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.     

Single bilayer vesicles prepared without sonication. Physico-chemical properties.

Single shelled lecithin vesicles of uniform size (diameter = 300 A) are prepared without sonication by solubilizing unsonicated lecithin dispersions with sodium cholate and removing the detergent from the mixed lecithin - cholate micelles by gel filtration on Sephadex G-50. A homogeneous population of pure lecithin single-bilayer vesicles free of multilamellar structures is obtained. The vesicle diameter is somewhat larger than the average diameter of sonicated vesicles. The curvature of the bilayer seems to be sufficiently large to allow for similar packing densities (areas/molecule) on the outer and inner layer of the bilayer. The morphology and some physico-chemical properties of these vesicles are described and compared with those of sonicated vesicles.     

The effect of free cholesterol on the solubilization of cholesteryl oleate in phosphatidylcholine bilayers: A 13C-NMR study

The solubilization of cholesteryl oleate in sonicated phosphatidylcholine vesicles containing between 0 and 50 mol% cholesterol was studied by 13C-NMR using isotopically enriched [carbonyl-13C]cholesteryl oleate. The carbonyl-13C chemical shift from cholesteryl oleate in the phospholipid/cholesterol bilayer was significantly downfield from that for cholesteryl oleate in an oil phase and the peak area, relative to that of the phospholipid carbonyl, was used to determine bilayer solubility of the ester. The solubility (with respect to phospholipid) in the phospholipid bilayer without cholesterol (2.9 mol%) was only moderately reduced (to 2.3 mol%) at cholesterol levels up to 33 mol% but showed a more marked reduction to 1.4 mol% at 40 mol% cholesterol or 1.2 mol% at 50 mol% cholesterol. Since the vesicles containing 50 mol% cholesterol were larger (520 +/- 152 A diameter) than those with no cholesterol (291 +/- 97 A diameter), we measured the solubility of cholesteryl oleate in large vesicles with no cholesterol, prepared by extrusion through polycarbonate membrane filters, and found it similar to that in small, sonicated vesicles with no cholesterol. Therefore, the larger size of vesicles was not the factor responsible for the decreased cholesteryl oleate solubility at high cholesterol contents. A more direct effect of cholesterol is envisioned where the ester becomes displaced to deeper regions of the bilayer.     

Vesicles from mixtures of bipolar archaebacterial lipids with egg phosphatidylcholine

Bipolar lipids from the membranes of archaebacterium Caldariella acidophila can form small unilamellar liposomes, when sonicated from lipid mixtures containing at least 25 mol% egg phosphatidylcholine. With increasing contents of archaebacterial lipid the inner radius of highly sonicated vesicles increases (from approx. 90 Å to approx. 160 Å) concomitant with an enhanced asymmetric distribution of the phosphatidylcholine molecules towards the outer face of the lipid bilayer membranes.     

Comparative action of lysophospholipases on acyl-oxyester and acyl-thioester substrates in micellar and membrane-bound form

Phospholipid analogs in which the acyl-oxyester bond is replaced by an acyl-thioester bond represent convenient substrates for sensitive assays of lipolytic enzymes. It has previously been found that such thioester substrates are hydrolyzed at higher rates than their oxyester counterparts. For bovine liver lysophospholipase II the preferential hydrolysis of thioesters appeared to be due to the thioester linkage per se rather than to the formation of preferred interfaces. The preferential hydrolysis of thioesters persisted when thioester and oxyester substrates were presented to the enzyme either as mixed micelles or incorporated in the bilayer of phospolipid vesicles. The transbilayer distribution of thioester and oxyester substrates in sonicated phospholipid vesicles is identical with no apparent indications for transbilayer movement of both substrates.     

Interaction of Adriamycin with Single and Multibilayer Dipalmitoylphosphatidylcholine Vesicles: Spin-Labeling and Calorimetric Study

Abstract

The effects of adriamycin on the organization of dipalmitoylphosphatidylcholine (DPPC) membranes alone or in the presence of 1 mol% cardiolipin (CL) in the form of single and multibilayer vesicles has been studied by spin-labeling, and by high sensitivity differential scanning calorimetry. With sonicated small unilamellar vesicles (SUVs), adriamycin increased the order parameter of 5-doxylstearate spin-labeled vesicles, an effect primarily observed in the gel phase of DPPC. thermal transition profiles, obtained by high-sensitivity differential scanning calorimetry and by 2,2,6,6-tetramethylpiperidinyl-l-oxy (TEMPO) partitioning studies, indicated that the drug induced aggregation and fusion of SUVs, especially at high drug-lipid molar ratios, and this phenomenon was further verified by negative-staining electron microscopy. the presence of J mol% CL in the lecithin bilayer markedly enhanced the effect of adriamycin on membrain order and fusion, especially under conditions of low ionic strength. the ordering effect of the drug was insensitive to the presence of other acidic phospholipids and was only partially inhibited by Ca²⁺ or Mg²⁺. In contrast to the small and highly-curved SUVs, however, the phase behavior of fused unilamellar vesicles (FUVs) or multilamellar vesicles (MLVs) was not significantly affected by the drug, suggesting that the bilayer curvature is an important factor in the interaction of the antibiotic with the corresponding bilayers. these findings demonstrate that changes in the bilayer packing configuration, due to differences in the radii of the curvature of the vesicles, must be considered in studies of adriamycin-lipid membrane interactions, as well as the phospholipid composition of the vesicles.     

Purification of acyl CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase

Acyl coenzyme A:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase (EC 2.3.1.23) is capable of forming lipid bilayer vesicles from its soluble substrates lysophosphatidylcholine (LPC) and oleoyl CoA. This suggested a purification method in which rat liver microsomes are first washed with deoxycholate to increase specific activity of the endogenous acyltransferase approximately fivefold, then solubilized by the detergent effect of excess LPC and oleoyl CoA in 1:1 stoichiometric ratios. As the LPC is converted to phosphatidylcholine by acyl group transfer, the detergent effect is lost and lipid vesicles containing the enzyme activity are produced. Other microsomal proteins are excluded from the vesicles. The vesicles may be separated by density gradient flotation and are found to contain acyltransferase with a specific activity of 9–10 µmol/mg/min. This reflects a purification of approximately 140-fold, about ten times greater than achieved in previous studies.     

Protein-mediated transbilayer movement of lysophosphatidylcholine in glycophorin-containing vesicles

1. Sonicated glycophorin-containing vesicles of dioleoyl phosphatidylcholine have been made. The outside-inside distributions of the lipid molecules in these vesicles was measured with NMR and was found to be comparable with that of protein-free vesicles. 2. The transbilayer distribution of palmitoyl lysophosphatidylcholine in these vesicles is such that they have a significantly higher content of the lyso-compound in the inner monolayer when compared with vesicles without glycophorin. 3. Lysophosphatidylcholine, added to pre-existing glycophorin-containing vesicles, is incorporated in the outer monolayer of these vesicles. Subsequently it is able to move to the inner monolayer with an estimated half time of about 1.5 h at 4 degrees C. This was measured with 13C-NMR using [N-13CH3]lysophosphatidylcholine. 4. Treatment of co-sonicated vesicles of phosphatidylcholine and lysophosphatidylcholine containing glycophorin with the enzyme lysophospholipase results in a complete degradation of the lyso-compound. A half time of transbilayer movement of lysophosphatidylcholine during this experiment was estimated to be about 1 h at 37 degrees C.     

Studies on the transverse localization of lysophospholipase in bovine liver microsomes using proteolytic enzymes.

1. Sonication of bovine liver microsomes completely solubilized the membrane-bound lysophospholipase II (EC 3.1.1.5). Co-chromatography with purified 125I-labelled lysophospholipase indicated that the enzyme was solubilized from microsomes in a lipid-free state. 2. In the presence of residual microsomal membranes, the solubilized lysophospholipase could only be partly degraded by trypsin (EC 3.4.21.4). Therefore, trypsin could not be used to study the transmembrane disposition of lysophospholipase in intact microsomes. 3. Chymotrypsin (EC 3.4.21.1) destroyed the solubilized lysophospholipase activity, even in the presence of residual microsomal membranes. 4. Lysophospholipase in intact microsomal vesicles was resistant to chymotrypsin digestion. 5. When microsomal vesicles were made leaky with lysophosphatidylcholine, chymotrypsin destroyed more than 95% of the lysophospholipase activity. 6. It is concluded from these experiments that at least the active center of lysophospholipase is located at the luminal side of the bovine liver microsomal membrane.     

Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.     

Localization of lysophosphatidylcholine in bovine chromaffin granules.

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1'-alkenyl glycerophosphocholine. The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.     

Comparison of bile acid binding to sinusoidal and bile canalicular membranes isolated from rat liver

Simon et al. (J. Clin. Invest., 70 (1982) 401) studied cholate binding to crude liver plasma membrane vesicles and suggested that the binding may represent mainly the binding to the receptor (carrier) on the canalicular membrane. This hypothesis was supported by finding a good correlation between the number of cholate binding sites on liver plasma membrane and the maximal rate of biliary secretion (Tm) for taurocholate. We studied bile acid binding to sinusoidal and canalicular membrane vesicles isolated from rat liver by a rapid filtration technique. Scatchard analysis of the saturation kinetics showed both [3H]cholate and [3H]chenodeoxycholate bind to two classes of binding site on each membrane. However, little difference was observed between the binding to sinusoidal and canalicular membrane vesicles for each bile acid (cholate, Kd1 = 10.4 and 19.8 microM, n1 = 31.0 23.6 pmol/mg protein, Kd2 = 1.32 and 1.73 mM, n2 = 13.1 and 23.4 nmol/mg protein; and chenodeoxycholate, Kd1 = 0.207 and 0.328 microM, n1 = 36.7 and 27.4 pmol/mg protein, Kd2 = 1.16 and 2.26 mM, and n2 = 20.6 and 24.2 nmol/mg protein; numbers show the mean values sinusoidal and canalicular membrane vesicles, respectively). Chenodeoxycholate binding to sinusoidal membrane vesicles was markedly inhibited by cholate but not by Rose bengal, an organic anion dye. These studies indicate that both membranes (sinusoidal and canalicular membrane vesicles) have two kinds of binding site for bile acids, although no clear difference in the binding properties was observed between the two membranes. Consequently, the cholate binding Simon detected may represent the binding not only to canalicular membrane vesicles but also to sinusoidal membrane vesicles.     

Selective removal of lipids from the outer membrane layer of human erythrocytes without hemolysis. Consequences for bilayer stability and cell shape

(1) Treatment of erythrocytes with phospholipase A₂ from bee venom cleaves about 55% of the phosphatidylcholine in the outer membrane lipid layer without changing the discoid shape of the cells. All of the fatty acids and 80% of the lysophosphatidylcholine produced under this conditions can be sequentially extracted by bovine serum albumin without hemolysis of the cells. (2) The cells remain discoid up to extraction of all of the fatty acids and 15% of the lysophosphatidylcholine. Removal of a higher fraction of lysophosphatidylcholine induces formation of stomatocytes and sphero-stomatocytes, probably going along with an internalization of membrane vesicles. Stomatocytosis can be explained on the basis of the ‘bilayer couple hypothesis’ (Sheetz, M.P. and Singer, S.J. (1974) Proc. Natl. Acad. Sci. 71, 4457–4461). The shape change will compensate for the differences in surface pressure between the two leaflets induced by selective removal of material from the outer leaf of the bilayer. (3) Increasing the shear modulus of the membrane by diamide prevents this compensatory shape change even after extraction of up to 80% of the lysophosphatidylcholine, which amounts to a loss of 34% of the phospholipids of the outer membrane layer or 22% of its area. This leads to the interesting situation of a membrane possibly having a strikingly diminished ratio of the numbers of phospholipid molecules in the outer to that in the inner lipid layer. A marked difference in surface pressures should arise in this situation, unless other compensatory mechanisms become operative. Evidence for a compensation for outer lipid loss by a constriction of the inner layer has been obtained. A compensation by transbilayer reorientation of phospholipids could not be demonstrated. This latter observation supports the concept of a stabilisation of the asymmetric phospholipid arrangement by proteins such as spectrin.     

Interaction of cholesterol with photoactivable phospholipids in sonicated vesicles

Photoactivable phospholipids containing either α-diazo-β-trifluoropropionyloxy or m-diazirinophenoxyl groups in the ω-positions of sn-2 fatty acyl chains were synthesized and incorporated into sonicated vesicles containing 33 mol% of cholesterol. Photolysis of the vesicles at 350 nm produced covalent cross-links between the synthetic phospholipids and cholesterol. The cross-linked products obtained using [¹⁴C]cholesterol were characterized by their chromatographic behavior, cleavage on phospholipase A₂ treatment, base-catalyzed transesterification and mass spectral measurements. The cross-linking was shown not to involve the 3-β-hydroxyl group of cholesterol, and it was concluded that the reactive carbene intermediates formed from the photolabels inserted into the hydrocarbon skeleton of cholesterol in the bilayer. The extent of cross-linking obtained was comparable to that observed previously using phospholipids alone, indicating that no lateral phase separation occurred. The present approach is promising for further precise studies of the molecular interactions between cholesterol and phospholipids in biological membranes.     

Solubilization of DMPC and DPPC vesicles by detergents below their critical micellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles

The interaction of sodium deoxycholate, sodium cholate and octyl glucoside with sonicated vesicles of L alpha-dimyristoyl-phosphatidylcholine (DMPC) and L alpha-dipalmitoylphosphatidylcholine (DPPC) at concentrations below the critical micellization concentration (cmc) of the detergents was studied by high-sensitivity DSC (hs-DSC), Fourier transform infrared spectroscopy (FT-IR) and freeze-fracture electron microscopy. The two phospholipids exhibited a striking different thermotropic behaviour in the presence of these detergents. For DPPC vesicles, the detergents were found to interact exclusively in the aqueous interface region of the bilayer below the membrane saturation concentration Rsat while in DMPC vesicles two coexisting interaction sites below this concentration persist. These are detergents which interact at the aqueous interface region (site 1) and in the acyl chain region (site 2) of the DMPC vesicles. The partition coefficients K of the detergents between DPPC vesicles and the water phase were calculated from the hs-DSC results at two detergent/phospholipid molar ratios Rtot less than or equal to Rsat as 0.35, 0.049 and 0.040 mol-1 for sodium deoxycholate, sodium cholate and octyl glucoside, respectively. In contrast, for DMPC the K values for Rtot less than or equal to Rsat were found to be dependent on Rtot due to the occupation of site 2 by the detergents above a certain Rtot. The model is discussed on the basis of the detergents free energies of transfer from the water phase to site 1 and site 2 of the vesicles, respectively. The solubilization behaviour of DPPC vesicles, dependent on whether the total detergent concentration is above or below the cmc at Rsat, differed significantly as revealed by hs-DSC. This suggests that in the latter case an additional hydrophobic effect could facilitate the formation of disc shaped mixed micelles. Moreover, this different behaviour was employed to measure the cmc values of the detergents studied in the presence of the vesicles by hs-DSC.     

The presence of lysophosphatidylcholine in chromaffin granules

Lysophosphatidylcholine is thought to be a characteristic component of the chromaffin granules in adrenal glands. By the use of a t.l.c. system that resolves minor phospholipids satisfactorily, this subcellular location was confirmed in the present study in bovine glands. However, phospholipid degradation was demonstrated in homogenates of the adrenal medulla and cortex under conditions similar to those of subcellular fractionation (incubation at 4°C for 90min). Phosphatidylethanolamine and cardiolipin were hydrolysed, but the concentration of lysophosphatidylcholine did not change, indicating that the latter was present in the medulla before this treatment. Attempts were made to decrease the time between death of the animal and the extraction of lipids. Lysophosphatidylcholine was easily demonstrable in lipid extracts of the dissected medulla and even in those of the whole bovine gland. For practical reasons it is not possible to decrease further the time lapse before extraction in the case of this animal. Adrenal glands were obtained from anaesthetized and untreated rabbits. These were frozen immediately in liquid N₂ and the lipids were extracted. In a control experiment, the glands from rabbit were dissected and treated in the same manner as with those of ox, and then the lipids were extracted. No lysophosphatidylcholine was detected in the extracts from glands frozen in liquid N₂ but lysophosphatidylcholine was observed in the controls. These results suggest that lysophosphatidylcholine is not a component of chromaffin granules, but is produced if the period between death of the animal and lipid extraction is unduly prolonged. To discover whether lysophosphatidylcholine affected the permeability barrier properties of chromaffin granules, sonicated liposomes of egg phosphatidylcholine alone or with lysophosphatidylcholine (15mol/100mol) were prepared. Both types were shown by electron microscopy to be largely made up of single bilayer vesicles. The exchange diffusion of [¹⁴C]dopamine was measured across their membranes. Both types of liposomes had similar capture volumes (0.5μl/μmol of phospholipid), and the activation energies of the exchange diffusion of dopamine were also similar (31kJ/mol). These results indicate that the presence of this proportion of lysophosphatidylcholine in chromaffin-granule membranes is not likely to influence their barrier properties towards catecholamines.     

Localization of lysophosphatidylcholine in bovine chromaffin granules

One of the unique features of the chromaffin granule membrane is the presence of about 17 mol% lysophosphatidylcholine. Lysophosphatidylcholine isolated from the granules could be degraded by approx. 94% by lysophospholipase. This result is consistent with chemical analyses data showing that about 9% of this lysophospholipid is 1′-alkenyl glycerophosphocholine.The localization of the acylglycerophosphocholine in the chromaffin granule membrane was studied by using pure bovine liver lysophospholipases. In intact granules only about 10% of the total lysophosphatidylcholine was directly available for enzymic hydrolysis. In contrast, when granule membranes (ghosts) were treated with lysophospholipases approx. 60% of the lysophosphatidylcholine was deacylated. These values did not increase after pre-treatment of intact granules or ghosts with trypsin. Added $\text{1-[1-}{}^{14}\text{C]palmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ did not mix with the endogenous lysophosphatidylcholine pool(s) and remained completely accessible to added lysophospholipases.     

Spontaneous vesiculation of uncharged phospholipid dispersions consisting of lecithin and lysolecithin

The work presented here demonstrates that the phenomenon of spontaneous vesiculation is not restricted to charged lipids and lipid mixtures, but occurs also in isoelectric phospholipid mixtures consisting of egg phosphatidylcholine (EPC) and egg lysophosphatidylcholine (lyso-EPC). 1H high-resolution NMR and freeze-fracture electron microscopy have been used to characterize the mixed EPC/lyso EPC dispersions in excess H2O. The predominant phase in these mixed phospholipid dispersions is smectic (lamellar) at least up to approximately 70% lysophosphatidylcholine. The type of phospholipid aggregate formed in excess H2O depends on the mole ratio diacyl to monoacyl phosphatidylcholine. The dispersive (lytic) action of lysophosphatidylcholine on phosphatidylcholine bilayers becomes effective at lysophospholipid contents in excess of approximately 10%. Large multilamellar liposomes are disrupted and replaced by smaller particles, mainly unilamellar vesicles. Between 30 and 70% lysophosphatidylcholine a significant proportion of the total phospholipid is present as small unilamellar vesicles (SUV) of a diameter of 23 nm (range: 20-70 nm). At even higher lysophosphatidylcholine contents the fraction of phospholipid present as small mixed micelles with a diameter smaller than about 14 nm grows at the expense of the vesicular structures. There is a second effect of increasing the quantity of lysophosphatidylcholine in phosphatidylcholine bilayers: the presence of lysophosphatidylcholine in excess of 10% renders the phospholipid bilayer more permeable to ions as compared to pure phosphatidylcholine bilayers. The key factor in inducing spontaneous vesiculation is probably not the charge but the wedge-like shape of the lysophospholipid molecule. The molecular shape may give rise to an asymmetric distribution of lysophosphatidylcholine between the two halves of the bilayer, thus stabilizing highly curved bilayers as present in SUV.