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J Wilschut

Publications and source records attributed to J Wilschut.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1985

Fusion of erythrocyte ghosts induced by calcium phosphate. Kinetic characteristics and the role of Ca2+, phosphate and calcium-phosphate complexes.

Using an assay which allows continuous monitoring of the mixing of aqueous contents during membrane fusion, we have investigated the kinetics of calcium-phosphate-induced fusion of erythrocyte ghosts. In the presence of 10 mM phosphate, the threshold concentration for Ca2+-induced fusion was 1.25 mM, while the optimal concentration was approx. 1.75 mM Ca2+. Further enhancement of the cation concentration (greater than or equal to 2 mM) inhibited fusion of the ghosts. Initiation of fusion required the addition of phosphate prior to the addition of Ca2+, indicating that the combined interaction of Ca2+ and phosphate in or at the plane of the bilayer was a prerequisite for the induction of fusion. Furthermore, fusion was greatly facilitated upon transformation of calcium phosphate in the bulk medium from an amorphous to a solid, crystalline phase. It is suggested that membrane aggregation, and hence fusion, is facilitated by the formation of crystalline calcium phosphate nucleating on the ghost membrane. La3+, Mg2+ and Mn2+ did not trigger the fusion process, although aggregation of the ghosts did occur. Under conditions where calcium phosphate precipitation was inhibited, lanthanum phosphate precipitates facilitated fusion after prior treatment of ghosts with phosphate and Ca2+. These results indicated that fusion-prone conditions were induced prior to calcium phosphate precipitation. It is proposed that prior to calcium phosphate precipitation membrane changes are induced by separate interaction of Ca2+ and phosphate with the ghost membrane. Such an interaction could then render the ghosts susceptible to fusion and as soon as conditions are provided allowing close contact between adjacent membranes, fusion will be observed.

Adenosine Triphosphate↗
PubMed1985

Modulation of membrane fusion by membrane fluidity: temperature dependence of divalent cation induced fusion of phosphatidylserine vesicles.

We have investigated the temperature dependence of the fusion of phospholipid vesicles composed of pure bovine brain phosphatidylserine (PS) induced by Ca2+ or Mg2+. Aggregation of the vesicles was monitored by 90 degrees light-scattering measurements, fusion by the terbium/dipicolinic acid assay for mixing of internal aqueous volumes, and release of vesicle contents by carboxyfluorescein fluorescence. Membrane fluidity was determined by diphenylhexatriene fluorescence polarization measurements. Small unilamellar vesicles (SUV, diameter 250 A) or large unilamellar vesicles (LUV, diameter 1000 A) were used, and the measurements were done in 0.1 M NaCl at pH 7.4. The following results were obtained: (1) At temperatures (0-5 degrees C) below the phase transition temperature (Tc) of the lipid, LUV (PS) show very little fusion in the presence of Ca2+, although vesicle aggregation is rapid and extensive. With increasing temperature, the initial rate of fusion increases dramatically. Leakage of contents at the higher temperatures remains limited initially, but subsequently complete release occurs as a result of collapse of the internal aqueous space of the fusion products. (2) SUV (PS) are still in the fluid state down to 0 degree C, due to the effect of bilayer curvature, and fuse rapidly in the entire temperature range from 0 to 35 degrees C in the presence of Ca2+. The initial rate of leakage is low relative to the rate of fusion. At higher temperatures (15 degrees C and above), subsequent collapse of the vesicles' internal space causes complete release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Fluorescence method for measuring the kinetics of fusion between biological membranes.

An assay is presented that allows continuous and sensitive monitoring of membrane fusion in both artificial and biological membrane systems. The method relies upon the relief of fluorescence self-quenching of octadecyl Rhodamine B chloride. When the probe is incorporated into a lipid bilayer at concentrations up to 9 mol% with respect to total lipid, the efficiency of self-quenching is proportional to its surface density. Upon fusion between membranes labeled with the probe and nonlabeled membranes, the decrease in surface density of the fluorophore results in a concomitant, proportional increase in fluorescence intensity, allowing kinetic and quantitative measurements of the fusion process. The kinetics of fusion between phospholipid vesicles monitored with this assay were found to be the same as those determined with a fusion assay based on resonance energy transfer [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099]. Octadecyl Rhodamine B chloride can be readily inserted into native biological membranes by addition of an ethanolic solution of the probe. Evidence is presented showing that the dilution of the fluorophore, occurring when octadecyl Rhodamine containing influenza virus is mixed with phospholipid vesicles at pH 5.0, but not pH 7.4, resulted from virus-vesicle fusion and was not related to processes other than fusion. Furthermore, by use of this method, the kinetics of fusion between Sendai virus and erythrocyte ghosts and virus-induced fusion of ghosts were readily revealed. Dilution of the probe was not observed upon prior treatment of fluorescently labeled Sendai virus with trypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte Membrane↗
PubMed1984

Ca2+-independent, protein-mediated fusion of chromaffin granule ghosts with liposomes.

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093-4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca2+, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.

Animals↗
PubMed1984

Modulation of membrane fusion by ionotropic and thermotropic phase transitions.

We have studied the relationship of ionotropic and thermotropic phase transitions to divalent cation induced fusion of large unilamellar phospholipid vesicles. Fusion was monitored by the Tb/dipicolinic acid fluorescence assay for the intermixing of internal aqueous contents of vesicles. The phase behavior of the membranes was followed by differential scanning calorimetry. (1) Sr2+ and Ba2+ shifted the phase transition temperature (Tc) of bovine brain phosphatidylserine vesicles from 6 to 27 and 31.5 degrees C, respectively. These cations induced vesicle fusion at temperatures above or below the Tc of that cation/phospholipid complex, indicating that an isothermal phase change from the liquid-crystalline to the gel phase is not a requirement for membrane fusion. (2) The temperature dependence of the initial rate of fusion of phosphatidylserine/dipalmitoylphosphatidylcholine (1:1) vesicles in the presence of Ca2+ exhibited a pronounced maximum at 17 degrees C, at the lower part of the broad phase transition endotherm whose Tc was about 25 degrees C; fusion was inhibited completely at 30 degrees C when the membrane was in the liquid-crystalline state. These observations suggest that molecular clusters rich in phosphatidylserine, formed when the membrane is in the phase transition region, allow the vesicles to fuse. (3) The fusion of phosphatidylserine/dimyristoylphosphatidylethanolamine (1:1) vesicles, whose Tc was also around 25 degrees C, had a different temperature dependence in that the initial rate increased sharply above the Tc, with a local maximum within the transition region. Phase separation of dimyristoylphosphatidylethanolamine was induced by Ca2+ but not by Mg2+, although both ions induced fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Exchange of phosphatidylcholine between small unilamellar liposomes and human plasma high-density lipoprotein involves exclusively the phospholipid in the outer monolayer of the liposomal membrane.

By making use of the capacity of phospholipase A2 to degrade selectively the phospholipid in the outer half of the lipid bilayer of small unilamellar phospholipid/cholesterol vesicles without affecting the retention of a vesicle-encapsulated solute, we demonstrated that the exchange of phosphatidylcholine between such vesicles and human high density lipoprotein involves exclusively the phosphatidylcholine present in the outer monolayer of the vesicle membrane.

Carbon Radioisotopes↗
PubMed1983

Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation.

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.

Animals↗
PubMed1983

Kinetics of calcium phosphate-induced fusion of human erythrocyte ghosts monitored by mixing of aqueous contents.

We have adapted the terbium fusion assay (Wilschut, J. and Papahadjopoulos, D. (1979) Nature 281, 690-692), which has proven to monitor the mixing of internal contents during phospholipid vesicle fusion in a reliable manner (Hoekstra, D. (1982) Biochim. Biophys. Acta 692, 171-175), to study the fusion of erythrocyte ghosts as induced by the combined action of Ca2+ and phosphate. Using this assay, it became possible to reveal, for the first time, the kinetics of fusion of a biological membrane vesicle system. The rate of fusion was critically dependent on the concentration of Ca2+ and phosphate. Prior addition of phosphate was essential for induction of fusion. Initial fusion was largely non-leaky, but in a process secondary to the fusion event the ghosts gradually released their contents. It is suggested that the experimental approach presented in this paper, would facilitate efforts to elucidate the mechanism of fusion of biological membranes.

Acetylcholinesterase↗
PubMed1982

Ca2+-induced fusion of cardiolipin/phosphatidylcholine vesicles monitored by mixing of aqueous contents.

The kinetics of Ca2+-induced fusion of large (0.1 micrometer) unilamellar cardiolipin/phosphatidylcholine (1:1) vesicles have been investigated by continuous monitoring of the mixing of the aqueous vesicle contents. In parallel, release of vesicle contents to the external medium has been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents to the external medium have been followed. Initial fusion of the vesicles is non-leaky, release of vesicle contents being largely a secondary phenomenon. The minimal Ca2+ concentration required for fusion in this system is approx. 9 mM. At higher Ca2+ concentrations fusion is extremely fast, occurring on the time scale of seconds.

Animals↗
PubMed1981

Calcium- and magnesium-induced fusion of mixed phosphatidylserine/phosphatidylcholine vesicles: effect of ion binding.

The aggregation, leakage, and fusion of pure PS (phosphatidylserine) and mixed PS/PC (phosphatidylcholine) sonicated vesicles were studied by light scattering, the release of encapsulated carboxyfluorescein, and a new fusion assay which monitors the mixing of the internal compartments of fusing vesicles. On a time scale of 1 min the extent of fusion was considerably greater than leakage. The Ca2+ and Mg2+ concentrations required to induce fusion increased when the PS content of the vesicles was decreased, and/or when the NaCl concentration was increased. Calculations employing a modified Gouy-Chapman equation and experimentally determined intrinsic binding constants of Na+ and Ca2+ to PS were shown to predict correctly the amount of Ca2+ bound in mixed PS/PC vesicles. For vesicles composed of either pure PS or of mixtures with PC in 100 mM NaCl (4:1 and 2:1 PS/PC); the induction of fusion (on a time scale of minutes) occurred when the amount of Ca or Mg bound/PS molecule exceeded 0.35-0.39. The induction of fusion for both pure PS and PS/PC mixed vesicles (with PS exceeding 50%) can be explained by assuming that destabilization of these vesicles requires a critical binding ratio of divalent cations to PS.

Binding Sites↗
PubMed1981

Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles.

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 micrometer diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca2+ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca2+ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca2+ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg2+ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg2+ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca2+ and also enhanced the rate and extent of fusion. Mg2+ had a synergistic effect on Ca2+-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles. We suggest that the role of phospholipids in membrane fusion is related to their ability to form dehydrated intermembrane complexes with divalent cations.

Calcium↗
PubMed1980

Studies on the mechanism of membrane fusion: kinetics of calcium ion induced fusion of phosphatidylserine vesicles followed by a new assay for mixing of aqueous vesicle contents.

We describe an assay for following the mixing of aqueous contents during fusion of phospholipid vesicles. Terbium is encapsulated as the Tb(citrate)3(6-) chelation complex in one population of vesicles, dipicolinic acid (DPA) in another. Vesicle fusion results in the formation of the fluorescent Tb(DPA)3(3-) chelation complex. The presence of EDTA (0.1 mM) and Ca2+ (greater than 1 mM) prevents the formation of the Tb/DPA complex in the external medium. We have studied the Ca2+-induced fusion of small or large unilamellar vesicles (SUV or LUV, respectively) composed of phosphatidylserine (PS). In addition, vesicle aggregation was monitored by light scattering, and release of vesicle contents was followed by carboxyfluorescein (CF) fluorescence enhancement. The addition of Ca2+ induced an immediate enhancement in Tb fluorescence with both SUV and LUV, which occurs on the same time scale as aggregation but much faster than the release of CF. The release of contents from LUV occurs with a considerable delay. It is estimated that the initial fusion of SUV is accompanied by 10% leakage of the internal volume per fusion event; in contrast, fusion of LUV is essentially nonleaky. Massive release of vesicle contents appears to be a secondary phenomenon related to the collapse of fused vesicles. The initial rate and the extent of Tb fluorescence enhancement are markedly dependent on the Ca2+ concentration. Threshold Ca2+ concentrations are 1.2 and 2.4 mM for SUV nd LUV, respectively. At saturating Ca2+ concentrations (greater than 10 mM), the rate of fusion of LUV is slightly lower than that of SUV at the same vesicle concentration. At any Ca2+ concentration, the rates of both SUV and LUV fusion are consistent with vesicle aggregation being rate limiting. When measured at a subsaturating Ca2+ concentration, fusion is essentially second order over a wide range of relatively low vesicle concentrations, whereas at higher vesicle concentrations the order is decreased. This suggests that at high vesicle concentrations (and at relatively low Ca2+ concentrations) aggregation may proceed faster than fusion.

Calcium↗
PubMed1980

Studies on the mechanism of membrane fusion: role of phosphate in promoting calcium ion induced fusion of phospholipid vesicles.

The role of phosphate in enhancing the calcium-induced fusion of phosphatidylserine (PS) vesicles has been examined by using the new fluorescent (terbium/dipicolinic acid) assay described by Wilschut et al. (1980) [Wilschut, J., Düzgüneş, N., Fraley, R., & Papahajopoulos, D. (1980) Biochemistry (preceding paper in this issue)]. In the presence of physiological levels of phosphate, the calcium concentration required for fusion of PS vesicles was lowered significantly (3-4-fold), and the rate of vesicle fusion was increased dramatically (up to 1000-fold). The fusion of PS vesicles by calcium and phosphate is shown to be specific and critically dependent on temperature, pH, ion concentrations, and the composition of the calcium phosphate crystalline phase present during the incubation. The results indicate that a significant enhancement in vesicle fusion occurs only when calcium phosphate precipitation is initiated in the presence of PS vesicles, suggesting that crystal nucleation on the vesicle surface is a prerequisite for fusion. Calcium and phosphate were shown to promote phospholipid phase separations and vesicle fusion under conditions (e.g., mixtures of PS and phosphatidylcholine) in which calcium alone is ineffective, indicating that formation of PS calcium phosphate complexes may facilitate the molecular segregation of PS into distinct domains. These experiments underline the important role that phosphate may play in calcium-mediated fusion phenomena in biological membranes.

Calcium↗