Lysine peptides induce lipid intermixing but not fusion between phosphatidic acid-containing vesicles

Penta(L-lysine) and poly(L-lysine) (but not di- or tri(-L-lysine)) were found to induce rapid and extensive phospholipid intermixing between unilamellar vesicles consisting largely of phosphatidic acid and phosphatidylethanolamine, without causing significant intermixing of, nor leakage from, the encapsulated aqueous spaces. Neither did the size of such vesicles increase after treatment with lysine peptide, as determined by gel chromatography. The results suggest that lysine oligo-(or poly-) peptides may induce reversible semifusion between vesicles, but not complete membrane fusion.

Pentalysine Polylysine Phosphatidic acid Lipid intermixing Membrane fusion     

Binding of CTP: phosphocholine cytidylyltransferase to large unilamellar vesicles

We have studied the binding of CTP: phosphocholine cytidylyltransferase from HeLa cell cytosol to large unilamellar vesicles of egg phosphatidylcholine (PC) or HeLa cell phospholipids that contain various amounts of oleic acid. A fatty acid/phospholipid molar ratio exceeding 10% was required for CTP: phosphocholine cytidylyltransferase binding to liposomes. At a fatty acid/phospholipid molar ratio of 1; 85% of the cytosolic CTP: phosphocholine cytidylyltransferase was bound. The enzyme also bound to liposomes with at least 20 mol% palmitic acid, monoolein, diolein or oleoylacetylglycerol. Oleoyl-CoA did not promote enzyme binding to liposomes. Binding to oleate-PC vesicles was blocked by Triton X-100 but not by 1 M KCl, and was reversed by incubation of the vesicles with bovine serum albumin. Cytidylyltransferase bound to egg PC vesicles that contained 33 mol% oleic acid equally well at 4 degrees C and 37 degrees C. The enzyme also bound to dimyristoyl- and dipalmitoylphosphatidylcholine vesicles containing oleic acid at temperatures below the phase transition for these liposomes. Binding of the cytidylyltransferase to egg PC vesicles containing oleic acid, monoolein, oleoylacetylglycerol or diolein resulted in enzyme activation, as did binding to dipalmitoylPC-oleic acid vesicles. However, binding to egg PC-palmitic acid vesicles did not fully activate the transferase. Various mechanisms for cytidylyltransferase interaction with membranes are discussed.     

Low pH induced membrane fusion of lipid vesicles containing proton-sensitive polymer

For the purpose of cytoplasmic delivery of aqueous content in liposomes through endosomes, we synthesized a pH-sensitive polymer, cetylacetyl(imidazol-4-ylmethyl)polyethylenimine (CAIPEI), which generates polycations at acidic pH. CAIPEI in its aqueous phase caused aggregation of sonicated vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) (molar ratio 1:4) when the pH of the solution was lowered. The polymer also induced membrane intermixing as measured by resonance energy transfer between vesicles containing N-(7-nitro-2,1,3-benz[d]oxadiazol-4-yl)phosphatidylethanolamine and those containing N-Rhodamine phosphatidylethanolamine at pH 4-5, while the addition of CAIPEI caused neither aggregation of PC vesicles nor the intermixing of liposomal membranes between PC and PC/PS vesicles at any pH. The CAIPEI-induced membrane intermixing was dependent on the polymer/vesicle ratio rather than on the polymer concentration. Then the polymer was incorporated into the bilayers of PC vesicles. These CAIPEI vesicles also caused membrane intermixing with liposomes containing PS under acidic conditions. The reconstituted CAIPEI did not reduce the trapping efficiency of vesicles or increase their permeability to glucose even at low pH. The vesicles caused the low pH induced aggregation and membrane intermixing with other negatively charged liposomes containing phosphatidic acid or phosphatidylglycerol. These results suggest that the protonation of the polymer at acidic pH endows the CAIPEI vesicles with the activity to fuse with negatively charged liposomes.     

Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium

Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.     

Lipase-catalyzed reactions in vesicles as an approach to vesicle self-reproduction

Abstract

As a continuation of our endeavor to find conditions under which bounded aggregate structures are able to self-reproduce, we have investigated the reactivity of lipase, both in free solution and vesicle-entrapped, against mixed oleic acid/oleate/ethyl oleate vesicles. Three types of vesicles have been prepared and characterized: (A) oleic acid/oleate vesicles; (B) oleic acid/oleate/ethyl oleate vesicles; and (C) lipase containing oleic acid/oleate vesicles. Long time stability studies by quasi elastic light scattering show that whereas (B) and (C) vesicles remain stable with a diameter of 110-130 nm and monodisperse for over a period of one month, vesicles (A) separated from an initial single population of 105 nm diameter into two populations, having respectively 70 nm diameter (more than 95% of the particles) and 180-210 nm diameter (less than 5% of the total population). In the case of vesicles (C), it could be shown that the enzyme remains localized inside the vesicles and it does not protrude into the water bulk phase. The enzymatic hydrolysis of ethyl oleate (which is water-insoluble) incorporated in the B-vesicles was studied under two configurations: (I) by adding lipase externally to the B-vesicles; (II) by mixing vesicles (B) and vesicles (C). In both cases, the reaction progressed to 100% hydrolysis. In the first case, the reaction was attended by an increase of the number of vesicles, and since this hydrolysis reaction takes place within the boundary of the parent vesicles, the criteria of autopoietic self-reproduction of vesicles are satisfied. In the case (II) instead, no increase of the population number of particles could be detected. The possible reasons for this difference are discussed.     

Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid

The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins. This pathway has been characterized further by studying the activation and incorporation of [9,10(n)-3H]oleic acid and transesterification of [1-14C]oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts. Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars. Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid. Compared to ghosts, the ability of inside-out vesicles to activate and incorporate [9,10(n)-3H]oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify [1-14C]oleoyl-CoA to phospholipid is diminished by over 50%. These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities. This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation.     

Growth and shape transformations of giant phospholipid vesicles upon interaction with an aqueous oleic acid suspension

The interaction of two types of vesicle systems was investigated: micrometer-sized, giant unilamellar vesicles (GUVs) formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and submicrometer-sized, large unilamellar vesicles (LUVs) formed from oleic acid and oleate, both in a buffered aqueous solution (pH 8.8). Individual POPC GUVs were transferred with a micropipette into a suspension of oleic acid/oleate LUVs, and the shape changes of the GUVs were monitored using optical microscopy. The behavior of POPC GUVs upon transfer into a 0.8 mM suspension of oleic acid, in which oleic acid/oleate forms vesicular bilayer structures, was qualitatively different from the behavior upon transfer into a 0.3 mM suspension of oleic acid/oleate, in which oleic acid/oleate is predominantly present in the form of monomers and possibly non-vesicular aggregates. In both cases, changes in vesicle morphology were observed within tens of seconds after the transfer. After an initial increase of the vesicle cross-section, the vesicle started to evaginate, spawning dozens of satellite vesicles connected to the mother vesicle with narrow necks or tethers. In 60% of the cases of transfer into a 0.8 mM oleic acid suspension, the evagination process reversed and proceeded to the point where the membrane formed invaginations. In some of these cases, several consecutive transitions between invaginated and evaginated shapes were observed. In the remaining 40% of the cases of transfer into the 0.8 mM oleic acid suspension and in all cases of vesicle transfer into the 0.3 mM oleic acid suspension, no invaginations nor subsequent evaginations were observed. An interpretation of the observed vesicle shape transformation on the basis of the bilayer-couple model is proposed, which takes into account uptake of oleic acid/oleate molecules by the POPC vesicles, oleic acid flip-flop processes and transient pore formation.     

The distribution of oleic acid between chylomicron-like emulsions, phospholipid bilayers, and serum albumin. A model for fatty acid distribution between lipoproteins, membranes, and albumin

In the process of lipoprotein lipolysis, masses of fatty acid are generated at the surface of the lipoprotein. The newly generated fatty acid may at least partly redistribute from the site of lipolysis to phospholipid-rich membranes and to albumin. We have studied the distribution of [1-13C]oleic acid in model systems consisting of chylomicron-like triacylglycerol-rich emulsions, unilamellar phosphatidylcholine vesicles, and bovine serum albumin. By using high resolution 13C NMR spectroscopy it was possible to distinguish fatty acid in each compartment (emulsion, vesicle, albumin) and quantitate the fatty acid distribution under various conditions of lipid compartment concentration and aqueous pH. When emulsions and vesicles were present in equivalent mass amounts, fatty acid exhibited a profound preference for the lipid bilayers. The release of oleic acid to phospholipid bilayers was presumably also a function of its high molar stoichiometry (5:1) with the albumin present. More equitable distributions of fatty acid between vesicles and emulsions were seen when higher concentrations of emulsion were used. The distribution of fatty acid between compartments was in good agreement with predictions made using the apparent ionization constant, expressed as pKapp, of 7.5 and the surface to core (phospholipid to triacylglycerol) distribution coefficient of 7.0, measured for unionized oleic acid in chylomicron particles (Spooner, P. J. R., Bennett Clark, S., Gantz, D. L., Hamilton, J. A., and Small, D. M. (1988) J. Biol. Chem. 263, 1444-1455). These results indicate that the affinities of fatty acid for phospholipid bilayer and chylomicron-like emulsion surfaces are equivalent. Redistribution of lipolytically generated fatty acid from chylomicron surface to cell membrane may simply be driven by the predominant quantity of the cell membrane surfaces.     

Uptake and utilization of free fatty acids supplied by liposomes to mammary tumor cells in culture.

Unesterified oleic acid was co-dispersed with various phospholipids in Ca-Mg-free saline and sonicated. Most of the fatty acid co-eluted with phospholipid vesicles when the sonicate was chromatographed on a column of Sepharose 4B. Cells from mouse mammary tumors, maintained in primary monolayer culture, were incubated with these phospholipid-oleic acid vesicles and optimal conditions for the uptake of oleic acid were established. When phosphatidylcholine and phosphatidylserine were used to make the vesicles, the oleic acid was taken up by cells more efficiently than was oleic acid complexed with albumin. The liposome-supplied unesterified oleic acid was rapidly incorporated into other cellular compounds, especially triglycerides and phospholipids. Growth of cells was measured after a 2-h pulse with the liposomes. The amount of DNA in treated cultures was 50% greater than in control cultures by 3 days after treatment. The magnitude of the growth response was sensitive to liposome dosage and to cell density in the treated cultures. This survey suggests that liposomes can be employed to insert materials directly into epithelial cells via their apical surface, that oleic acid so supplied is readily metabolized, and that solutes from liposomes can participate in functioning of the cells.     

Retention of aqueous contents during divalent cation-induced fusion of phospholipid vesicles

The relative kinetics of intermixing and release of liposome aqueous contents during Ca²⁺-induced membrane fusion has been investigated. Fusion was monitored by the Tb-dipicolinic acid (DPA) fluorescence assay. Release was followed by the relief of self-quenching of carboxyfluorescein or by Tb fluorescence, with essentially identical results. Fusion of large unilamellar vesicles (LUV) made of phosphatidylserine (PS) in 100 mM NaCl (pH 7.4) at 25°C was initially non-leaky, whereas the fusion of small unilamellar vesicles (SUV) was accompanied by partial release of contents. After several rounds of fusion, the internal aqueous space of the vesicles collapsed. The rate of intermixing of lipids, measured by a resonance energy transfer assay, and the rate of coalescence of aqueous contents during fusion were similar over a range of Ca²⁺ concentrations. Most of the aqueous contents were retained after the fusion of SUV (PS) in 5 mM NaCl and 1 mM Ca²⁺. LUV made of a 1:1 mixture of Bacillus subtilis cardiolipin and dioleoylphosphatidylcholine went through about two rounds of fusion in the presence of Ca²⁺ at 10°C, with complete retention of contents. Similar results were obtained with vesicles composed of phosphatidate/PS/phosphatidylethanolamine/cholesterol (1:2:3:2) in the presence of Ca²⁺ and synexin at 25°C. These results emphasize the diversity of the relative kinetics of fusion and release in different phospholipid vesicle systems under various ionic conditions, and indicate that the initial events in the fusion of LUV are in general, non-leaky.     

l-Glutamic Acid Fermentation

A study was made on the differences between Brevibacterium thiogenitalis No. 653 and its oleic acid-requiring mutant D-248 in some physiological characteristics.

The most important difference of the characteristics was found in their intracellular fatty acid contents. Namely, the cellular oleic acid content of D-248 was scarcely affected by biotin but limited by the oleic acid which was added to the medium.

On the other hand, various enzyme activities and rates of oxygen uptake for several organic acids were found to be slightly different between the two strains.

These observations suggest that oleic acid has an important role for the production of l-glutamic acid.

The effect of biotin and oleic acid on the cellular fatty acid contents, and the relation between the cellular fatty acid contents and the productivity of l-glutamic acid were investigated using Brevibacterium thiogenitalis No. 653 and its oleic acid-requiring mutant, D-248.

While the synthesis of palmitic acid in D-248 was stimulated by biotin and competitively reversed by oleic acid added to the culture medium, the level of cellular oleic acid was scarcely affected by biotin but regulated by oleic acid in the medium.

For the productivity of L-glutamic acid, the most important factor was the level of cellular oleic acid, and the effect of cellular palmitic acid was considerably weak. This relation was subjected to a figuration and able to be expressed on the whole as one exponential-like curve. An amount of over 70 per cent of cellular fatty acids was distributed in the phospholipid fraction and its fatty acid composition was almost the same as that of whole cells.     

Evidence for synthesis in vitro of neutral lipids by isolated oocytes of Perinereis cultrifera (Annelida,Polychaeta). A biochemical and autoradiographic study

Summary

Isolated oocytes of Perinereis cultrifera have been incubated in culture media with added [³H]glycerol, [¹⁴C]butyric acid or [¹⁴C]oleic acid. The principal neutral lipid synthesized was triacylglycerol, although incorporation of radioactivity into other lipid categories (sterol, fatty acid, wax ester) was also observed. A more significant percentage of triacylglycerol was labelled after incubation with [³H]glycerol and [¹⁴C]oleic acid than with [¹⁴C]butyric acid. With this precursor, monoacylglycerol appears to be the class of lipid compartment which initially show the most radioactivity. Electron microscopic autoradiography has revealed that labelling after incorporation of glycerol was mainly localized on the lipid droplets but not on the yolk granules. A second metabolic pathway is represented by phospholipid membrane synthesis.     

Effects of carbon sources on extracellular lipase production and lipA transcription in Acinetobacter calcoaceticus

  The effects of lactic acid, oleic acid, gum arabic and their mutual interactions, on the production of extracellular lipase and the regulation of the expression of the lipase encoding gene (lipA) in Acinetobacter calcoaceticus were investigated. Formation of extracellular lipase was measured in culture supernatants of wild-type strain BD413 and expression of the lipA gene was monitored in vivo with a chromosomal fusion of lipA to lacZ. At the level of lipA expression only oleic acid had a significant effect; it lowered expression. Neither gum arabic nor lactic acid had any effect on lipA expression. On the other hand, the yield of extracellular lipase increased 2–5 times by the addition of gum arabic, possibly due to the release of cell surface-bound lipase. An interaction between oleic acid and lactic acid was also detected. Journal of Industrial Microbiology & Biotechnology (2000) 24, 25–30. Received 03 May 1999/ Accepted in revised form 04 September 1999     

Proton-induced fusion of oleic acid-phosphatidylethanolamine liposomes

Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become destabilized, and fuse below pH 6.5 in 150 mM NaCl. Fusion is monitored by (i) the intermixing of internal aqueous contents of liposomes, utilizing the quenching of aminonaphthalene-3,6,8-trisulfonic acid (ANTS) by N,N'-p-xylylenebis(pyridinium bromide) (DPX) encapsulated in two separate populations of vesicles, (ii) a resonance energy transfer assay for the dilution of fluorescent phospholipids from labeled to unlabeled liposomes, (iii) irreversible changes in turbidity, and (iv) quick-freezing freeze-fracture electron microscopy. Destabilization is followed by the fluorescence increase caused by the leakage of coencapsulated ANTS/DPX or of calcein. Ca2+ and Mg2+ also induce fusion of these vesicles at 3 and 4 mM, respectively. The threshold for fusion is at a higher pH in the presence of low (subfusogenic) concentrations of these divalent cations. Vesicles composed of phosphatidylserine/phosphatidylethanolamine or of oleic acid/phosphatidylcholine (3:7 mole ratio) do not aggregate, destabilize, or fuse in the pH range 7-4, indicating that phosphatidylserine and phosphatidylcholine cannot be substituted for oleic acid and phosphatidylethanolamine, respectively, for proton-induced membrane fusion. Freeze-fracture replicas of oleic acid/phosphatidylethanolamine liposomes frozen within 1 s of stimulation with pH 5.3 display larger vesicles and vesicles undergoing fusion, with membrane ridges and areas of bilayer continuity between them. The construction of pH-sensitive liposomes is useful as a model for studying the molecular requirements for proton-induced membrane fusion in biological systems and for the cytoplasmic delivery of macromolecules.     

The effect of lipids on the enzymatic activity of 6-phosphofructo-1-kinase from B. stearothermophilus

6-Phosphofructo-1-kinase (PFK-1), a major regulatory enzyme in the glycolysis pathway, is a cytoplasmic enzyme with complicated allosteric kinetics. Here we investigate the effects of lipids on the activity of PFK from Bacillus stearothermophilus (BsPFK), to determine whether BsPFK shares any of the membrane binding or lipid binding properties reported for some mammalian PFKs. Our results show that large unilamellar vesicles (LUVs) composed of either the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or of 1:1 (mole ratio) DOPC and the fatty acid, oleic acid (OA), cause a three-fold increase in V_max, depending on the lipid concentration and vesicle composition, but no change in K_m. Further studies show lipids do not reverse the allosteric inhibitory effects of phosphoenolpyruvate (PEP) on BsPFK. SDS/PAGE studies do not show significant binding of the BsPFK tetramer to the surface of the phospholipid vesicles, suggesting that modulation of catalytic activity is due to binding of lipid monomers. By simulating the kinetics of BsPFK interaction with vesicles and lipid monomers we conclude that the change in BsPFK catalytic activity with respect to lipid concentration is consistent with monomer abstraction from vesicles rather than direct uptake of lipid monomers from solution.     

Ca(2+)-induced fusion of phospholipid vesicles containing free fatty acids: modulation by transmembrane pH gradients.

The influence of a transmembrane pH gradient on the Ca(2+)-induced fusion of phospholipid vesicles, containing free fatty acids, has been investigated. Large unilamellar vesicles composed of an equimolar mixture of cardiolipin, dioleoylphosphatidylcholine, and cholesterol, containing 20 mol % oleic acid, were employed. Fusion was measured using a kinetic assay for lipid mixing, based on fluorescence resonance energy transfer. At pH 7.5, but not at pH 6.0, in the absence of a pH gradient, oleic acid stimulates the fusion of the vesicles by shifting the Ca2+ threshold concentration required for aggregation and fusion of the vesicles from about 13 mM to 10 mM. In the presence of a pH gradient (at an external pH of 7.5 and a vesicle interior pH of 10.5), the vesicles exhibit fusion characteristics similar to vesicles that do not contain oleic acid at all, consistent with an effective sequestration of the fatty acid to the inner monolayer of the vesicle bilayer induced by the imposed pH gradient. The kinetics of the fusion process upon simultaneous generation of the pH gradient across the vesicle bilayer and initiation of the fusion reaction show that the inward movement of oleic acid in response to the pH gradient is extremely fast, occurring well within 1 s. Conversely, dissipation of an imposed pH gradient, by addition of a proton ionophore during the course of the fusion process, results in a rapid enhancement of the rate of fusion due to reequilibration of the oleic acid between the two bilayers leaflets.     

Model systems of precursor cellular membranes: long-chain alcohols stabilize spontaneously formed oleic acid vesicles

Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid.     

Uniform preparations of large unilamellar vesicles containing anionic lipids

A general procedure for the preparation of large unilamellar vesicles of selected sizes has been developed. The procedure consists of dissolving the lipid in organic solvent, washing with mild acid, removing the solvent, adding salt (0.15 M KCl) solution, and adjusting the pH (raising it to about pH 10 and lowering it immediately to pH 7.55). The procedure takes less than 30 min. The resulting unilamellar vesicles are of a single size with a rather low standard deviation. The sizes of these preparations range between 150 and 1000 nm in diameter. Sizes and polydispersities were measured to within 1-2% by photon correlation spectroscopy. Vesicle size varies with the phospholipid structure, the composition of the phospholipid mixture, the ionic strength of the medium, the alkyl chain composition, the cholesterol content of the phospholipid mixture, and the timing of the pH adjustment procedure. Uniform preparations of vesicles have been obtained from the dioleoyl esters of phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine, from diphytanyl ethers of glycolipid sulfate, phosphatidylglycerol, phosphatidylglycerol phosphate, and phosphatidylglycerol sulfate, from bovine liver phosphatidylinositol, from Escherichia coli phosphatidylethanolamine, from membrane lipid extracts from E. coli and Holabacterium cutirubrum, and from dodecanesulfonate-alkanol mixtures and free oleic acid. The preparation of unilamellar vesicles from oleic acid is novel, and the size range is 600-3000 nm; the preparations are relatively uniform. Vesicles of phospholipids in which sucrose and trehalose replace salt as the impermeant do not differ significantly from those prepared in pentaerythritol.     

Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between binding of the protein and the subsequent destabilization of the phospholipid vesicles a set of experiments was performed using phospholipid monolayers and vesicles. Using microelectrophoresis the binding of lysozyme to phospholipid vesicles made of PS was determined. At low ionic strength and mild acidic pH of the solution lysozyme reduced the magnitude of the negative zeta potential of PS vesicles at lower concentrations compared to neutral pH and high ionic strength. In contrast, the bound fraction of lysozyme to PS vesicles was nearly constant at acidic and neutral pH. At low pH, the binding of lysozyme was accompanied by a strong aggregation of the vesicles. Lysozyme binding to PS vesicles is accompanied by its penetration into the PL monolayer. This was measured by surface tension and film balance measurements at low pH and low ionic strength. The interaction of lysozyme with negatively charged vesicles lead to a decrease of the vesicle surface hydration as measured by the shift of the emission peak of the fluorescent probe DPE. The binding of bis-ANS increased at low pH after addition of lysozyme to the vesicles. This indicates that more hydrophobic patches of the lysozyme-PS complex are exposed at low pH. At low pH the binding process of lysozyme to PS vesicles was followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the aqueous content of vesicles.     

An in vitro ESR study of uncatalyzed and rat liver protein-catalyzed spin-labeled phosphatidylcholine exchange

ESR spectrometry has been used to study fatty acid spin-labeled phosphatidylcholine exchange from single bilayer donor vesicles to various acceptor systems, such as intact or differently treated mitochondria, phospholipid multilamellar vesicles or single bilayer vesicles. This exchange is catalyzed by soluble non-specific rat liver protein, first investigated by Bloj and Zilversmit in 1977 (J. Biol. Chem. 252, 1613–1619). Non-catalyzed phosphatidylcholine exchange has also been studied. Full inhibition of both mechanisms occurs with lipid-depleted acceptor mitochondria, while $\text{N-}\text{ethylmaleimide-treated}$ mitochondria behave as good acceptors during catalyzed exchange but are in no way effective during spontaneous exchange. Non-catalyzed exchange does not take place with phospholipase D-treated mitochondria as acceptors, while the pure catalyzed mechanism is inhibited by 28%. Neither multilamellar nor single bilayer phospholipid vesicles exchange spin-labeled phosphatidylcholine in the absence of protein, the former being a poorer acceptor system than the latter during catalyzed exchange, when this activity is 31 and 80%, respectively, of that of intact mitochondria. The hypothesis is made that the spontaneous mechanism is active among intact natural membranes and could be of some importance in vivo. Furthermore, the biomembrane protein moiety is assumed to be involved in the catalyzed exchange more as a phospholipid spacer than as a binder between the exchange protein and the membrane involved. Phospholipids, on the contrary, appear to be important for both functions.