Multiple factors influence the binding of a soluble, Ca2+-independent, diacylglycerol kinase to unilamellar phosphoglyceride vesicles

We studied the influence of membrane lipids, MgCl2, and ATP on the ability of a soluble diacylglycerol kinase to bind to 100-nm lipid vesicles. The enzyme did not bind detectably to vesicles that contained phosphatidylcholine alone or to vesicles that contained 50 mol % phosphatidylcholine + 50 mol % phosphatidylethanolamine. But it did bind to vesicles that contained anionic phosphoglycerides, and maximal binding occurred (in the presence of MgCl2) when the vesicles contained anionic phosphoglycerides alone. When increasing amounts of phosphatidylcholine were included in phosphatidylserine-containing vesicles, enzyme binding to the vesicles decreased by as much as 1000-fold. However, when increasing amounts of phosphatidylethanolamine were included in phosphatidylserine-containing vesicles, little change in binding occurred until the concentration of phosphatidylserine was reduced to below 25 mol %. These results and results obtained with vesicles that contained various mixtures of anionic phosphoglycerides, phosphatidylcholine, phosphatidylethanolamine, and unesterified cholesterol provided evidence that anionic phosphoglycerides were positive effectors of binding, phosphatidylcholine was a negative effector, and phosphatidylethanolamine and unesterified cholesterol were essentially neutral diluents. Other experiments showed that diacylglycerol and some of its structural analogues also were important, positive effectors of enzyme binding and that addition of ATP to the medium increased their effects. The combined results of the study suggest that the enzyme may bind to vesicles via at least two types of binding sites: one type that requires anionic phospholipids and is enhanced by Mg2+ but inhibited by phosphatidylcholine, and one type that requires diacylglycerol and is enhanced by ATP.     

Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 μm diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca²⁺ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca²⁺ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca²⁺ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg²⁺ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg²⁺ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca²⁺ and also enhanced     

Phospholipid transfer between vesicles. Dependence on presence of cytochrome P-450 and phosphatidylcholine-phosphatidylethanolamine ratio

The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicle was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they was transferred to the nonspin-labeled acceptor vesicles. A lower transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles for a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a 31P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.     

Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase.

Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for 125I-insulin (Kd = 0.12 nM versus 0.76 nM). Inclusion of 8.3-16.7% phosphatidylserine into the reconstituted vesicles restores 40-50% of the insulin-sensitivity to the receptor kinase. An elevated apparent affinity for 125I-insulin of insulin receptors in vesicles containing phosphatidylcholine and phosphatidylethanolamine is also restored to the value observed in detergent solution by the inclusion of phosphatidylserine in the reconstituted system. The effect of phosphatidylserine on insulin receptor kinase appears specific, because cholesterol, phosphatidylinositol and phosphatidic acid are all unable to restore insulin-sensitivity to the receptor kinase. Autophosphorylation sites on the insulin receptor as analysed by h.p.l.c. of tryptic 32P-labelled receptor phosphopeptides are not different for insulin receptors autophosphorylated in detergent solution or for the reconstituted vesicles in the presence or absence of phosphatidylserine. These data indicate that the phospholipid environment of insulin receptors can modulate its binding and kinase activity, and phosphatidylserine acts to restore insulin-sensitivity to the receptor kinase incorporated into phosphatidylcholine/phosphatidylethanolamine vesicles.     

On the substrate specificity of rat liver phospholipase A1

The substrate specificity of purified phospholipase A1 was studied using mixed micelles of phospholipid and Triton X-100. The kinetic analysis employed determined Vmax, Ks (a dissociation constant for the phospholipase A1-mixed micelle complex), and Km (the Michaelis constant for the catalytic step which reflects the binding of the enzyme to the substrate in the interface). The order of Vmax values was phosphatidic acid greater than phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine. The order of Ks values was phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidic acid greater than phosphatidylserine; the order of Km values was phosphatidic acid greater than phosphatidylethanolamine = phosphatidylserine greater than phosphatidylcholine. When present together, phosphatidylcholine inhibited the hydrolysis of phosphatidylethanolamine but phosphatidylethanolamine did not affect the hydrolysis of phosphatidylcholine. Sphingomyelin, phosphatidylcholine plasmalogen, and phosphatidylethanolamine plasmalogen had no effect on the hydrolysis of phosphatidylethanolamine. The effects of the reaction products, lysolipids and/or fatty acids, were also considered for their influence on phosphatidylethanolamine hydrolysis catalyzed by phospholipase A1. Free fatty acid was found to inhibit, whereas lysophospholipids stimulated hydrolysis of phosphatidylethanolamine. In a mixture of 1,2- and 1,3-diacylglycerides in mixed micelles, only the acyl chain at the sn-1 position of the 1,2 compound was hydrolyzed. Surface charge did not modulate the hydrolysis of phosphatidylcholine vesicles or mixed micelles. In conclusion, it is hypothesized that steric hindrance at position 3 of the glycerol regulates substrate binding in the active site and that an acyl group in position 1 is favored over a vinyl ether linkage for binding.     

Effect of spectrin on structure properties of lipid bilayers formed from mixtures of phospholipids. Fluorescence and microcalorimetric studies.

Effect of spectrin from human erythrocytes on structure properties of lipid bilayers formed from a mixture of phosphatidylethanolamine/phosphatidylserine (PE/PS) and/or phosphatidylethanolamine/phosphatidylcholine (PE/PC) was studied with the use of fluorescence and microcalorimetric methods. Spectrin did not affect the order parameter of lipids in PE/PS vesicles. However, spectrin binding to liposomes did influence temperature, half-width and enthalpy of phase transitions in mixtures of dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidylcholine (DMPC), and this effect was dependent on DMPE to DMPC weight ratio. A change in miscibility of the components in the presence of spectrin was observed and it might be due to spectrin-PE interactions.     

Chemical modification of methionine residues of the phosphatidylcholine transfer protein from bovine liver. A spin-label study

The role of methionine residues in the interaction of the phosphatidylcholine transfer protein from bovine liver with phospholipid vesicles was investigated by specific modification of these residues with iodoacetamide. The modified protein was digested with cyanogen bromide in order to determine which methionine residues had become resistant to this cleavage. Automated Edman degradation on the digest indicated that after 72 h of reaction, Met-1 was modified for 80%, Met-73 for 50%, Met-109 for 20%, whilst Met-173 and Met-203 were found to be unmodified. This distinct modification did not result in any loss of phosphatidylcholine transfer activity. The interaction of the phosphatidylcholine transfer protein with phospholipid vesicles was investigated by making use of electron spin resonance spectroscopy. The interaction of unmodified protein with vesicles composed of phosphatidylcholine/phosphatidic acid/spin-labeled phosphatidylethanolamine (79:16:5, mol%) or composed of phosphatidylserine/spin-labeled phosphatidylethanolamine (95:5, mol%), gave an increase of about 50% in the rotation correlation time. A similar increase was observed with the modified protein. This interaction was further investigated by labeling Met-1 and Met-73 in the transfer protein with iodoacetamidoproxyl spin-label. Spin-labeling did not inactivate the transfer protein. In addition, the electron spin resonance spectra of the spin-labeled protein were not affected upon addition of vesicles composed of phosphatidylcholine/phosphatidic acid (80:20, mol%). These experiments strongly suggest that Met-1 and Met-73 are not part of the site that interacts with the membrane.     

Phospholipid transfer between vesicles: Dependence on presence of cytochrome P-450 and phosphatidylcholine-phosphatidylethanolamine ratio

The rate of transfer of spin-labeled phospholipid from donor vesicles of sonicated 1-acyl-2-(10-doxylstearoyl)-sn-glycero-3-phosphocholine to other vesicles was determined as a function of content of cytochrome P-450 and the phosphatidylcholine/phosphatidylethanolamine ratio in the acceptor vesicles. The transfer rate was measured as an increase in intensity that resulted from a decrease in the line width in the EPR spectrum of the spin-labeled phospholipids as they were transferred to the nonspin-labeled acceptor vesicles. A lowe transfer rate was observed for acceptor vesicles of pure egg phosphatidylcholine vesicles than for vesicles of a mixture of phosphatidylcholine and phosphatidylethanolamine. The presence of cytochrome P-450 in the acceptor vesicles further increased the transfer rate. Those alterations in the mole ratios of the protein and the two phospholipids that made the bilayer of the reconstituted vesicles more like the membrane of the endoplasmic reticulum resulted in an increase in phospholipid-transfer rate. The mole ratios of components that produce high phospholipid-transfer rates were similar to those that in an earlier study produced a ³¹P-NMR spectrum characteristic of a nonbilayer phase. These findings suggest that, in the membrane of the endoplasmic reticulum, phospholipid exchange may be an important element in function and interaction with other intracellular organelles.     

Sodium-induced aggregation of phosphatidic acid and mixed phospholipid vesicles

Sodium-induced aggregations of sonicated vesicles prepared from synthetic phosphatidic acid and from its 1 : 1 mixtures with synthetic phosphatidylethanolamine and phosphatidylcholine were studied by turbidimetric measurements. The aggregation reactions were almost completely reversible on change in the Na⁺ concentration, pH or temperature. The threshold concentrations of Na⁺ for aggregation of pure dipalmitoylphosphatidic acid vesicles and mixed dipalmitoylphosphatidylenolamine- and dimyristoylphosphatidylcholine-dipalmitoylphosphatidic acid vesicles were found to be 200, 310 and 550 mM, respectively, at 25° and pH 7.2. The hydrocarbon chain lengths of phosphatidic acid and phosphatidylethanolamine had little effect on the threshold concentrations. The threshold concentrations for phospholipid vesicles composed of phosphatidic acid alone or its 1 : 1 mixture with phosphatidylethanolamine were changed by varying either the pH or temperature, while that for phosphatidylcholine-phosphatidic acid vesicles was almost independent of the pH and temperature, implying that aggregation of the latter vesicles is induced by a somewhat different mechanism.     

Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers

Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/microgram of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kd value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenase-sensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of poly(ethylene glycol)-induced fusion by membrane hydration: importance of interbilayer separation.

Large unilamellar vesicles composed of lipids with different hydration properties were prepared by the extrusion technique. Vesicles were composed of dioleoylphosphatidylcholine in combination with either 0.5 mol % monooleoylphosphatidylcholine or different molar ratios of dilauroylphosphatidylethanolamine. Fusion was revealed via a fluorescence assay for contents mixing and leakage, a fluorescent lipid probe assay for membrane mixing, and quasi-elastic light scattering to detect vesicle size growth. As the percentage of poorly hydrating phosphatidylethanolamine increased, the concentration of poly(ethylene glycol) (PEG) required to induce fusion decreased. From differential scanning calorimetry studies of membrane-phase behavior and X-ray diffraction monitoring of phase structure in PEG, it was concluded that PEG did not induce a hexagonal-phase transition or lamellar-phase separation. Electron density profiles derived from X-ray diffraction studies of multi- and unilamellar vesicles indicated that the water layer between vesicles had a thickness of approximately 5 A at PEG concentrations at which vesicles were first induced to fuse. At this distance of separation, the choline headgroups from apposing bilayers are in near-molecular contact. Since pure phosphatidylcholine vesicles did not fuse at this interbilayer spacing, a reduction in the interbilayer water layer to a critical width of approximately 2 water molecules may contribute to but is not sufficient to produce PEG-mediated fusion of phospholipid membranes. Comparison of these results with other results from this laboratory also indicates that, while close contact between bilayers promotes fusion, near-molecular contact is apparently not absolutely necessary to bring about fusion. A tentative model is presented to account for these results.     

Stimulation by phospholipid vesicles of proteolysis of egg white lysozyme by chymotrypsin

Egg white lysozyme was rapidly and extensively hydrolyzed by chymotrypsin in the presence of negatively charged phospholipid vesicles. The extent of hydrolysis of lysozyme by chymotrypsin depended on the amount of phospholipid present. The optimum amount of phospholipid varied with the amounts of both lysozyme and chymotrypsin. The proteolysis was strongly inhibited at high ionic strength. The amidolytic activity of chymotrypsin against a synthetic substrate was inhibited by phospholipid. Purified phosphatidic acid and phosphatidylethanolamine from egg yolk induced susceptibility of lysozyme to chymotrypsin, whereas synthetic dimyristoyl phosphatidylcholine did not. The extent of the hydrolysis was smaller with phosphatidic acid and phosphatidylethanolamine than with phospholipid mixture, indicating that vesicles of phospholipid mixture were more effective than those of phosphatidic acid or phosphatidylethanolamine in enhancing the proteolysis of lysozyme by chymotrypsin.     

Synthesis and properties of radioiodinated phospholipid analogues that spontaneously undergo vesicle-vesicle and vesicle-cell transfer

An efficient method for the synthesis and purification of a variety of iodinated phospholipid analogues is described. 1-Acyl-2-[[[3-(3-[125I]iodo-4-hydroxyphenyl)- propionyl]amino]caproyl]phosphatidylcholine (125I-PC) was prepared by alkylation of 1-acyl-2-(aminocaproyl)phosphatidylcholine with monoiodinated Bolton-Hunter reagent. 125I-Labeled phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine were produced from 125I-PC by phospholipase D catalyzed base exchange in the presence of ethanol-amine or L-serine. All of these lipid analogues transferred readily from donor vesicles into recipient membranes. When an excess of acceptor vesicles was mixed with a population of donor vesicles containing the iodinated analogues, approximately 50% of the 125I-labeled lipids transferred to the acceptor vesicle population. In addition, under appropriate incubation conditions, these lipids were observed to transfer from vesicles to mammalian cells. Autoradiographic analysis of 125I-labeled lipids extracted from the cells after incubation with vesicles at 2 degrees C for 60 min revealed that a large proportion of the 125I-labeled phosphatidic acid was metabolized to 125I-labeled diglyceride and 125I-labeled phosphatidylcholine, whereas no metabolism of exogenously supplied 125I-labeled phosphatidylethanolamine or 125I-labeled phosphatidylcholine could be detected.     

Functional reconstitution of the integral membrane proteins of influenza virus into phospholipid liposomes

The integral membrane proteins of influenza virus, a hemagglutinin and a neuraminidase, have been incorporated into liposomes composed of either phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylethanolamine (2:1 w/w) using detergent dialysis. The virus spike glycoproteins for reconstitution were selectively solubilized by using cetyltrimethylammonium bromide to leave a "core particle", which lacked a lipid bilayer but possessed quaternary structure as observed by electron microscopy. The viral spike proteins were combined with exogenous phospholipid in excess sodium cholate followed by exhaustive dialysis for 150 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that only the viral glycoproteins were associated with all the complexes formed. The level of sodium cholate remaining after dialysis was shown to be reduced to less than 1 molecule per 80 protein molecules. Viral proteins reconstituted into dimyristoylphosphatidylcholine liposomes were shown to have retained hemagglutination, low-pH-dependent hemolysis, and neuraminidase activities and were associated with a lipid bilayer in two types of complexes with average lipid to protein mole ratios after sucrose density gradient purification of either 590:1 or 970:1. The bilayer vesicles formed were of similar sizes and were shown by negative-stain electron microscopy to be 150-300 nm in diameter with well-defined spikes on their surface. Reconstituted liposomes of dimyristoylphosphatidylcholine were found to be unstable with respect to their trapped volume and therefore were unsuitable for fusion studies, unlike complexes formed with phosphatidylcholine or a mixture of phosphatidylcholine/phosphatidylethanolamine derived from hen eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 microM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 microM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 microM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.     

Reconstitution of the sodium-calcium exchanger from cardiac sarcolemmal vesicles

The Na+-Ca2+ exchanger was extracted from cardiac sarcolemmal vesicles and reconstituted into phospholipid vesicles by a cholate-dialysis method. Reconstitution was attempted with different phospholipids. Phosphatidylcholine alone was ineffective, whereas phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) showed high activity, but a significant Ca2+ uptake in the absence of Na+ gradient. Optimal reconstitution was obtained with a mixture of phosphatidylcholine and phosphatidylserine (9:1, mol/mol). The reconstituted proteoliposomes showed an ouabain-sensitive (Na+ + K+)-ATPase activity and a Na+-Ca2+ exchange with a specific activity comparable to that of the original vesicles. The specificity toward Na+ was also recovered. A partial purification of the exchanger was obtained by the method of transport-specificity fractionation ( Goldin , S.M. and Rhoden , V. (1978) J. Biol. Chem. 253, 2575-2583). When proteoliposomes were reconstituted with sodium oxalate inside and incubated with calcium in the presence of an outwardly directed Na+ gradient, the vesicles containing the Na+-Ca2+ exchanger specifically accumulated calcium which precipitated inside as calcium oxalate. The resulting increase in density allowed separation of the proteoliposomes containing the Na+-Ca2+ exchanger from the rest of the vesicles on a sucrose density gradient.     

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.     

Calcium-induced fusion of proteoliposomes and protein-free liposomes Effect of their phosphatidylethanolamine content on the structure of fused vesicles

The acidic phospholipid cardiolipin was shown to be very efficient in promoting calcium-induced fusion of proteoliposomes. The degree of fusion was dependent on the phosphatidylethanolamine content of the vesicles. Addition of CaCl₂ to proteoliposomes containing phosphatidylcholine and cardiolipin but without phosphatidylethanolamine did not induce fusion. Fusion of cytochrome oxidase vesicles, containing less than 50 mol% phosphatidylethanolamine resulted in monolamellar vesicles with a diameter of about 200 nm. The vesicles could be induced to fuse further by establishing an osmotic pressure across their membranes. When proteoliposomes containing more than 50 mol% phosphatidylethanolamine were fused, large vesicles with a diameter exceeding 1 μm were formed. They appeared in the electron microscope as a mixture of multilamellar and monolamellar vesicles. Fusion of corresponding liposomes resulted in formation of even larger structures appearing as dense multilamellar bodies and paracrystalline honeycomb-like lattices.     

Interactions of divalent cations or basic proteins with phosphatidylethanolamine vesicles

Small unilamellar vesicles have been prepared from phosphatidylethanolamine by sonication of the lipid in aqueous buffers of low ionic strength and high pH. These vesicles and their interactions with various di- and trivalent cations have been characterized using freeze-fracture electron microscopy. Phosphatidylethanolamine from 4 sources was examined: Hens' yolk phosphatidylethanolamine, human grey matter phosphatidylethanolamine, Escherichia coli phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine. The phosphatidylethanolamine from natural sources formed spherical, uniform 20–40 nm vesicles while dimyristoyl phosphatidylethanolamine formed larger, 70 × 25 nm, disc-shaped vesicles when sonicated above the phase transition temperature. Fusion of the unilamellar egg phosphatidylethanolamine, E. coli phosphatidylethanolamine and human grey matter phosphatidylethanolamine vesicles was induced by dialysis against buffers containing 2.0 nM Ca⁺ or 3.0 mM Mg²⁺. The fusion of the vesicles resulted in the precipitation of the lipid and the formation of multilamellar and, in some cases, hexagonal II structures. Dimyristoyl phosphatidylethanolamine vesicles were precipitated at 55°C by 1.0 mM Ca⁺ or 2.0 mM Mg²⁺. Treatment of the calcium- and magnesium-precipitated vesicles of hen's egg yolk phosphatidylethanolamine, E. coli phosphatidylethanolamine, human grey matter phosphatidylethanolamine and dimyristoyl phosphatidylethanolamine with EDTA resulted in resuspension of the lipid. The specific size and shape of the vesicles formed in this manner depends on the type of phosphatidylethanolamine and ion involved. Dialysis of the Ca⁺- and Mg²⁺-precipitated egg phosphatidylethanolamine vesicles against buffer containing no Ca⁺, Mg²⁺ or EDTA also resulted in dissociation of the precipitate and formation again of a new vesicle population. This evidence indicates that the Ca⁺ and Mg²⁺ are not strongly bound to the phosphatidylethanolamine.Egg phosphatidylethanolamine vesicles would fuse in the presence of many di- and trivalent ions. Egg phosphatidylethanolamine vesicles were precipitated by beryllium, aluminum, chromium, manganese, cobalt, nickel, copper, zinc, strontium, cadmium, barium, lanthanium, mercury and lead. The amount of ion required to precipitate the vesicles and the type of structure resulting from the fusion of the vesicles was found to be unique for each ion.Small unilamellar vesicles prepared from egg phosphatidylethanolamine were reacted with several basic proteins (cytochrome c, basic protein from human myelin, protamine, poly-l-lysine and cationically-modified ferritin). The basic proteins also initiated the fusion of egg phosphatidylethanolamine vesicles but these proteins did not fuse egg phosphatidylcholine vesicles nor did normal ferritin initiate fusion. Human myelin basic protein initiated the fusion of dimyristoyl phosphatidylethanolamine vesicles above and below the phase transition of this lipid.     

Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities

The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion.