Fusion of negatively charged phospholipid vesicles by alpha-latrotoxin

alpha-Latrotoxin-induced fusion of liposomes has been described using large unilamellar vesicles composed of phosphatidylcholine/phosphatidylethanolamine/cardiolipin at a molar ratio of 2:3:5. Vesicle fusion was monitored by terbium/dipicolinic acid assay as well as by fluorescence energy transfer measurement. The enhancement of the fusogenic effect of LTX by low concentrations (0.1-3 mM) of CaCl2 has been demonstrated. The efficiency of other divalent cations on the LTX fusogenic activity was shown to decrease in the sequence Ca greater than Cd greater than Sr greater than Mg greater than Ba. LTX-induced fusion was accompanied by the increase of vesicle size measured by laser correlation spectroscopy. It is concluded that fusogenic action of LTX may be involved in its effect on synaptic apparatus.     

Lysozyme induced fusion of negatively charged phospholipid vesicles

Lysozyme promotes fusion of negatively charged phospholipid vesicles prepared by ethanolic injection. Vesicle fusion was a leaky process as revealed by the release of encapsulated carboxyfluorescein or Tb-DPA complex. Extensive proteolysis of lysozyme inhibited the fusion process. The fusion process was critically dependent on the medium ionic strength; 100 mM of any salt was sufficient to inhibit totally the fusion activity of the protein. The high efficiency of lysozyme (80% RET) was almost constant in the pH range from 4.0 to 9.0, but it was sharply diminished when the pH of the medium was at the isoelectric point of the protein (pI 11.0). Fusion induced by chemically modified lysozyme, showed that the pH profile changed according to the isoelectric point of the protein derivative. These observations stress the importance of electrostatic interactions in the process of fusion induced by lysozyme.     

Insulin-mediated fusion of negatively charged phospholipid vesicles at low pH

Fusion of negatively charged phospholipid vesicles by bovine insulin was studied. The fusion induced by the hormone was demonstrated by resonance energy transfer, sepharose chromatography, light scattering and electron microscopy. The insulin effect was more effective when the pH was in the range of 3.6 - 3.9. The action of insulin also depends on the phosphatidylcholine: phosphatidic acid molar ratio, and buffer and vesicles concentration. At optimal conditions, half-maximal effect was obtained at 2 X 10(-8)M. The insulin-mediated fusion is non specific. The potential importance of these studies is discussed.     

Penetratin-induced aggregation and subsequent dissociation of negatively charged phospholipid vesicles

The interaction of the cellular delivery vector penetratin with a model system consisting of negatively charged phospholipid vesicles has been studied. Above a certain peptide to lipid molar ratio, the cationic oligopeptide induces vesicle aggregation. Interestingly, the aggregation is followed by spontaneous disaggregation, which may be related to membrane translocation of the peptide. Circular dichroism (CD) measurements indicate a conformational transition, from alpha-helix to antiparallel beta-pleated sheet, which is simultaneous with the aggregation process. The potential influence of spectroscopic artifacts on CD data due to the drastically increased turbidity during aggregation is discussed.     

Fusion of negatively charged phospholipid vesicles by insulin. Relationship with lipid fluidity

We have studied, by fluorescence methods, the association of insulin to liposomes, the modification of lipid fluidity, and the fusion of vesicles induced by insulin. All parameters showed a similar dependence on pH and ionic strength of the medium and on negative charges in liposomes. The influence of temperature indicated that the association of insulin to liposomes per se was not sufficient to produce a decrease in lipid fluidity and fusion of liposomes. The modification of lipid fluidity induced by insulin in biological membranes is discussed as a possible general event in the action of the hormone.     

Water permeability of phospholipid vesicles

Summary The water permeability of phospholipid vesicles 0.5 to 10 in diameter bounded by one or by several lipid bilayers was measured by following the change in turbidity of a suspension after mixing in a stopped flow apparatus. A semi-empirical formulation for evaluating volume changes of vesicles with a broad size range by measurement of turbidity is developed. The rate of flow is analyzed in terms of reaction rate theory. The water permeability coefficients for phosphatidylcholine vesicles were approximately 44 /sec at 25°C and 70 /sec at 37°C. The activation energy for water transport was 8.25 kcal/mole. The results were consistent with the view that water permeates by dissolution and diffusion in the membrane.     

Tumor necrosis factor enhances the neutrophil-dependent increase in endothelial permeability

We examined the effect of tumor necrosis factor alpha (TNF alpha) on the increase in pulmonary microvascular endothelial monolayer permeability induced by activated neutrophils (PMN). Layering of PMN onto endothelial monolayers followed by activation of PMN with phorbol 12-myristate 13-acetate (PMA) increased 125I-albumin clearance rate across the monolayers. Pretreatment of endothelial monolayers for 6 hr with TNF alpha (200 U/ml) potentiated the PMN-dependent increase in endothelial permeability, whereas 1 hr or 6 hr pretreatment of endothelial monolayers with 200 U/ml and 100 U/ml, respectively, TNF alpha did not enhance the response. Adherence of PMN to the endothelial cells was increased at 1 and 6 hr after TNF alpha (200 U/ml) treatment, but the adherence response was markedly greater following 6 hr of TNF alpha. The TNF alpha treatment of endothelial cells did not enhance neutrophil activation responses to PMA. Pretreatment of PMN with IB4, a MAb to the CD18 integrin, the common beta subunit of the adhesion proteins LFA-1, Mac-1, and p150,95 of PMN, reduced the increases in PMN adherence and the endothelial monolayer permeability induced by the 6 hr TNF alpha treatment. In contrast, pretreatment of PMN with OKM-1, a MAb to the CD11b epitope (alpha-subunit), had no effect on the adherence and the potentiation of the increase in permeability. The potentiation of the PMN-dependent permeability increase and enhanced endothelial adhesivity at 6 hr after TNF alpha priming of endothelial cells was dependent on protein synthesis. The results indicate that protein synthesis-dependent expression of an endothelial ligand for CD18 and resultant endothelial hyperadhesiveness potentiates the PMN-mediated increase in endothelial permeability after TNF alpha activation of endothelial cells. The priming of endothelial cells by TNF alpha may be a critical step in the mediation of endothelial injury.     

Fusion of negatively charged liposomes with clathrin-uncoated vesicles

The interaction of lipid vesicles with uncoated vesicles from bovine brain has been studied by fluorescence energy transfer between fluorescent lipid analogs (NBD-PE, Rh-DOPE), by loss of fluorescence self-quenching (NBD-PE, carboxyfluorescein) and by freeze-fracture electron microscopy. The fluorescence techniques monitor the mixing of membranous lipids and the induced release of encapsulated material. The results demonstrate a mixing of the negatively charged lipid (PA, PS) vesicles with the uncoated vesicles. In parallel with the lipid mixing a release of intravesicularly encapsulated material takes place. Lipid vesicles composed of zwitterionic lipids (PC, DOPC, PC:PE) do not specifically interact with uncoated vesicles. The electron micrographs reveal single fusion events. Studies on the kinetics are consistent with a fusional mechanism of the negatively charged lipid vesicles with uncoated vesicles.     

Covalent binding of negatively charged phospholipid to Na,K-ATPase

Phosphatidylglycolaldehyde and its lyso derivative were applied as probes in order to study lipid-protein interactions with purified, membrane-bound Na,K-ATPase. Reduction with [3H]NaCNBH3 led to formation of a stable chemical derivative between added lipid and protein. The extent of modification of the two subunits of Na,K-ATPase was similar. Extensive tryptic digestion of derivatized ATPase resulted in cleavage of the alpha-subunit without hydrolysis of the beta-subunit.     

Tumor necrosis factor-induced alterations in circulating leukocyte populations

Tumor necrosis factor is a potent agent possessing diverse biological functions. We investigated the effects of intravenous administration of human recombinant tumor necrosis factor (TNF) on immune cell populations in CBA/J mice. The animals developed a significant lymphopenia and neutrophilia both reaching a maximum at 4 hours post-injection with a trend towards resolution to normal values by 6 hours. The lymphopenia was both relative and absolute. Similarly, the neutrophilia was both relative and absolute and was due to the presence of both immature and mature neutrophils. As the neutrophilia and lymphopenia occurred concomitantly, there was no difference at any time point in the total number of peripheral blood white cells. Extensive controls were done to rule out LPS contamination in the TNF preparation. These data demonstrate the potent effects of intravenous administration of human recombinant tumor necrosis factor on peripheral blood constituents.     

Binding of ricin A-chain to negatively charged phospholipid vesicles leads to protein structural changes and destabilizes the lipid bilayer

Ricin is a heterodimeric protein toxin in which a catalytic polypeptide (the A-chain or RTA) is linked by a disulfide bond to a cell-binding polypeptide (the B-chain or RTB). During cell entry, ricin undergoes retrograde vesicular transport to reach the endoplasmic reticulum (ER) lumen, from where RTA translocates into the cytosol, probably by masquerading as a substrate for the ER-associated protein degradation (ERAD) pathway. In partitioning studies in Triton X-114 solution, RTA is predominantly found in the detergent phase, whereas ricin holotoxin, native RTB, and several single-chain ribosome-inactivating proteins (RIPs) are in the aqueous phase. Fluorescence spectroscopy and far-UV circular dichroism (CD) demonstrated significant structural changes in RTA as a result of its interaction with liposomes containing negatively charged phospholipid (POPG). These lipid-induced structural changes markedly increased the trypsin sensitivity of RTA and, on the basis of the protein fluorescence determinations, abolished its ability to bind to adenine, the product resulting from RTA-catalyzed depurination of 28S ribosomal RNA. RTA also released trapped calcein from POPG vesicles, indicating that it destabilized the lipid bilayer. We speculate that membrane-induced partial unfolding of RTA during cell entry may facilitate its recognition as an ERAD substrate.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.     

Structure of the Alzheimer beta-amyloid peptide (25-35) and its interaction with negatively charged phospholipid vesicles.

The secondary structure of amyloid betaAP(25-35) peptide was studied in pure form and in the presence of different phospholipid vesicles, by using Fourier transform infrared spectroscopy (FT-IR). Pure peptide aggregated with time, forming fibrils with beta-structure. Phospholipid vesicles formed by negatively charged phospholipids such as 1,2-dimyristoyl-sn-glycerol-3-phospho-L-serine (Myr2PtdSer), 1,2-dimyristoyl-sn-glycerol-3-phospho-rac-1-glycerol (Myr2PtdGro) and 1,2-dimyristoyl-sn-glycerol 3-phosphate (Myr2PtdH), greatly accelerated the aggregation of the peptide. However, the presence of vesicles formed by the zwitterionic phospholipid, 1, 2-dimyristoyl-sn-glycerol-3-phosphocholine (Myr2PtdCho), slowed down the aggregation process. Differential scanning calorimetry (DSC) measurements showed that the effect of betaAP(25-35) on the gel to crystal liquid phase transition was small at neutral pH for negatively charged phospholipids and practically nil for Myr2PtdCho. In the case of Myr2PtdSer the effect was also zero at pH 9 but the effect was large at pH 3. The effect on Myr2PtdH was not, however, very dependent on pH. These results were fully confirmed by the observation through FT-IR of the change with temperature of the CH2 antisymmetric stretching vibration. The case of Myr2PtdGro was special as this phospholipid presents polymorphism giving solid quasicrystalline phases when it is not sufficiently hydrated, and it is remarkable that betaAP(25-35) was able to induce the formation of crystalline phases in samples prepared through a method which ensure a good hydration of phospholipid. These results show that the interaction of amyloid betaAP(25-35) peptide with phospholipids is based on electrostatic interactions, that these interactions favour the aggregation of the peptides, and that the presence of the aggregates may disturb the lipid-water interphase of the membrane.     

Tumor necrosis factor-induced downregulation of its receptors in HeLa cells

Tumor necrosis factor (TNF) induced loss of TNF receptors in HeLa cells was studied using acid elution technique, which could distinguish surface occupancy and real loss of receptors. Exposure of HeLa cells to TNF resulted in a rapid reduction in the number of TNF receptors without affecting the apparent binding affinity. The binding of transferrin after treatment with unlabeled TNF was unaffected. The TNF-mediated decrease in receptor number on the cells was reversible. Following removal of TNF from growth medium, binding activity was restored within 3 h. Cycloheximide prevented the restoration of TNF receptors, suggesting that de novo synthesis of receptors was required to restore the binding activity.     

Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes

To obtain cationic liposomes of which affinity to negatively charged membranes can be controlled by temperature, cationic liposomes consisting of 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol and dioleoylphosphatidylethanolamine were modified with poly(N-acryloylpyrrolidine), which is a thermosensitive polymer exhibiting a lower critical solution temperature (LCST) at ca. 52 degrees C. The unmodified cationic liposomes did not change its zeta potential between 20-60 degrees C. The polymer-modified cationic liposomes revealed much lower zeta potential values below the LCST of the polymer than the unmodified cationic liposomes. However, their zeta potential increased significantly above this temperature. The unmodified cationic liposomes formed aggregates and fused intensively with anionic liposomes consisting of egg yolk phosphatidylcholine and phosphatidic acid in the region of 20-60 degrees C, due to the electrostatic interaction. In contrast, aggregation and fusion of the polymer-modified cationic liposomes with the anionic liposomes were strongly suppressed below the LCST. However, these interactions were enhanced remarkably above the LCST. In addition, the polymer-modified cationic liposomes did not cause leakage of calcein from the anionic liposomes below the LCST, but promoted the leakage above this temperature as the unmodified cationic liposomes did. Temperature-induced conformational change of the polymer chains from a hydrated coil to a dehydrated globule might affect the affinity of the polymer-modified cationic liposomes to the anionic liposomes.     

The interaction of neuropeptide Y with negatively charged and zwitterionic phospholipid membranes

The interaction of the 36 amino acid neuropeptide Y (NPY) with liposomes was studied using the intrinsic tyrosine fluorescence of NPY and an NPY fragment comprising amino acids 18–36. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the experimentally measured binding curves, the standard Gibbs free energy for the peptide transfer from aqueous solution to the lipid membrane was calculated to be around ?30 kJ/mol for membrane mixtures containing physiological amounts of acidic lipids at pH 5. The effective charge of the peptide depends on the pH of the buffer and is about half of its theoretical net charge. The results were confirmed using the fluorescence of the NPY analogue [Trp³²]-NPY. Further, the position of NPY’s α-helix in the membrane was estimated from the intrinsic tyrosine fluorescence of NPY, from quenching experiments with spin-labelled phospholipids using [Trp³²]-NPY, and from ¹H magic-angle spinning NMR relaxation measurements using spin-labelled [Ala³¹, TOAC³²]-NPY. The results suggest that the immersion depth of NPY into the membrane is triggered by the membrane composition. The α-helix of NPY is located in the upper chain region of zwitterionic membranes but its position is shifted to the glycerol region in negatively charged membranes. For membranes composed of phosphatidylcholine and phosphatidylserine, an intermediate position of the α-helix is observed.     

Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes

We have investigated the binding of a new dansylcadaverine derivative of substance P (DNC-SP) with negatively charged small unilamellar vesicles composed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycerol (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniques. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic strengths. The experimental association isotherms were analyzed using two binding approaches: (i) the Langmuir adsorption isotherm and the partition equilibrium model, that neglect the activity coefficients; and (ii) the partition equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP binding curve at different lipid composition was achieved by means of the Gouy-Chapman approach using a peptide effective interfacial charge (v) value of (0.95 +/- 0.02), which is lower than the physical charge of the peptide. For PC/PG membranes, the partition equilibrium constant were 7.8 x 10(3) M(-1) (9/1, mol/mol) and 6.9 x 10(3) M(-1) (7/3, mol/mol), whereas for PC/PS membranes an average value of 6.8 x 10(3) M(-1) was estimated. These partition equilibrium constants were similar to those obtained for the interaction of DNC-SP with neutral PC membranes (4.9 x 10(3) M(-1)), as theoretically expected. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge density of the vesicles. Also, the potential of fluorescent dansylated SP analogue in studies involving interactions with cell membranes is discussed.     

Glucosylceramide,a neutral glycosphingolipid anticoagulant cofactor,enhances the interaction of human- and bovine-activated protein C with negatively charged phospholipid vesicles

The effect of glucosylceramide (GlcCer) on activated protein C (APC)-phospholipid interactions was examined using fluorescence resonance energy transfer. Human APC, labeled with either fluorescein (Fl-APC) or dansyl (DEGR-APC) donor, bound to phosphatidylcholine/phosphatidylserine (PC/PS, 9:1 w/w) vesicles containing octadecylrhodamine (OR) acceptor with a K(d) (app) = 16 micro g/ml, whereas Fl-APC (or DEGR-APC) bound to PC/PS/GlcCer(OR) (8:1:1) vesicles with a K(d) (app) = 3 micro g/ml. This 5-fold increase in apparent affinity was not species-specific since bovine DEGR-APC also showed a similar GlcCer-dependent enhancement of binding of APC to PC/PS vesicles. From the efficiency of fluorescence resonance energy transfer, distances of closest approach of approximately 63 and approximately 64 A were estimated between the dansyl on DEGR-APC and rhodamine in PC/PS/GlcCer(OR) and PC/PS(OR), respectively, assuming kappa(2) = 2/3. DEGR-APC bound to short chain C8-GlcCer with an apparent K(d) of 460 nm. The presence of C8-GlcCer selectively enhanced the binding of C16,6-NBD-phosphatidylserine but not C16,6-7-nitrobenz-2-oxa-1,3-diazole (NBD)-phosphatidylcholine to coumarin-labeled APC. These data suggest that APC binds to GlcCer, that PC/PS/GlcCer vesicles like PC/PS vesicles bind to the N-terminal gamma-carboxyglutamic acid domain of APC, and that one mechanism by which GlcCer enhances the activity of APC is by increasing its affinity for membrane surfaces containing negatively charged phospholipids.