect of the altered polar head group of phosphatidylcholine (PC) on its transbilayer distributions in small unilamellar vesicles containing sphingomyelin (SM) was ascertained with phospholipase A2 as the external membrane probe. These vesicles were formed by sonication and fractionated by centrifugation. The vesicle size was determined by gel-permeation chromatography and solute entrapment. Experiments were done to confirm that phospholipase A2 treatments did not induce fusion, lyse the vesicles, or cause PC to migrate across the vesicle bilayer. The complete degradation of external PC in intact vesicles was assured by carrying out the enzyme reactions in the absence as well as in the presence of 9.2 X 10(-5) M bovine serum albumin. In small vesicles comprised of SM and 30 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC preferentially distributed in the inner monolayer. This preference of DPPC in these vesicles disappeared upon introducing one C2H5 group at the carbon atom adjacent to the quaternary ammonium residue in its polar head group and was reversed when the C2H5 group was replaced by C6H5 and C6H5CH2 substituents or when the P-N distance was increased. These results indicate that the effective polar head-group volume is an important factor in determining the phospholipid distributions across the small vesicle bilayer.     

Relationship of upper body fat distribution to serum glucose and lipids in a Costa Rican population

Present models of the relation between subcutaneous fat distribution and serum biochemistries have been based largely on U.S. White populations. To determine interpopulational differences in that relation, we measured 68 clinically normal adult Costa Ricans aged 17-32. Data collected included six skinfolds: triceps, subscapular, suprailiac, umbilical, anterior mid-thigh, and medial calf; height, weight, and four fasting serum parameters: glucose, triglyceride, cholesterol, and high-density lipoprotein (HDL). Correlations between standardized skinfold ratios and biochemistries were highest--on the order of 0.40-0.50--for upper-lower body contrasts to triglyceride and cholesterol in males and to glucose and HDL in females. Canonical correlation analysis, with body mass index partialed out, found significant correlations for the first male variate and the first two female variates. The first male variate was positively weighted on subscapular fatness and on triglyceride and cholesterol, respectively. The two female skinfold variates were positively weighted on subscapular and on outer limbs, respectively, while their corresponding biochemical variates were weighted on glucose and triglyceride and on cholesterol and HDL, respectively. These findings are generally consistent with those based on U.S. populations but suggest that in non-Anglo populations, upper trunk fatness may be more relevant than anterior waist fatness to biochemical dysfunction.