Aggregation of ligand-modified liposomes by specific interactions with proteins. I: Biotinylated liposomes and avidin

The aggregation of biotin-modified phospholipid vesicles (liposomes) induced by binding the protein avidin in solution is analyzed experimentally and theoretically. Avidin has four binding sites that can recognize biotin specifically, and is able to cross-link the liposomes to form large aggregates. The aggregation kinetics were followed using quasi-elastic light scattering (QLS) to measure the mean particle size, and by measuring the solution turbidity. The rate and extent of aggregation were determined as a function of vesicle concentration, protein concentration, and the biotin density on the surface of the liposomes. A model based on Smoluchowski kinetics, fractal concepts, and Rayleigh and Mie light scattering theory was developed to analyze the experimental observations. Small aggregates (<7800 A diameter) may be treated as globular; however, the fractal nature of larger particles must be taken into account. Parameters in the model are taken from molecular simulations, or fit to the experimental observations. The aggregation kinetics are primarily determined by the biotin density on the liposome surface, the stoichiometric ratio of avidin molecules to liposomes, and the liposome concentration. Good agreement is found between the model and the experimental results. (c) 1996 John Wiley & Sons, Inc.     

Aggregation of hapten-bearing liposomes mediated by specific antibodies.

We studied specific membrane-membrane interactions mediated by ligand-receptor binding in a model system, which consisted of (a) FG3P, the fluorescein hapten attached to a phospholipid by a peptidyl spacer as described previously (Petrossian, A., A.B. Kantor, and J.C. Owicki. 1985. J. Lipid Res. 26:767-773), (b) antifluorescein monoclonal antibodies (MAbs), and (c) phospholipid vesicles (liposomes) into which the FG3P was incorporated. The aggregation of the hapten-bearing liposomes by four MAbs was studied by differential centrifugation. The ability of the MAbs to induce vesicle aggregation varied considerably and correlated inversely with affinity. Aggregation by one of the MAbs was studied in more detail by turbidimetry and freeze-fracture electron microscopy of samples frozen throughout the course of the aggregation. Rapid freezing was achieved with a double propane-jet apparatus. The aggregate morphologies and the time evolution of the aggregate size distribution were obtained from the two-dimensional fracture views with a stereological correction. The aggregation kinetics were simulated by considering dynamical aggregation according to a mass-action model with two parameters, the rate constants for antibody-mediated vesicle aggregation and disaggregation. Both rate constants were orders of magnitude lower than the rate constants for the corresponding interactions of antibodies with haptens either in solution or on vesicles under nonaggregating conditions.     

Affinity precipitation of an antibody by ligand-modified phospholipids

A new method for the selective precipitation of proteins is applied to the isolation and purification of an antibody. Ligand-modified phospholipids (LMPs) are solubilized by the nonionic ethoxylated alcohol detergent, resulting in small (50 to 100 A) micellar aggregates of LMPs and surfactant. When introduced into protein solutions containing an antibody for which the LMP has specific affinity, the ligand binds to the protein. Hydrophobic interactions between phospholipid tail groups bound to the protein molecules result in an insoluble precipitate. Polyclonal and monoclonal antibiotin antibody (pABA and mABA) are shown to be selectively precipitated using ratios of dimyristoylphosphatidylethanolamidobiotin (DMPE-B) to ABA ranging from 1:1 to 19:1. The kinetics and yield of the precipitation achieve a maximum at a ratio of DMPE-B to ABA of approximately 7:1. The kinetics and magnitude of the turbidity change are modeled using the Mie theory of light scattering coupled with the smoluchowski theory of aggregation. The kinetics are shown to be enhanced significantly by the addition of salt. In particular, the addition of 0.5 M ammonium sulfate salt increases the rate of precipitation by more than an order of magnitude. It is demonstrated that pABA can be recovered with total activity yields of 60% to 70% from mixtures containing nonspecific lgG antibodies in very high purity (>99%). (c) 1994 John Wiley & Sons, Inc.     

Affinity precipitation of proteins by surfactant-solubilized, ligand-modified phospholipids.

The use of ligand-modified phospholipids solubilized in aqueous solution by nonionic surfactant for affinity precipitation of proteins is described. Avidin was precipitated by contact with solutions in which dimyristoylphosphatidylethanolamine (DMPE) functionalized with biotin (DMPE-B) was solubilized in octaethylene glycol mono-n-dodecyl ether (C12E8) solutions. The nonionic surfactant solubilizes the phospholipid in micelles above its critical micelle concentration (CMC) and in small submicellar aggregates below this concentration. At C12E8 concentrations significantly exceeding its CMC, determined to be about 100 microM, precipitation of avidin by solubilized DMPE-B is not observed. In this regime, binding of protein by DMPE-B was monitored by a hyperchroic shift in the protein's UV-visible spectrum at 231.5 nm. The data were analyzed using a model that considers the four binding sites on the protein to be independent and identical in binding strength for DMPE-B. Below the CMC of C12E8, precipitation is observed and is monitored by increasing turbidity of the solution. The kinetics of precipitation and the aggregate size measured by quasielastic light scattering were analyzed using Smoluchowski kinetics and the Mie scattering theory. These results help establish more completely the factors that influence the precipitation of proteins by ligand-modified phospholipids, and they are helpful in specifying conditions for the precipitation of other proteins.     

Biotinylated peptides/proteins. II. Identification of biotinylated lysyl phenylthiohydantoins

The identification and characterization of biotinylated lysyl residues in a polypeptide chain by automated sequence analysis is described. An in depth analytical study was conducted for the delineation of N epsilon modification of lysyl residues with N-hydroxysuccinimide esters of biotin and 6-aminohexanoic biotin. Confirmation of the structure of the phenylthiohydantoin derivatives of N epsilon biotinylated lysine was achieved by mass spectrometry. The analytical study focused on the identification of biotinylated lysine-4 of neuropeptide Y which served as a model peptide for the analytical procedures detailed.     

Head group and structure specific interactions of enkephalins and dynorphin with liposomes: investigation by hydrophobic photolabeling

The interaction of the opioid enkephalins and endorphins with lipid bilayers is largely unknown. Such interactions might, however, be important for understanding the molecular mechanisms of biological action. We have therefore studied the interaction of several enkephalins and of dynorphin-(1-13)-tridecapeptide (dynorphin1-13) with model membrane systems, using the extremely hydrophobic photolabel of J. Brunner and G. Semenza, (Biochemistry 20, 7174-7182 (1981)) 3-trifluoromethyl-3-(m[125I]iodophenyl)diazirine. By observing several limitations of the method, it was possible to characterize hydrophobic interactions of opioid peptides with liposomes prepared from egg yolk phosphatidylcholine (PC) plus dipalmitoylphosphatidic acid (PA), from brain phosphatidylserine (PS) alone, and from brain cerebroside sulfate (CS) alone, Dynorphine1-13 exhibited strong hydrophobic interactions through its N-terminal "message" segment which were potentiated by the "address" that itself remained in the aqueous phase. This behavior was consistent with the reported pharmacological potentiation. The enkephalins were generally weakly labeled in the PC/PA and PS systems. However, in the CS systems the preferentially mu agonists were labeled very strongly whereas the preferentially delta agonists were labeled more weakly yet. The kappa agonist, dynorphin1-13, was strongly, but more equally labeled in the three systems. Thus, there was a head group specificity that, in our series of compounds, correlated with opiate receptor sub-type specificity. The results were consistent with the behavior of the mu agonist, morphine.     

Lectin-receptor interactions in liposomes II. Interaction of wheat germ agglutinin with phospatidylcholine liposomes containing incorporated monosialoganglioside

Bovine brain gangliosides incorporated into phospholipid liposomes provide receptors for wheat germ agglutinin. Purified monosialogangliosides were mixed with egg phosphatidylcholine, and unilamellar liposomes were generated. Addition of wheat germ agglutinin induced the liposomes to fuse, and gel filtration analysis revealed that the lectin was incorporated into the fused liposomes. The fusion process was studied by following the changes in the 190° light scattering. Increasing the proportion of the monosialoganglioside in the liposomes was found to increase both the extent of the lectin-induced liposome fusion and the rate of the reaction; below a threshold of approx. 5 mol %, the process was extremely slow. The increase in light scattering could be prevented by the addition of the hapten inhibitor, N-acetyl-d-glucosamine (1 mM). Addition of the inhibitor, subsequent to the lectin, caused a partial decrease in light scattering due to the dissociation of unfused vesicle aggregates. Electron microscopic examination revealed that the ganglioside-containing liposomes were vesicles, 244±25 Å (S.D.) in diameter. Upon addition of wheat germ agglutinin, the vesicles appeared to fuse to form larger vesicles, corresponding to dimers and trimers of the initial vesicles. Inhibition studies with a variety of monosaccharides indicated that the sialic acid moieties present in the gangliosides acted as the lectin-receptor sites. This was confirmed by the observation that wheat germ agglutinin did not interact with phosphatidylcholine vesicles containing desialyated ganglioside.     

Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies

Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG.     

Aggregation of cardiolipin liposomes induced by monovalent cations

Monovalent ion induced aggregation of the cardiolipin bilayer liposomes is studied. Derived threshold concentrations (Ck) stimulating fast aggregation testify that the order of effectiveness for monovalent cations to cause this process is: H+ greater than Na+ greater than Li+ greater than K+. The Ck is shown to be nonmonotonously dependent on the temperature discovering a maximum in the range approximately 30-40 degrees C. It is also shown that the liposomes preliminary temperature processing for two hours at approximately 70 degrees C as well as the liposomes incubation for several days at approximately 5 degrees C affect the Ck value. In both cases a considerable Ck increase is accompanied by almost two-fold increase of the lipid oxidation index. The studied process is reversible to both electrolyte concentration dilution and temperature changes. However, unlike the phosphatidylserine (PS) and phosphatidic acid (PA) liposomes the observed changes in the cardiolipin case proceeding considerably slower possibly indicate that the potential must be lower in its depth than that in the case of PS and/or PA.     

Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts.

Lecithin and lecithin/cholesterol liposomes formed in aqueous solutions of DNA entrap covalently closed circular, open circular and linear DNA molecules of size up to at least 13 kilobases. The sequestered DNA molecules are efficiently protected against exogenous deoxyribonuclease action although nicking and linearization of circular DNA can be observed. The size of these liposomes ranges from approximately 0.5 to 7.5 mu with an average of 2.5--4 mu. DNA filled liposomes strongly interact with plant protoplasts under conditions inducing protoplast fusion. Results suggest that sequestered plasmid DNA can be transferred to protoplast nuclei.     

Interactions of liposomes with the reticuloendothelial system: II. Nonspecific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages

Liposomes are taken up as intact vesicles by mouse peritoneal macrophages in a process which is temperature sensitive and is affected by inhibitors of glycolytic metabolism and of microfilament activity. Macrophages take up negatively charged vesicles more readily than positively charged vesicles (2-fold) or neutral vesicles (4-fold). Macrophages take up similar amounts of multilamellar liposomes, reversed phase liposomes and small unilamellar liposomes in terms of lipid, however this corresponds to vastly different numbers of particles and amounts of trapped volume. Coating the liposomes with macromolecular ligands capable of interacting with macrophage surface receptors can markedly promote liposome uptake. Thus, formation of an IgG-antigen complex on the liposome surface results in a 10²-fold enhancement of liposome uptake, while coating the vesicles with fibronectin results in a 10-fold augmentation of uptake. Uptake via IgG-mediated and fibronectin-mediated processes seem to be independent since excess unlabelled, IgG-coated liposomes will inhibit the uptake of radioactively-labelled IgG-coated liposomes much more effectively than the uptake of radioactively-labelled fibronectin-coated liposomes. Cell-bound liposomes can readily be visualized on and inside of the macrophages using fluorescence microscopy techniques.     

Aggregation of isolated myofibrils stimulated by their contraction. II. Aggregation mechanism

Aggregation of contracted myofibrils was studied as a function of experimental conditions at myofibril contraction. The aggregation rate increased at higher concentrations of suspensions and at increased ionic strength of the medium to achieve the maximum at 0.1 M KCL in the last case. The aggregate sizes grow with an increase of ionic strength and concentration of MgATP and reduce with addition of F-actin. Aggregation of myofibrils develops only in the case of their complete or significant contraction. It was suggested that aggregation is stimulated by dehydration of myofibril at contraction.     

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.     

Development of amphotericin B liposomes bearing antibody specific to Candida albicans

Summary Liposomes expressing external antibody specific for Candida albicans and encapsulating amphotericin B were developed and characterized in this study. Antibody was first modified by the covalent attachment of palmitic acid residues. Liposomes were produced by reverse-phase evaporation and modified antibody was incorporated into these liposomes via the hydrophobic interaction between the palmitic acid and the phospholipids composing the liposomes. The liposomes were characterized as to the amount of amphotericin B by spectroscopy and for the presence of antibody by protein analysis and secondary immunolabeling by fluorescent and electron microscopic methods. Immunogold labeling showed that the antibody was being expressed externally on the liposomes in the electron microscopic studies and the specificity of these liposomes for C. albicans was observed by secondary immunofluorescence.     

Plant–eriophyoid mite interactions: specific and unspecific morphological alterations. Part II

The paper presents recent advances related to both specific and unspecific morphological alterations of plant organs caused by eriophyoid mites. Based on old and new case studies, the diversity of plant malformations, such as galls, non-distortive feeding effects and complex symptoms induced by eriophyoids and/or pathogens vectored by them, is analysed and summarised.     

Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization

A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-epsilon Ahx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-aminohexanoic acid spacer arm argue strongly for the NHS-epsilon Ahx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.