The interaction of leucocidin with the cell membrane of the polymorphonuclear leucocyte

1. When leucocidin is incubated with leucocytes it is inactivated in solution and only a little adsorption takes place. This reaction has been used to purify the cell membrane. 2. The interaction of the membrane with leucocidin is very complex and at least three phenomena occur: (a) An inactivation of leucocidin in solution by large amounts of membrane which is synergistic between the two components of leucocidin, is thermolabile and is not inhibited by electrolyte. (b) An adsorption of leucocidin which is synergistic between the two components of leucocidin, does not proceed to the same extent as the inactivation in solution and is a function of the phospholipid components. Phospholipids isolated from the membrane adsorb leucocidin but the adsorption requires the presence of several molecular species. (c) Polymerization of leucocidin induced by tenfold smaller amounts of membrane than are required to bring about the first two interactions. The polymerization is reversed by adjustment of the ionic strength. It is due to the presence of the lipid components of the membrane. Different lipids are equally effective in inducing the polymerization. 3. Each component of leucocidin will polymerize in the absence of membranes and lose biological activity at low ionic strength. This is reversed by electrolyte and it does not proceed to the same extent as in the presence of membranes. 4. The nature of the interaction of leucocidin with cells, membranes and lipids and the spontaneous polymerization indicate that each component of leucocidin can adopt different isomeric forms. 5. The relationship of the interaction with the membrane to the cytotoxic effect of leucocidin is discussed.     

Aggregation of liposomes by dextrans of high molecular weight

Sonicated dispersions (liposomes) of natural and synthetic phospholipids are aggregated reversibly by Dextrans 40, 110 and 500. The dextran concentration required for aggregation is dependent on chain length, lipid composition of the liposome and, for ionically-charged phospholipids, the ionic strength of the medium. The results indicate that adsorption of dextrans to the erythrocyte surface can occur by interaction with surface phospholipid substituents.     

Interaction of nominally soluble proteins with phospholipid monolayers at the air-water interface

The interactions of carbonmonoxyhemoglobin (HbCO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and polyhistidine with phospholipid monolayers at the air-water interface were studied at physiological pH and ionic strength. HbCO and GAPDH both interact more strongly with monolayers containing negatively charged lipids. The interaction of HbCO and GAPDH with lipid monolayers decreases with increasing pH. Both the HbCO-monolayer and the GAPDH-monolayer interactions can be modeled as diffusion-limited processes, with kinetic data fit to a stretched exponential equation. The significance of these kinetics are discussed. Polyhistidine interacts only with monolayers containing lipids with negatively charged headgroups. In total, the results presented are consistent with an HbCO-lipid interaction with a large electrostatic component, a GAPDH-lipid interaction with comparable electrostatic and hydrophobic components, and a polyhistidine-lipid interaction that is solely electrostatic.     

Kinetics and efficiency of excitation energy transfer from chlorophylls,their heavy metal-substituted derivatives,and pheophytins to singlet oxygen

The interactions of carbonmonoxyhemoglobin (HbCO), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and polyhistidine with phospholipid monolayers at the air-water interface were studied at physiological pH and ionic strength. HbCO and GAPDH both interact more strongly with monolayers containing negatively charged lipids. The interaction of HbCO and GAPDH with lipid monolayers decreases with increasing pH. Both the HbCO-monolayer and the GAPDH-monolayer interactions can be modeled as diffusion-limited processes, with kinetic data fit to a stretched exponential equation. The significance of these kinetics are discussed. Polyhistidine interacts only with monolayers containing lipids with negatively charged headgroups. In total, the results presented are consistent with an HbCO-lipid interaction with a large electrostatic component, a GAPDH-lipid interaction with comparable electrostatic and hydrophobic components, and a polyhistidine-lipid interaction that is solely electrostatic.     

Differential lipid specificities of the repeated domains of annexin IV

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.     

Rhodopsin-detergent micelles aggregate upon activation of cyclic guanosine monophosphate phosphodiesterase

In the presence of G protein and phosphodiesterase, GTP induces aggregation of phospholipid-free rhodopsin-detergent micelles or rhodopsin reconstituted in phospholipid vesicles. The net electrical charge of the vesicle is not critical to the aggregation process since this phenomenon is not altered by reconstitution with phospholipids with different charge. The aggregation process is observed by monitoring changes in the light-scattering properties of the detergent micelles or vesicle suspension and by phase-contrast microscopy. The lowest light intensity which triggers the aggregation process and concomitant light-scattering changes in a rhodopsin-detergent micellar suspension bleaches 6% rhodopsin. Under these conditions, the signal saturates at 30% rhodopsin bleaching. The aggregation process appears likely to depend on the protein-protein interaction, and the presence of a disk membrane is not necessary for this process.     

Self-assembly of laminin induced by acidic pH

The supramolecular architecture of the basement membrane is provided by two enmeshed networks of collagen IV and laminin. The laminin network is maintained exclusively by interactions among individual laminin molecules and does not depend on the presence of other extracellular matrix components. Laminin polymers can be obtained in vitro either in solution or in association with the surface of bilayers containing acidic lipids. In this work, we have tested the hypothesis that the negative charges present on acidic lipids establish an acid microenvironment that is directly responsible for inducing laminin aggregation. Using light-scattering measurements, we show that laminin does not aggregate on vesicles of neutral lipids, whereas instantaneous aggregation occurs to progressively greater extents as the proportion of acidic phospholipids in the vesicles is increased. Aggregation of laminin induced by vesicles containing acidic phospholipids occurs very rapidly, so that maximal aggregation for each condition is reached within 1 min after laminin dilution. Aggregation depends on the presence of Ca(2+) ions, is reversed by increasing ionic strength, and can be detected at laminin concentrations as low as 6 nM. In addition, we show that, in the absence of vesicles, acidification of the bulk solution can also induce laminin self-polymerization through a process that exhibits the same properties as lipid-induced polymerization. The fact that there is a correspondence between the processes of self-polymerization of laminin in acidic medium and in neutral medium but in the presence of vesicles containing negatively charged lipids leads us to propose that the microenvironment of an acidic surface may trigger the assembly of laminin networks. In vivo, such an acidic microenvironment would be provided by negatively charged sialic acid and sulfate groups present in the glycocalyx surrounding the cells.     

Interaction of the cytoskeletal component vinculin with bilayer structures analyzed with a photoactivatable phospholipid

The cytoskeletal component vinculin has been proposed to act as an actin-plasma membrane linker. In order to demonstrate a possible direct interaction of vinculin with bilayers, photolabeling with a phospholipid generating a highly reactive carbene was used. This phosphatidylcholine analogue (1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[3H] 9-oxaundecanoyl]-sn-glycero-3-phosphocholine), with the photoactivatable diazirine group on its apolar portion, has been shown to label selectively membrane-embedded domains of membrane proteins. Vinculin is significantly labeled upon incubation and photolysis with liposomes containing trace amounts of this photoactivatable phospholipid, but only when the liposomes also contain acidic phospholipids. Labeling of vinculin is markedly increased (5-17-fold) by all acidic phospholipids tested so far (30%, w/w), compared to labeling in neutral phospholipids. Labeling is high at low ionic strength, but significant vinculin labeling can still be observed at physiological salt concentrations and acidic phospholipid content of the membrane. Our results provide evidence that vinculin inserts into the hydrophobic part of the bilayer by interacting with acidic phospholipids. A similar interaction may be of importance in vivo.     

On the role of polysialoglycosphingolipids as tetanus toxin receptors. A study with lipid monolayers

Lipid monolayers of different compositions were used to study the interaction of tetanus toxin with membrane lipids and to evaluate the role of polysialoglycosphingolipids as membrane receptors. At neutral pH, the toxin binds to dioleoylglycerophosphocholine monolayers and inserts into the phospholipid layer. This effect is potentiated by acidic phospholipids without an apparent preference for a single class of phospholipids. Polysialoglycosphingolipids further increase the fixation and penetration of tetanus toxin in lipid monolayers, but no specific requirement for a particular ganglioside was identified. The ganglioside effect is abolished in the presence of other nervous tissue lipids: cerebrosides and glycosphingolipid sulfates are partially responsible for this effect. The penetration of tetanus toxin in the lipid monolayer is pH dependent. It increases with lowering pH, it is facilitated by acidic phospholipids and by glycosphingolipid sulfates and it is mediated both by hydrophobic and electrostatic interactions as deduced from an analysis of the effect of ionic strength. Fragment B of tetanus toxin the low-pH-driven lipid interaction of the toxin. On the basis of the present findings, the possible role of polysialoglycosphingolipids in the neurospecific binding of tetanus toxin is discussed.     

Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles.

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.     

Effect of the state of association of melittin and phospholipids on their reciprocal binding

In solutions of increasing ionic strength, the molecular weight of melittin varies from 2840 (monomeric melittin) to 11 200. This polymerization, concomitant with an important change in conformation (Talbot, J.C., Dufourcq, J., De Bony, J., Faucon, J.F. and Lussan, C. (1979) FEBS Lett. 102, 191–193), is accompanied by a significant alteration in the partial specific volume of the molecule. The binding of melittin to phospholipids (phosphatidylserine, lysolecithin, dihexanoyl-, dioctanoyl- and lysolauroylphosphatidylcholine) depends on the state of association of the toxin and on the critical micelle concentration of lipids. No interaction is observed between monomeric melittin and free lipids, whereas tetrameric melittin can bind free lipids to form mixed micelles. At phospholipid concentrations above the critical micelle concentration, melittin in any state of self-association can bind lipids. The mixed micelles formed at saturation appear to be independent of the initial state of association of melittin.     

Interaction of the water-soluble protein aprotinin with liposomes: gel-filtration, turbidity studies, and 31P NMR studies

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; 31P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV-aprotinin complexes was determined. Lipid-protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. 31P NMR spectroscopy of the MLV-aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

The cell wall of Bacillus brevis producing gramicidin S, a cyclodecapeptide antibiotic

Gramicidin S is tritiated in Bacillus brevis G.-B. intact cells by activated tritium atoms (which is indicative of its surface localisation). The cell wall protein is tritiated very weakly, which shows that it is screened, apparently, by gramicidin adsorbed on its surface. The cell wall protein is not a glycoprotein and its interaction with exogenous gramicidin S causes cell aggregation. As follows from the Rf value after the chromatographic separation of B. brevis lipids, the reaction of staining, and the data of H-NMR spectroscopy for the fraction of phospholipids, the main membrane phospholipid is an anionic acetylated phosphatidyl ethanolamine.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Membrane insertion and lipid-protein interactions of bovine seminal plasma protein PDC-109 investigated by spin-label electron spin resonance spectroscopy

The interaction of the major acidic bovine seminal plasma protein, PDC-109, with dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by spin-label electron spin resonance spectroscopy. Studies employing phosphatidylcholine spin labels, bearing the spin labels at different positions along the sn-2 acyl chain indicate that the protein penetrates into the hydrophobic interior of the membrane and interacts with the lipid acyl chains up to the 14th C atom. Binding of PDC-109 at high protein/lipid ratios (PDC-109:DMPC = 1:2, w/w) results in a considerable decrease in the chain segmental mobility of the lipid as seen by spin-label electron spin resonance spectroscopy. A further interesting new observation is that, at high concentrations, PDC-109 is capable of (partially) solubilizing DMPC bilayers. The selectivity of PDC-109 in its interaction with membrane lipids was investigated by using different spin-labeled phospholipid and steroid probes in the DMPC host membrane. These studies indicate that the protein exhibits highest selectivity for the choline phospholipids phosphatidylcholine and sphingomyelin under physiological conditions of pH and ionic strength. The selectivity for different lipids is in the following order: phosphatidylcholine approximately sphingomyelin > or = phosphatidic acid (pH 6.0) > phosphatidylglycerol approximately phosphatidylserine approximately and rostanol > phosphatidylethanolamine > or = N-acyl phosphatidylethanolamine > cholestane. Thus, the lipids bearing the phosphocholine moiety in the headgroup are clearly the lipids most strongly recognized by PDC-109. However, these studies demonstrate that this protein also recognizes other lipids such as phosphatidylglycerol and the sterol androstanol, albeit with somewhat reduced affinity.     

Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles

Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC). The effects of phospholipid vesicle composition, calcium concentration, pH, and ionic strength on the equilibrium constants of factor Va- and factor Va-LC-phospholipid interaction were studied. Factor Va and factor Va-LC binding to phospholipid requires the presence of negatively charged phospholipids. It is further demonstrated that the following occur: (a) Calcium ions compete with factor Va and factor Va-LC for phospholipid-binding sites. (b) The dissociation constant of protein-phospholipid interaction increases with the ionic strength, whereas the maximum protein-binding capacity of the phospholipid vesicle was not affected by ionic strength. (c) The dissociation constant for factor Va-phospholipid interaction depends on pH when the vesicle consists of phosphatidic acid. It is concluded that factor Va-phospholipid interaction is primarily electrostatic in nature, where positively charged groups on the protein directly interact with the phosphate group of net negatively charged phospholipids. The results suggest that factor Va, like factor Xa and prothrombin, has the characteristics of an extrinsic membrane protein.     

Interaction of the Water-Soluble Protein Aprotinin with Liposomes: Gel-Filtration,Turbidity Studies,and 31P NMR Studies

Abstract

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; ³¹P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV–aprotinin complexes was determined. Lipid–protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. ³¹P NMR spectroscopy of the MLV–aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

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.     

Binding of recombinant rat liver fatty acid-binding protein to small anionic phospholipid vesicles results in ligand release: a model for interfacial binding and fatty acid targeting

A number of intracellular proteins bind to negatively charged phospholipid membranes, and this interfacial binding results in a conformational change that modulates the activity of the protein. Using a fluorescent fatty acid analogue, 11-[5-(dimethylamino)naphthalenesulfonyl]undecanoic acid (DAUDA), it is possible to demonstrate the release of this ligand from recombinant rat liver FABP in the presence of phospholipid vesicles that contain a significant proportion of anionic phospholipids. The ligand release that is observed with anionic phospholipids is sensitive to the ionic strength of the assay conditions and the anionic charge density of the phospholipid at the interface, indicating that nonspecific electrostatic interactions play an important role in the process. The stoichiometric relationship between anionic phospholipid and liver FABP suggests that the liver FABP coats the surface of the phospholipid vesicle. The most likely explanation for ligand release is that interaction of FABP with an anionic membrane interface induces a rapid conformational change, resulting in a reduced affinity of DAUDA for the protein. The nature of this interaction involves both electrostatic and nonpolar interactions as maximal release of liver FABP from phospholipid vesicles with recovery of ligand binding cannot be achieved with high salt and requires the presence of a nonionic detergent. The precise interfacial mechanism that results in the rapid release of ligand from L-FABP remains to be determined, but studies with two mutants, F3W and F18W, suggest the possible involvement of the amino-terminal region of the protein in the process. The conformational change linked to interfacial binding of this protein could provide a mechanism for fatty acid targeting within the cell.     

Interaction of protein kinase C with phosphatidylserine. 2. Specificity and regulation.

The roles of specific and nonspecific interactions in the regulation of protein kinase C by lipid have been examined. Binding and activity measurements reveal two mechanisms by which protein kinase C interacts with membranes: (1) a specific binding to the activating lipid phosphatidylserine and (2) a nonspecific binding to nonactivating, acidic lipids. The specific interaction with phosphatidylserine is relatively insensitive to ionic strength, surface charge, and the presence of nonactivating lipids. The two second messengers of the kinase, diacylglycerol and Ca2+, increase markedly the affinity of the kinase for phosphatidylserine. In contrast, the nonspecific interaction is sensitive to ionic strength and surface charge, and is unaffected by diacylglycerol. These results suggest that electrostatic interactions promote the binding of protein kinase C to membranes but the cooperative and selective binding of phosphatidylserine is the dominant driving force in a productive protein-lipid interaction.