Aggregation Of Lipid Vesicles Research Articles

Polyamine-RNA-membrane interactions: From the past to the future in biology

Biogenic polyamines (PAs), spermine, spermidine and putrescine are widely spread amino acid derivatives, present in living cells throughout the whole evolutionary scale. Their amino groups confer them a marked basic character at the cellular pH. We have tested the interaction of PAs with negatively-charged phospholipids in the absence and presence of nucleic acids (tRNA was mainly used for practical reasons). PAs induced aggregation of lipid vesicles containing acidic phospholipids. Aggregation was detected using both spectroscopic and fluorescence microscopy methods (the latter with giant unilamellar vesicles). PA-liposome complexes were partially disaggregated when nucleic acids were added to the mixture, indicating a competition between lipids and nucleic acids for PAs in a multiple equilibrium phenomenon. Equivalent observations could be made when vesicles composed of oleic acid and 1-decanol (1:1mol ratio) were used instead of phospholipid liposomes. The data could evoke putative primitive processes of proto-biotic evolution. At the other end of the time scale, this system may be at the basis of an interesting tool in the development of nanoscale drug delivery.

Citric Acid and the RNA World

RNA world: The chelation of magnesium ions by citric acid prevents the magnesium-induced aggregation of lipid vesicles and RNA degradation while promoting nonenzymatic RNA oligomerization. These findings suggest a unique role for this simple organic acid in supporting key processes in RNA world organisms.

Exploring New Biological Functions of Amyloids: Bacteria Cell Agglutination Mediated by Host Protein Aggregation

Antimicrobial proteins and peptides (AMPs) are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala) can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.

Effect of repetitive lysine–tryptophan motifs on the bactericidal activity of antimicrobial peptides

Previous studies identified lysine- and tryptophan-rich sequences within various cationic antimicrobial peptides. In the present study, we synthesized a series of peptides composed of lysine (K)-tryptophan (W) repeats (KW)n (where n equals 2, 3, 4 or 5) with amidation of the C-terminal to increase cationicity. We found that increases in chain length up to (KW)4 enhanced the peptides’ antibacterial activity; (KW)5 exhibited somewhat less bactericidal activity than (KW)4. Cytotoxicity, measured as lysis of human red blood cells, also increased with increasing chain length. With (KW)5, reduced antibacterial activity and increased cytotoxicity correlated with greater hydrophobicity and self-aggregation in the aqueous environment. The peptides acted by inducing rapid collapse of the bacterial transmembrane potential and induction of membrane permeability. The mode of interaction of the peptides and the phosphate groups of lipopolysaccharide was dependent upon the peptides’ ability to permeate the membrane. Longer peptides [(KW)4 and (KW)5] but not shorter peptides [(KW)2 and (KW)3] strongly bound and partially inserted into negatively charged, anionic lipid bilayers. These longer peptides also induced membrane permeabilization and aggregation of lipid vesicles. The peptides had a disordered structure in aqueous solution, and only (KW)4 and (KW)5 displayed a folded conformation on lipid membranes. Moreover, (KW)4 destroyed and agglutinated bacterial cells, demonstrating its potential as an antimicrobial agent. Collectively, the results show (KW)4 to be the most efficacious peptide in the (KW)n series, exhibiting strong antibacterial activity with little cytotoxicity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-012-1388-6) contains supplementary material, which is available to authorized users.

Binding of Islet Amyloid Polypeptide to Supported Lipid Bilayers and Amyloid Aggregation at the Membranes

Amyloid deposition of human islet amyloid polypeptide (hIAPP) in the islets of Langerhans is closely associated with the pathogenesis of type II diabetes mellitus. Despite substantial evidence linking amyloidogenic hIAPP to loss of β-cell mass and decreased pancreatic function, the molecular mechanism of hIAPP cytotoxicity is poorly understood. We here investigated the binding of hIAPP and nonamyloidogenic rat IAPP to substrate-supported planar bilayers and examined the membrane-mediated amyloid aggregation. The membrane binding of IAPP in soluble and fibrillar states was characterized using quartz crystal microbalance with dissipation monitoring, revealing significant differences in the binding abilities among different species and conformational states of IAPP. Patterned model membranes composed of polymerized and fluid lipid bilayer domains were used to microscopically observe the amyloid aggregation of hIAPP in its membrane-bound state. The results have important implications for lipid-mediated aggregation following the penetration of hIAPP into fluid membranes. Using the fluorescence recovery after photobleaching method, we show that the processes of membrane binding and subsequent amyloid aggregation are accompanied by substantial changes in membrane fluidity and morphology. Additionally, we show that the fibrillar hIAPP has a potential ability to perturb the membrane structure in experiments of the fibril-mediated aggregation of lipid vesicles. The results obtained in this study using model membranes reveal that membrane-bound hIAPP species display a pronounced membrane perturbation ability and suggest the potential involvement of the oligomeic forms of hAPP in membrane dysfunction.

Kinetic analysis of the interaction between poly(amidoamine) dendrimers and model lipid membranes

We used fluorescence spectroscopy and surface tensiometry to study the interaction between low-generation (G1 and G4) poly(amidoamine) (PAMAM) dendrimers, potential vehicles for intracellular drug delivery, and model lipid bilayers. Membrane association of fluorescently labeled dendrimers, measured by fluorescence anisotropy, increased with increasing size of the dendrimer and with increasing negative charge density in the membrane, indicating the electrostatic nature of the interaction. When the membrane was doped with pyrene-labeled phosphatidyl glycerol (pyrene-PG), pyrene excimer fluorescence demonstrated a dendrimer-induced selective aggregation of negatively charged lipids when the membrane was in the liquid crystalline state. A nonlinear Stern–Volmer quenching of dendrimer fluorescence with cobalt bromide suggested a dendrimer-induced aggregation of lipid vesicles, which increased with the dendrimer's generation number. Surface tensiometry measurements showed that dendrimers penetrated into the lipid monolayer only at subphysiologic surface pressures (<30mN/m). We conclude that the low-generation PAMAM dendrimers associate with lipid membranes predominantly electrostatically, without significantly compromising the bilayer integrity. They bind stronger to membranes with higher fluidity and lower surface pressure, which are characteristic of rapidly dividing cells.

Lipid Vesicle Aggregation Induced by Cooling

Lipid bilayer fusion is a complex process requiring several intermediate steps. Initially, the two bilayers are brought into close contact following removal of intervening water layers and overcoming electrostatic repulsions between opposing bilayer head groups. In this study we monitor by light scattering the reversible aggregation of phosphatidylcholine single shell vesicles during which adhesion occurs but stops prior to a fusion process. Light scattering measurements of dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in water show that lowering the temperature of about 0.14 micron single shell vesicles of DPPC (from 20 °C to 5 °C) and about 2 micron vesicles of DSPC (from 20 °C to 15 °C), but not of 1 micron vesicles of DMPC, results in extensive aggregation within 24 hours that is reversible by an increase in temperature. Aggregation of DSPC vesicles was confirmed by direct visual observation. Orientation of lipid head groups parallel to the plane of the bilayer and consequent reduction of the negative surface charge can account for the ability of DPPC and DSPC vesicles to aggregate. Retention of negatively charged phosphates on the surface and the burial of positively charged cholines within the bilayer offer an explanation for the failure of DMPC vesicles to aggregate. Lowering the temperature of 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) vesicles from 20 °C to 5 °C failed to increase aggregation within 24 hours at Mg++/DPPS ratios that begin to initiate aggregation and fusion.

Bactericidal and membrane disruption activities of the eosinophil cationic protein are largely retained in an N-terminal fragment

ECP (eosinophil cationic protein) is an eosinophil secretion protein with antipathogen activities involved in the host immune defence system. The bactericidal capacity of ECP relies on its action on both the plasma membrane and the bacterial wall. In a search for the structural determinants of ECP antimicrobial activity, we have identified an N-terminal domain (residues 1-45) that retains most of ECP's membrane-destabilizing and antimicrobial activities. Two sections of this domain, ECP-(1-19) and ECP-(24-45), have also been evaluated. All three peptides bind and partially insert into lipid bilayers, inducing aggregation of lipid vesicles and leakage of their aqueous content. In such an environment, the peptides undergo conformational change, significantly increasing their alpha-helix content. The bactericidal activity of the three peptides against Escherichia coli and Staphylococcus aureus has been assessed at both the cytoplasmic membrane and the bacterial envelope levels. ECP-(1-45) and ECP-(24-45) partially retain the native proteins ability to bind LPS (lipopolysaccharides), and electron microscopy reveals cell damage by both peptides. Interestingly, in the E. coli cells agglutination activity of ECP is only retained by the longest segment ECP-(1-45). Comparative results suggest a task distribution, whereby residues 1-19 would contribute to membrane association and destabilization, while the 24-45 region would be essential for bactericidal action. Results also indicate that ECP cytotoxicity is not uniquely dependant on its membrane disruption capacity, and that specific interactions at the bacteria wall are also involved.

An α-Helical Conformation of the SEVI Peptide, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion

A peptide ubiquitous in human seminal fluid has been recently described that dramatically enhances the infectivity of the HIV virus (3-5 orders of magnitude by some measures). Previous studies have shown that this peptide, a fragment of human Prostatic Acid Phosphatase (PAP248-286) referred to as SEVI (Semen-derived Enhancer of Viral Infection), is amyloidogenic and the enhancement of viral infectivity is dependent on the aggregation state of the peptide. To complement these previous in vivo studies we have performed in vitro assays to investigate the physical mechanisms by which the PAP248-286 promotes the interaction with lipid bilayers. Our results indicate a strong interaction of freshly dissolved PAP248-286 with lipid bilayers but a weaker interaction with the amyloid form of PAP248-286, as measured by the tendency of freshly dissolved PAP248-286 to induce aggregation of lipid vesicles and membrane fusion. The amyloid form of PAP248-286 had little effect on either vesicle aggregation or fusion. To further investigate this effect we have solved the structure of PAP248-286 in SDS micelles. A largely α-helical conformation of PAP248-286, lying parallel to the membrane surface, is implicated in promoting bridging interactions between membranes by the screening of the electrostatic repulsion that occurs when two membranes are brought into close contact. This suggests non-specific binding of small oligomeric forms of SEVI in an α-helical conformation to lipid membranes may be an additional mechanism by which SEVI enhances the infectivity of the HIV virus.

Topography Studies on the Membrane Interaction Mechanism of the Eosinophil Cationic Protein

The eosinophil cationic protein (ECP) is an antipathogen protein involved in the host defense system. ECP displays bactericidal and membrane lytic capacities [Carreras et al. (2003) Biochemistry 42, 6636-6644]. We have now characterized in detail the protein-membrane interaction process. All observed fluorescent parameters of the wild type and single-tryptophan-containing mutants, as well as the results of decomposition analysis of protein fluorescence, suggest that W10 and W35 belong to two distinct spectral classes I and III, respectively. Tryptophan residues were classified and assigned to distinct structural classes using statistical approaches based on the analysis of tryptophan microenvironment structural properties. W10 belongs to class I and is buried in a relative nonpolar, nonflexible protein environment, while W35 (class III) is fully exposed to free water molecules. Tryptophan solvent exposure and the depth of the protein insertion in the lipid bilayer were monitored by the degree of protein fluorescence quenching by KI and brominated phospholipids, respectively. Results indicate that W35 partially inserts into the lipid bilayer, whereas W10 does not. Further analysis by electron microscopy and dynamic light scattering indicates that ECP can destabilize and trigger lipid vesicle aggregation at a nanomolar concentration range, corresponding to about 1:1000 protein/lipid ratio. No significant leakage of the vesicle aqueous content takes place below that protein concentration threshold. The data are consistent with a membrane destabilization "carpet-like" mechanism.

Studies on canthaxanthin in lipid membranes

Polar carotenoid pigment – canthaxanthin – has been found to interfere with the organization of biological membranes, in particular of the retina membranes of an eye of primates. The organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) and egg yolk phosphatidylcholine containing canthaxanthin was studied by means of several techniques including: electronic absorption spectroscopy, linear dichroism, X-ray diffractometry, 1H-NMR spectroscopy and FTIR spectroscopy. It appears that canthaxanthin present in the lipid membranes at relatively low concentration (below 1 mol% with respect to lipid) modifies significantly physical properties of the membranes. In particular, canthaxanthin (i) exerts restrictions to the segmental molecular motion of lipid molecules both in the headgroup region and in the hydrophobic core of the bilayer, (ii) promotes extended conformation of alkyl lipid chains, (iii) modifies the surface of the lipid membranes (in particular in the gel state, L β´) and promotes the aggregation of lipid vesicles. It is concluded that canthaxanthin incorporated into lipid membranes is distributed among two pools: one spanning the lipid bilayer roughly perpendicularly to the surface of the membrane and one parallel to the membrane, localized in the headgroup region. The population of the horizontal fraction increases with the increase in the concentration of the pigment in the lipid phase. Such a conclusion is supported by the linear dichroism analysis of the oriented lipid multibilayers containing canthaxanthin: The mean angle between the dipole transition moment and the axis normal to the plane of the membrane was determined as 20 ± 3° at 0.5 mol% and 47 ± 3° at 2 mol% canthaxanthin. The analysis of the absorption spectra of canthaxanthin in the lipid phase and 1H-NMR spectra of lipids point to the exceptionally low aggregation threshold of the pigment in the membrane environment (∼1 mol%). All results demonstrate a very strong modifying effect of canthaxanthin with respect to the dynamic and structural properties of lipid membranes.

Myelin proteolipid protein-induced aggregation of lipid vesicles: efficacy of the various molecular species.

The different molecular species that form the myelin proteolipid protein family were isolated by size-exclusion and ion-exchange chromatography in organic solvents and their adhesive properties were tested using a vesicle aggregation assay. Addition of the major proteolipid (PLP) to phosphatidylcholine-cholesterol vesicles caused their clustering as determined by increase in O.D.(450 nm) and by transmission electron microscopy. A small fraction of the aggregated vesicles underwent fusion as determined by resonance energy transfer experiments. Vesicle aggregation by PLP, but not the dissociation of the aggregates, was influenced by pH suggesting that electrostatic interactions are important only during cluster formation. Cleavage of disulfide bonds and methylation of carboxyl groups in PLP greatly reduced the aggregating activity, indicating that the process is dependent on the protein's conformation. Unexpectedly, the proteolipid DM-20 was also effective at inducing the clustering of neutral lipid vesicles. In contrast, three protein fractions comprising the naturally-occurring PLP fragments 1-107/112, 113/125-276 and 129/131-276, bearing different net charges, displayed a much lower activity. In addition, trypsin digestion of PLP resulted in a progressive decrease in the protein's ability to induce vesicle aggregation which coincided with the disappearance of the full-length molecule. Together, these results suggest that even large PLP fragments cannot fulfill the adhesive function of the intact protein.

Highly deiminated isoform of myelin basic protein from multiple sclerosis brain causes fragmentation of lipid vesicles

Myelin basic protein (MBP) occurs as a number of charge isomers due to phosphorylation, deamidation, and deimination of arginine to citrulline. All of these modifications decrease the net positive charge of the protein and its ability to cause aggregation of negatively charged lipid vesicles. This is used as a model system for the ability of MBP to cause adhesion of the cytosolic surfaces of myelin. Therefore, the effect of two deiminated forms of MBP on lipid vesicles was compared with that of the unmodified, most positively charged isomer, C1, to determine how loss of positively charged arginines would affect the function of MBP. The deiminated forms were the isomer isolated from normal human brains, in which only 6 Arg are deiminated to citrulline (MBP-Cit(6)), and an isomer isolated from the brain of a patient who died with acute, fulminating multiple sclerosis (Marburg type), in which 18 of the 19 Arg were deiminated (MBP-Cit(18)). Whereas C1 caused aggregation of lipid vesicles, resulting in an increase in absorbance due to light scattering, MBP-Cit(18) caused a decrease in absorbance of the lipid vesicles. Size exclusion chromatography and negative staining electron microscopy showed that this was due to fragmentation of the large multilayered vesicles into much smaller vesicles. MBP-Cit(6) caused less aggregation of lipid vesicles than did C1. However, no fragmentation of the vesicles into smaller ones in the presence of C1 and MBP-Cit(6) was detected by size exclusion chromatography or electron microscopy. The membrane fragmentation caused by MBP-Cit(18) is dramatically different from the effects of other forms of MBP from normal brain and may indicate a pathogenic effect of this charge isomer, which may have contributed to the severity of the Marburg type of multiple sclerosis. Alternatively, the deimination may have been a secondary effect resulting from the disease process. Regardless of the role of MBP-Cit(18) in multiple sclerosis, the effect of this modification indicates that, when most of the arginines of MBP are modified to an uncharged amino acid, the protein acquires properties similar to an apolipoprotein; thus, it may take up an amphipathic structure when bound to lipid. A partly amphipathic character may also be related to the role of MBP-Cit(6) in normal immature myelin, where it is the predominant charge isomer.

Highly deiminated isoform of myelin basic protein from multiple sclerosis brain causes fragmentation of lipid vesicles

Myelin basic protein (MBP) occurs as a number of charge isomers due to phosphorylation, deamidation, and deimination of arginine to citrulline. All of these modifications decrease the net positive charge of the protein and its ability to cause aggregation of negatively charged lipid vesicles. This is used as a model system for the ability of MBP to cause adhesion of the cytosolic surfaces of myelin. Therefore, the effect of two deiminated forms of MBP on lipid vesicles was compared with that of the unmodified, most positively charged isomer, C1, to determine how loss of positively charged arginines would affect the function of MBP. The deiminated forms were the isomer isolated from normal human brains, in which only 6 Arg are deiminated to citrulline (MBP-Cit6), and an isomer isolated from the brain of a patient who died with acute, fulminating multiple sclerosis (Marburg type), in which 18 of the 19 Arg were deiminated (MBP-Cit18). Whereas C1 caused aggregation of lipid vesicles, resulting in an increase in absorbance due to light scattering, MBP-Cit18 caused a decrease in absorbance of the lipid vesicles. Size exclusion chromatography and negative staining electron microscopy showed that this was due to fragmentation of the large multilayered vesicles into much smaller vesicles. MBP-Cit6 caused less aggregation of lipid vesicles than did C1. However, no fragmentation of the vesicles into smaller ones in the presence of C1 and MBP-Cit6 was detected by size exclusion chromatography or electron microscopy. The membrane fragmentation caused by MBP-Cit18 is dramatically different from the effects of other forms of MBP from normal brain and may indicate a pathogenic effect of this charge isomer, which may have contributed to the severity of the Marburg type of multiple sclerosis. Alternatively, the deimination may have been a secondary effect resulting from the disease process. Regardless of the role of MBP-Cit18 in multiple sclerosis, the effect of this modification indicates that, when most of the arginines of MBP are modified to an uncharged amino acid, the protein acquires properties similar to an apolipoprotein; thus, it may take up an amphipathic structure when bound to lipid. A partly amphipathic character may also be related to the role of MBP-Cit6 in normal immature myelin, where it is the predominant charge isomer. J. Neurosci. Res. 57:529–535, 1999. © 1999 Wiley-Liss, Inc.

Interaction and aggregation of lipid vesicles (DLVO theory versus modified DLVO theory)

Interaction and aggregation of acidic phospholipid (phosphatidylserine) vesicles were studied with variation of cation species and their concentrations in vesicle suspensions, and of vesicle sizes. Aggregation was determined by measuring turbidity of vesicle suspension. The experimental results of aggregation of vesicles induced by monovalent cations (Na +, K +, Cs + and TMA +) were explained well in terms of the interaction energy of two interacting vesicles using the ordinary Derjaguin–Landau–Verwey–Overbeek (DLVO) theory for both small and large lipid vesicles. However, the experimental results of aggregation of vesicles induced by divalent cations (Ca 2+, Mg 2+ and Ba 2+) were not explained by the ordinary DLVO theory. In order to explain the experimental results of these vesicle aggregation phenomena, it was necessary to modify the theory by including hydration interaction energies which are due to hydrated water at membrane surfaces, and their magnitude and sign depend upon the nature (hydrophobicity) of the membrane surface.

The Copines, a Novel Class of C2 Domain-containing, Calciumdependent, Phospholipid-binding Proteins Conserved from Paramecium to Humans

In an attempt to identify proteins that might underlie membrane trafficking processes in ciliates, calcium-dependent, phospholipid-binding proteins were isolated from extracts of Paramecium tetraurelia. The major protein obtained, named copine, had a mass of 55 kDa, bound phosphatidylserine but not phosphatidylcholine at micromolar levels of calcium but not magnesium, and promoted lipid vesicle aggregation. The sequence of a 920-base pair partial cDNA revealed that copine is a novel protein that contains a C2 domain likely to be responsible for its membrane active properties. Paramecium was found to have two closely related copine genes, CPN1 and CPN2. Current sequence data bases indicate the presence of multiple copine homologs in green plants, nematodes, and humans. The full-length sequences reveal that copines consist of two C2 domains at the N terminus followed by a domain similar to the A domain that mediates interactions between integrins and extracellular ligands. A human homolog, copine I, was expressed in bacteria as a fusion protein with glutathione S-transferase. This recombinant protein exhibited calcium-dependent phospholipid binding properties similar to those of Paramecium copine. An antiserum raised against a fragment of human copine I was used to identify chromobindin 17, a secretory vesicle-binding protein, as a copine. This association with secretory vesicles, as well the general ability of copines to bind phospholipid bilayers in a calcium-dependent manner, suggests that these proteins may function in membrane trafficking.

The C Terminus of SecA Is Involved in Both Lipid Binding and SecB Binding

Using C-terminal deletion mutations in secA, we localized the previously proposed (Breukink, E., Keller, R.C. A., and de Kruijff, B. (1993), FEBS Lett. 331, 19-24) second lipid binding site on SecA. Since removal of these residues completely abolished the property of SecA to cause aggregation of negatively charged phosphatidyl-glycerol vesicles, we conclude that the C-terminal 70 amino acid residues of SecA are involved in lipid-binding. The C-terminal 70 amino acid residues of SecA are important for efficient in vitro translocation of the SecB-dependent precursor of PhoE across inverted inner membrane vesicles. Moreover, in vivo studies showed that this region is essential for growth. SecB and a SecB-precursor complex were shown to inhibit the SecA-mediated lipid vesicle aggregation, suggesting that the overall acidic SecB protein binds at or near the second lipid binding site on SecA. This together with the observation that the SecA mutant protein lacking the C-terminal 70 residues had a strongly reduced ability to mediate binding of SecB-precursor complexes to inverted inner membrane vesicles demonstrates that the C terminus of SecA is also involved in SecB binding.

Phosphorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.

Annexin II tetramer (A-IIt) is a member of the annexin family of Ca2+ and phospholipid-binding proteins. The ability of this protein to aggregate both phospholipid vesicles and chromaffin granules has suggested a role for the protein in membrane trafficking events such as exocytosis. A-IIt is also a major intracellular substrate of both pp60src and protein kinase C; however, the effect of phosphorylation on the activity of this protein is unknown. In the current report we have examined the effect of phosphorylation on the lipid vesicle aggregation activity of the protein. Protein kinase C catalyzed the incorporation of 2.1 +/- 0.8 mol of phosphate/mol of A-IIt. Phosphorylation of A-IIt caused a dramatic decrease in the rate and extent of lipid vesicle aggregation without significantly effecting Ca(2+)-dependent lipid binding by the phosphorylated protein. Phosphorylation of A-IIt increased the A50%(Ca2+) of lipid vesicle aggregation from 0.18 microM to 0.65 mM. Activation of A-IIt phosphorylation, concomitant with activation of lipid vesicle aggregation, inhibited both the rate and extent of lipid vesicle aggregation but did not cause disassembly of the aggregated lipid vesicles. These results suggest that protein kinase C-dependent phosphorylation of A-IIt blocks the ability of the protein to aggregate phospholipid vesicles without affecting the lipid vesicle binding properties of the protein.

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.

Abscisic acid promotes fusion of phospholipid vesicles

The plant hormone abscisic acid (ABA) is shown to promote the fusion of phospholipid small unilamellar vesicles under compositional and pH conditions similar to those previously reported for ABA-induced enhancement of membrane permeability and lipid vesicle aggregation. Single component phosphatidylcholine (PC) vesicles were resistant to ABA-induced fusion. Upon the incorporation of 10 membrane mol per cent phosphatidylethanolamine (PE) to the PC membranes the vesicles were shown to fuse as followed by two different techniques, one monitoring the intermixing of the membranes (florescence resonance energy transfer) and the other the mixing of the sequestered aqueous compartments. Both synthetic saturated and natural unsaturated phospholipids were tested. Fusion was dependent on the concentration of the minor (PE) membrane component as well as on the concentration of ABA. Membranes made of PC's having different acyl chains in which liquid crystal and gel states coexist, were also fused by ABA. The process was pH dependent as only non-dissociated hormone promoted fusion. Fusion is also shown to be inhibited by incorporation of plant sterols into the bilayers. Some selectivity was evident as the biologically active cis-trans geometric isomer of ABA was fusogenic while the biologically inactive trans-trans isomer was not. These experiments are consistent with a lipid site of action on membranes for ABA.