Biomedical subjects

D Hoekstra

Publications and source records attributed to D Hoekstra.

At least 109 records · Page 6Linked to original sources

PubMed1988

Effect of poly(ethylene glycol) on the Ca2+-induced fusion of didodecyl phosphate vesicles.

This paper reports a study of the effect of the dehydrating agent poly(ethylene glycol) (PEG) on didodecyl phosphate (DDP) bilayers and on the fusion activity of DDP vesicles as a function of the molecular weight of PEG. PEG 8K in a concentration of 10 wt % does not induce fusion. However, Ca2+-induced fusion is promoted as reflected by a lowering of the Ca2+ threshold concentration. This effect can most likely be attributed to the dehydrating capacity of the polymer. Interestingly, low concentrations (0.1 wt %) of PEG 20 K induce a moderate fusion capacity. At higher concentrations (0.5 wt %) fusion is inhibited, irrespective of the presence of Ca2+. These molecular weight dependent effects can be rationalized by taking into account that the clouding temperature differs for PEGs of different molecular weights. In the case of PEG 20K a microscopic phase separation will occur at the bilayer-water interface because PEG-PEG interactions and presumably PEG-DDP interactions are favored over PEG-water interactions. As a consequence, the DDP vesicle surface becomes covered with PEG 20K, resulting in a steric stabilization of the vesicles. This will impede or prevent, depending on the polymer concentration, the vesicles from approaching each other sufficiently close for fusion to occur.

Calcium↗

PubMed1988

Membrane penetration of Sendai virus glycoproteins during the early stages of fusion with liposomes as determined by hydrophobic photoaffinity labeling.

The hydrophobic photoaffinity label 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to label Sendai virus proteins during fusion with cardiolipin and phosphatidylserine liposomes. Preferential labeling of the viral fusion protein during the initial stages of fusion demonstrated that this protein interacts with the hydrophobic core of the target membrane as an initiating event of virus-liposome fusion. Labeling showed time, temperature, and pH dependence consistent with earlier fluorescent measurements of fusion kinetics. The present method provides conclusive evidence supporting the hypothesis that hydrophobic interaction of the fusion protein with the target bilayer is an initial event in the fusion mechanism of viral membranes.

Affinity Labels↗

PubMed1987

Calcium-induced fusion of didodecylphosphate vesicles: the lamellar to hexagonal II (HII) phase transition.

Electron microscopic techniques have been employed to investigate the ability of didodecylphosphate vesicles (diameter approx. 900 A) to fuse in the presence of Ca2+. As revealed by negative staining, Ca2+ induces extensive fusion and large vesicles with diameters up to 7000 A are formed. In a process secondary to fusion, the fused vesicles display a tendency to flatten and are subsequently transformed into extended tubular structures. Freeze-fracture electron microscopy, in conjunction with 31P NMR and selected area electron diffraction measurements indicate that the tubes are packed in a hexagonal (HII) array and that the amphiphiles are converted from the lamellar to the hexagonal HII phase. The relationship between membrane fusion and the lamellar-to-hexagonal phase transition is discussed in terms of formation and abundance of transiently stable inverted micellar intermediates at contact regions between two interacting membranes. A model for the conversion of the (vesicular) lamellar into the (tubular) hexagonal HII phase is presented, taking into account the molecular shape of the amphiphile. The relevance of using simple synthetic amphiphiles as models for phospholipid bilayers and complex biomembrane behavior is briefly discussed.

Calcium↗

PubMed1987

Anatomy and differential growth of the lateral line system of the mottled sculpin, Cottus bairdi (Scorpaeniformes: Cottidae).

Scanning electron microscopy and standard histological techniques were used to describe the basic anatomy and postembryonic growth of the lateral line system of Cottus bairdi. The gross anatomy and distribution of lateral line neuromasts in canals and superficially on the skin are similar to what has been reported for many primitive actinopterygian fishes. Both canal and superficial neuromasts showed postembryonic growth in the number of hair cells and size of neuromasts, but canal neuromasts grew more elongate whereas superficial neuromast grew symmetrically. Neuromasts in the mandibular canal grew significantly faster than any other neuromasts and superficial neuromasts on the head showed the least amount of growth. Differential growth of the sculpin lateral line system may be related to the feeding behavior of the animal and to differences in the ambient levels of water noise to which larval and adult fish are exposed.

Animals↗

PubMed1986

Parameters affecting fusion between Sendai virus and liposomes. Role of viral proteins, liposome composition, and pH.

A kinetic and quantitative characterization of the fusion process between Sendai virus and phospholipid vesicles is presented. Membrane fusion was monitored in a direct and continuous manner by employing an assay which relies on the relief of fluorescence self-quenching of the probe octadecylrhodamine B chloride which was located in the viral membrane. Viral fusion activity was strongly dependent on the vesicle lipid composition and was most efficient with vesicles solely consisting of acidic phospholipids, particularly cardiolipin (CL). This result implies that the fusion of viruses with liposomes does not display an absolute requirement for specific membrane receptors. Incorporation of phosphatidylcholine (PC), rather than phosphatidylethanolamine (PE), into CL bilayers strongly inhibited fusion, suggesting that repulsive hydration forces interfere with the close approach of viral and target membrane. Virus-liposome fusion products were capable of fusing with liposomes, but not with virus. In contrast to fusion with erythrocyte membranes, fusion between virus and acidic phospholipid vesicles was triggered immediately, did not strictly depend on viral protein conformation, and did not display a pH optimum around pH 7.5. On the other hand, with vesicles consisting of PC, PE, cholesterol, and the ganglioside GD1a, the virus resembled more closely the fusogenic properties that were seen with erythrocyte target membranes. Upon decreasing the pH below 5.0, the viral fusion activity increased dramatically. With acidic phospholipid vesicles, maximal activity was observed around pH 4.0, while with GD1a-containing zwitterionic vesicles the fusion activity continued to increase with decreasing pH down to values as low as 3.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Mass action analysis of kinetics and extent of fusion between Sendai virus and phospholipid vesicles.

The kinetics and extent of fusion between Sendai virus particles and liposomes were investigated with an assay for lipid mixing based on the relief of self-quenching of fluorescence. The measurements, which were carried out at pH 7.4 and 5.0, included liposomes of three compositions, cardiolipin (CL), CL/dioleoylphosphatidylcholine (CL/DOPC 1:1), and phosphatidylserine (PS). Liposomal lipid concentrations varied from 2.5 to 50 microM. In addition, the effect of low concentrations of the dehydrating agent poly(ethylene glycol) (PEG) on fusion between the virus and the liposomes at pH 7.4 was studied. The results were analyzed in terms of a mass action kinetic model which views the overall fusion reaction as a sequence of a second-order process of virus-liposome adhesion or aggregation, followed by the first-order fusion reaction itself. The fusion products were shown to consist of a single virus particle and several liposomes. Analytical solutions were found for the final extent of fusion and increase in fluorescence intensity following the fusion of fluorescently labeled virus particles with liposomes. The final extents of fluorescence intensity were explained by assuming an essentially irreversible binding of liposomes to inactive virus particles. The percents of active virus particles and the rate constants of fusion and aggregation were larger at pH 5 than at pH 7.4, increased when PEG was included in the medium, and varied with liposomal lipid composition according to the sequence CL greater than CL/DOPC greater than PS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Fusion activity of influenza virus. A comparison between biological and artificial target membrane vesicles.

We have investigated the pH-dependent fusion activity of influenza virus toward human erythrocyte ghosts, utilizing a recently developed fluorescence assay, which permits continuous monitoring of the fusion reaction. The rate of fusion is negligible at neutral pH but shows a sharp increase at pH values just below 5.5. This pH dependence profile closely corresponds to that of virus-induced hemolysis. Fusion is rapidly inactivated by a low-pH preincubation of the virus alone either at 37 or at 0 degrees C. The presence of ghosts during this low-pH preincubation, carried out at 0 degree C under which condition there is hardly any fusion, causes a significant protection of the viral fusion activity against inactivation. Fusion initiated at low pH can be arrested instantaneously by readjustment of the pH to neutral. The characteristics of fusion of influenza virus with ghosts deviate from those of fusion with cardiolipin liposomes (Stegmann, T., Hoekstra, D., Scherphof, G., and Wilschut, J. (1985) Biochemistry 24, 3107-3113). Fusion with ghosts is consistent with a requirement of the well-documented pH-dependent conformational change in the viral hemagglutinin, whereas fusion with cardiolipin liposomes does not exhibit a strict dependence on the conformational change. Rather, the negative surface charge on the liposomes plays a critical role, as zwitterionic liposomes containing gangliosides show fusion behavior similar to that of erythrocyte ghosts.

Cold Temperature↗

PubMed1986

Kinetics and extent of fusion between Sendai virus and erythrocyte ghosts: application of a mass action kinetic model.

The kinetics and extent of fusion between Sendai virus and erythrocyte ghosts were investigated with an assay for lipid mixing based on the relief of self-quenching of fluorescence. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of virus-cell adhesion followed by the first-order fusion reaction itself. The fluorescence development during the course of the fusion process was calculated by numerical integration, employing separate rate constants for the adhesion step and for the subsequent fusion reaction. Dissociation of virus particles from the cells was found to be of minor importance when fusion was initiated by mixing the particles at 37 degrees C. However, besides the initiation of fusion, extensive dissociation does occur after a preincubation of a concentrated suspension of particles at 4 degrees C followed by a transfer of the sample to 37 degrees C. The conclusion drawn from the levels of fluorescence increase obtained after 20 h of incubation is that in principle most virus particles can fuse with the ghosts at 37 degrees C and pH 7.4. However, the number of Sendai virus particles that actually fuse with a single ghost is limited to 100-200, despite the fact more than 1000 particles can bind to one cell. This finding may imply that 100-200 specific fusion sites for Sendai virus exist on the erythrocyte membrane. A simple equation can yield predictions for the final levels of fluorescence for a wide range of ratios of virus particles to ghosts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Ricinus communis agglutinin-mediated agglutination and fusion of glycolipid-containing phospholipid vesicles: effect of carbohydrate head group size, calcium ions, and spermine.

The glycolipids galactosylcerebroside (GalCer), lactosylceramide (LacCer), and trihexosylceramide (Gb3) were inserted into phospholipid vesicles, consisting of phosphatidylethanolamine and phosphatidic acid. The extent to which their carbohydrate head groups protruded beyond the vesicle surface and their interference with membrane approach were examined by determining vesicle susceptibility toward type I Ricinus communis agglutinin (RCA1) induced agglutination and Ca2+- and spermine-induced aggregation and fusion either in the presence or in the absence of the lectin. The initial agglutination rates increased in the order GalCer much less than LacCer less than Gb3, while a reversed order was obtained for Ca2+- and spermine-induced aggregation and fusion, indicating an enhanced steric interference on close approach of bilayers with increasing head group size. The lectin-mediated agglutination rates for LacCer- and Gb3-containing vesicles increased by an order of magnitude when Ca2+ was also included in the medium, at a concentration that did not induce aggregation per se. Charge neutralization could not account for this observation as the polyvalent cation spermine did not display this synergistic effect with RCA1. Addition of Ca2+ to preagglutinated vesicles substantially reduced the threshold cation concentration for fusion (micromolar vs. millimolar). Quantitatively, this concentration decreased with decreasing carbohydrate head group size, indicating that the head group protrusion determined the interbilayer distance within the vesicle aggregate. The distinct behavior of Ca2+ vs. spermine on RCA1-induced agglutination on the one hand and fusion on the other indicated that Ca2+ regulates the steric orientation of the carbohydrate head group, which appears to be related to its ability to dehydrate the bilayer. As a result, lectin agglutinability becomes enhanced while fusion will be interrupted as the interbilayer distance increases, the threshold head group size being three carbohydrate residues (Gb3). Finally, GalCer-containing vesicles were not agglutinated by RCA1 at ambient temperature, irrespective of the presence of Ca2+. Above 25 degrees C, RCA1 facilitated Ca2+-induced fusion of the vesicles, which was abolished by the haptenic sugar lactose. Since Gb3- and LacCer-containing vesicles displayed a similar behavior, a temperature-induced alteration in the supporting lipid matrix is suggested, which apparently affects lectin/glycolipid interaction.

Agglutination↗

PubMed1986

Use of a fluorescence assay to monitor the kinetics of fusion between erythrocyte ghosts, as induced by Sendai virus.

The kinetics of the fusion process between erythrocyte ghosts, as induced by Sendai virus, were readily revealed by a simple fluorescence procedure previously employed to characterize the fusion of viruses with biological membranes. The method relies on the relief of fluorescence selfquenching of the membrane-inserted probe octadecyl Rhodamine B chloride (R18) as occurs when labeled membranes fuse with unlabeled counterparts. The kinetics of R18 insertion into ghost membranes, the non-exchangeable properties of the fluorophore and the kinetics, and some characteristics of Sendai virus-induced fusion of ghosts, are described. We propose that the experimental approach may be particularly advantageous to obtain insight into the efficiency and mechanism of a wide range of fusogens, capable of inducing fusion of erythrocyte membranes.

Cell Fusion↗

PubMed1986

Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion.

A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost.

Cold Temperature↗

PubMed1985

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing.

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.

Animals↗

PubMed1985

Low pH-induced fusion of liposomes with membrane vesicles derived from Bacillus subtilis.

We have investigated the pH-dependent interaction between large unilamellar phospholipid vesicles (liposomes) and membrane vesicles derived from Bacillus subtilis, utilizing a fluorescent assay based on resonance energy transfer (RET) (Struck, D. K., Hoekstra, D., and Pagano, R. E. (1981) Biochemistry 20, 4093-4099). Efficient interaction occurs only with negatively charged liposomes, containing cardiolipin or phosphatidylserine, as revealed by the dilution of the RET probes from the liposomal bilayer into the bacterial membrane. The initial rate of fluorophore dilution increases steeply with decreasing pH. The interaction involves a process of membrane fusion, as indicated by the proportional transfer of cholesteryl-[1-14C]oleate, 14C-labeled egg PC, and the RET probes from the liposomes to the bacterial vesicles, the formation of interaction products with an intermediate buoyant density, and the appearance of colloidal gold, initially encapsulated in the liposomes, in the internal volume of fused structures as revealed by thin-section electron microscopy. Treatment of B. subtilis vesicles with trypsin strongly inhibits the fusion reaction, indicating the protein dependence of the process. Vesicles derived from Streptococcus cremoris or from the inner membrane of Escherichia coli also show low pH-dependent fusion with liposomes. The fusion process described in this paper may well be of considerable importance to studies on the mechanisms of membrane fusion and to studies on the structure and function of bacterial membranes. In addition, the fusion reaction could be utilized to deliver foreign substances into bacterial protoplasts.

Bacillus subtilis↗

PubMed1985

Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes.

A novel fluorescence assay [Hoekstra, D., De Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-5681] has been used to characterize the fusogenic properties of Sendai virus, using erythrocyte ghosts and liposomes as target membranes. This assay involves the incorporation of the "fusion-reporting" probe in the viral membrane, allowing continuous monitoring of the fusion process in a very sensitive manner. Fusion was inhibited upon pretreatment of Sendai virus with trypsin. Low concentrations of the reducing agent dithiothreitol (1 mM) almost completely abolished viral fusion activity, whereas virus binding was reduced by ca. 50%, indicating that the fusogenic properties of Sendai virus are strongly dependent on the integrity of intramolecular disulfide bonds in the fusion (F) protein. Pretreatment of erythrocyte ghosts with nonlabeled Sendai virus inhibited subsequent fusion of fluorophore-labeled virus irrespective of the removal of nonbound virus, thus suggesting that the initial binding of the virus to the target membrane is largely irreversible. As a function of pH, Sendai virus displayed optimal fusion activity around pH 7.5-8.0. Preincubation of the virus at suboptimal pH values resulted in an irreversible diminishment of its fusion capacity. Since virus binding was not affected by the pH, the results are consistent with a pH-induced irreversible conformational change in the molecular structure of the F protein, occurring under mild acidic and alkaline conditions. In contrast to virus binding, fusion appeared to be strongly dependent on temperature, increasing ca. 25-fold when the temperature was raised from 23 to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte Membrane↗

PubMed1985

Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion.

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗

PubMed1985

Kinetics of pH-dependent fusion between influenza virus and liposomes.

The pH-dependent fusion between influenza virus and liposomes (large unilamellar vesicles) has been investigated as a model for the fusion step in the infectious entry of the virus into cells. Fusion was monitored continuously, with a fluorescence assay based on resonance energy transfer (RET) [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which allows an accurate quantitation of the fusion process. Evidence is presented indicating that the dilution of the RET probes from the liposomal bilayer into the viral membrane is not due to transfer of individual lipid molecules. The initial rate and final extent of the fusion reaction increase dramatically with decreasing pH, fusion being virtually complete within 1 min at pH 4.5-5.0. From experiments in which the ratio of virus to liposomes was varied, it is concluded that virus-liposome fusion products continue to fuse with liposomes, but not with virus. Fusion is most efficient with liposomes consisting of negatively charged phospholipids, while phosphatidylcholine and sphingomyelin are inhibitory. The reaction is completely blocked by an antiserum against the virus and inhibited by pretreatment of the virus with trypsin. The effect of proteolytic pretreatment at pH 7.4 is enhanced after preincubation of the virus at pH 5.0, consistent with the occurrence of a low pH induced, irreversible, conformational change in the viral fusion protein, the hemagglutinin (HA), exposing trypsin cleavage sites. When, after initiation of the fusion reaction at pH 5.0, the pH is readjusted to neutral, the process is arrested instantaneously, indicating that the low pH induced conformational change in the HA protein, in itself, is not sufficient to trigger fusion activity.

Animals↗

PubMed1985

Reconstitution and fusogenic properties of Sendai virus envelopes.

Sendai virus membranes were reconstituted by detergent dialysis, using the non-ionic detergents Triton X-100 and octyl glucoside. Membrane reassembly was determined by measuring the surface-density-dependent efficiency of resonance energy transfer between two fluorescent phospholipid analogues, which were co-reconstituted with the viral envelopes. The functional incorporation of the viral proteins was established by monitoring the ability of the reconstitution products to fuse with erythrocyte membranes, utilizing assays based on either resonance energy transfer or on relief of fluorescence selfquenching. The persistent adherence of residual Triton X-100 with the reconstituted membrane was revealed by an artificial detergent-effect on the resonance energy transfer efficiency and the occurrence of hemolysis of human erythrocytes under conditions where fusion does not occur. Properly reconstituted Sendai virus envelopes were obtained with octyl glucoside. The fusion activity of the viral envelopes was dependent on the initial concentration of octyl glucoside used to disrupt the virus and the rate of detergent removal. Rapid removal of detergent by dialysis against large volumes of dialysis buffer (ratio 1:850) or by gel filtration produced reconstituted membranes capable of inducing hemagglutination but significant fusion activity was not detected. By decreasing the volume ratio of dialysate versus dialysis buffer to 1:250 or 1:25, fusogenic viral envelopes were obtained. The initial fusion kinetics of the reconstituted viral membrane and the parent virus were different in that both the onset and the initial rate of fusion of the reconstituted membranes were faster, whereas the extents to which both particles eventually fused with the target membrane were similar. The differences in the initial fusion kinetics lead us to suggest that the details of the fusion mechanism between Sendai virus and the target membrane involve factors other than the mere presence of glycoproteins F and HN in the viral bilayer. Finally, the results also indicate that determination of the viral fusion activity in a direct manner, rather than by an indirect assay, such as hemolysis, is imperative for a proper evaluation of the functional properties retained upon viral reconstitution.

Chromatography, Gel↗

PubMed1985

Agglutination and fusion of globoside GL-4 containing phospholipid vesicles mediated by lectins and calcium ions.

We have investigated the interaction of five N-acetylgalactosamine (GalNAc) specific lectins with the glycosphingolipid globoside GL-4, inserted into phospholipid vesicles composed of phosphatidyl-ethanolamine and phosphatidic acid, with respect to their ability to induce vesicle agglutination, fusion, and destabilization. The following lectins were used: soybean agglutinin (SBA); Sophora japonica agglutinin (SJA); Helix pomatia agglutinin (HPA); Ricinus communis agglutinin II (RCAII); and Codium fragile agglutinin (CFA). SBA and SJA caused rapid vesicle agglutination while HPA, CFA, and RCAII were ineffective. However, in the presence of RCAII, but not HPA and CFA, the addition of Ca2+ caused vesicle agglutination which was specifically inhibited by the haptenic sugar GalNAc, while ethylenediaminetetraacetic acid (EDTA) dissociated the vesicle complex. RCAII/Ca2+-induced vesicle agglutination was accomplished by binding of Ca2+ to RCAII after the lectin/receptor interaction. The rate of SBA-induced vesicle agglutination was increased in the presence of Ca2+, independent of the order of Ca2+ addition, and was not reversed by EDTA, indicating that the mechanism by which Ca2+ stimulated agglutination in this case was different from that observed in the presence of RCAII. In contrast to RCAII/Ca2+, SBA/Ca2+ induced of the vesicles, which occurred only when Ca2+ was added after lectin addition. Close approach of adjacent bilayers was accomplished by nonspecific interactions of SBA with the bilayer after lectin binding to the receptor as revealed by a limited extent of SBA-induced fusion and an enhanced membrane permeability upon lectin binding. The phenomena observed can be explained in terms of a Ca2+-modulated reorientation of the carbohydrate head group, causing it to adopt a more perpendicular orientation with respect to the plane of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylgalactosamine↗