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D Hoekstra

Publications and source records attributed to D Hoekstra.

At least 127 records · Page 7Linked to original sources
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

Lectins facilitate calcium-induced fusion of phospholipid vesicles containing glycosphingolipids.

Ca2+-induced fusion of phospholipid vesicles containing globoside (GL-4) or disialoganglioside (GDla) is several-fold slower than the fusion of the pure phospholipid vesicles. Lectins specific for these glycosphingolipids, soybean agglutinin and wheat germ agglutinin, respectively, enhance the rate of fusion when added to the vesicle suspension before the introduction of Ca2+. The enhancement depends on the lectin concentration and the time of preincubation with the lectin. We propose that lectins facilitate membrane fusion by inducing intermembrane contact, which is the first step in the overall process of membrane fusion, or by laterally phase separating the inhibitory glycolipids.

Calcium↗
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↗
PubMed1983

Topographical distribution of a membrane-inserted fluorescent phospholipid analogue during cell fusion.

Potential alterations in the transbilayer distribution of lipid molecules during cell-cell fusion were studied, using the fluorescent phospholipid analogue 1-acyl, 2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC). The fluophore was inserted into the outer leaflet of the plasma membrane of Chinese hamster fibroblasts from an exogenous source and cell-cell fusion was induced either with Sendai virus or polyethylene glycol (PEG). After fusion, the cells were examined under a fluorescence microscope and the pool of tagged lipid molecules in the external monolayer was determined quantitatively. The results showed that in contrast to PEG-induced cell fusion, substantial redistribution of the lipid marker occurred when cell fusion was induced by Sendai virus and it was estimated that approx. 40% of exogenously supplied lipid was internalized. The possible mechanism causing lipid redistribution in the case of Sendai virus-induced cell fusion is discussed.

Animals↗
PubMed1982

Transbilayer redistribution of phosphatidylethanolamine during fusion of phospholipid vesicles. Dependence on fusion rate, lipid phase separation, and formation of nonbilayer structures.

The effect of membrane fusion on the transbilayer distribution of dioleoyl- and dipalmitoylphosphatidylethanolamine (DOPE and DPPE, respectively) in phosphatidylserine (PS) vesicles was investigated. A 7-fold increase in the external pool of DOPE, as determined by labeling of the vesicle surface with 2,4,6-trinitrobenzenesulfonic acid, was observed when multilamellar vesicles (MLV) consisting of PS and DOPE were incubated with small unilamellar vesicles (SUV) of PS in the presence of Ca2+. In contrast, no significant redistribution of DPPE was seen when similar experiments were performed by using PS bilayers that contained DPPE instead of DOPE. Redistribution of neither DOPE nor DPPE could be detected during SUV-SUV fusion. By using the resonance energy transfer fusion assay for mixing of membrane lipids [Struck, D.K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099; Hoekstra, D. (1982) Biochemistry 21, 2833-2840], it was demonstrated that fusion between SUV and MLV had actually occurred. The results also suggested that (partial) fusion of internal bilayers within the multilamellar system must have occurred. Although Mg2+-induced fusion between SUV and MLV was also observed, no redistribution of DOPE was seen in this case. It is concluded that the observed translocation of DOPE during fusion was probably mediated via inverted micellar structures, which were formed when the lipid was converted to the hexagonal (HII) phase resulting from lipid phase separation between PS and DOPE. However, induction of the hexagonal phase per se, i.e., in the absence of fusion, did not cause substantial transbilayer redistribution of DOPE, suggesting that fusion was intimately involved in this phenomenon. It is suggested that fusion represents the "driving force" for transbilayer DOPE redistribution, requiring a (partial) overlap between the kinetics of phase separation and fusion.

Calcium↗
PubMed1982

Role of lipid phase separations and membrane hydration in phospholipid vesicle fusion.

The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, which occurs when the local concentration of the probe increases upon Ca2+-induced phase separation in phosphatidylserine (PS) bilayers [Hoekstra, D. (1982) Biochemistry 21, 1055-1061]. Fusion was determined by using the resonance energy transfer fusion assay [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which monitors the mixing of fluorescent lipid donor and acceptor molecules, resulting in an increase in energy transfer efficiency. The results show that in the presence of Ca2+, fusion proceeds much more rapidly (t 1/2 less than 5 s) than the process of phase separation (T 1/2 congruent to 1 min). Mg2+ also induced fusion, albeit at higher concentrations than Ca2+. Mg2+-induced phase separation were not detected, however. Subthreshold concentrations of Ca2+ (0.5 mM) or Mg2+ (2 mM) induced extensive fusion of PS-containing vesicles in poly(ethylene glycol) containing media. This effect did not appear to be a poly(ethylene glycol)-facilitated enhancement of cation binding to the bilayer, and consequently Ca2+-induced phase separation was not observed. The results suggest that macroscopic phase separation may facilitate but does not induced the fusion process and is therefore, not directly involved in the actual fusion mechanism. The fusion experiments performed in the presence of poly(ethylene glycol) suggest that the degree of bilayer dehydration and the creation of "point defects" in the bilayer without rigorous structural rearrangements in the membrane are dominant factors in the initial fusion events.

Calcium↗
PubMed1982

Fluorescence method for measuring the kinetics of Ca2+-induced phase separations in phosphatidylserine-containing lipid vesicles.

The effects of Ca2+ and Mg2+ on the fluorescence behavior of the phospholipid analogues 1-acyl-2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl]phosphatidylcholin e and N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine in small unilamellar vesicles consisting of phosphatidylserine, mixtures of phosphaitdylserine/phosphatidylcholine, and mixtures of phosphatidylserine/cholesterol were studied. Fluorescence quenching was observed when Ca2+, but not Mg2+, was added to phosphatidylserine vesicles containing 5 mol% fluorescent lipid. The quenching process, which could be monitored continuously, was virtually complete within 5-6 min at Ca2+ concentrations greater than or equal to 1.5 mM and resulted in a decrease of fluorescence intensity of approximately 60%. Fluorescence quenching did not occur in the presence of 0.5 mM Ca2+; however, simultaneous addition of 6 mM Mg2+ initiated a quenching process similar in rate and extent to that observed at higher concentrations of Ca2+ alone. This quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence is best explained in terms of Ca2+-induced separation of lipid phases that leads to an increase in local concentration of NBD-lipid in the bilayer and hence to self-quenching of NBD fluorescence. The kinetics of Ca2+-induced phase separation were also studied in several mixed lipid systems containing phosphatidylserine. In the case of mixtures of phosphatidylserine/cholesterol, the results indicate the presence of phase-separated regions as an intrinsic property of the vesicles in the absence of Ca2+. Finally, results are presented indicating that the kinetics of phase separation is slow compared to vesicle-vesicle fusion.

Calcium↗
PubMed1981

Use of resonance energy transfer to monitor membrane fusion.

An assay for vesicle--vesicle fusion involving resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl), the energy donor, and rhodamine, the energy acceptor, has been developed. The two fluorophores are coupled to the free amino group of phosphatidylethanolamine to provide analogues which can be incorporated into a lipid vesicle bilayer. When both fluorescent lipids are in phosphatidylserine vesicles at appropriate surface densities (ratio of fluorescent lipid to total lipid), efficient energy transfer is observed. When such vesicles are fused with a population of pure phosphatidylserine vesicles by the addition of calcium, the two probes mix with the other lipids present to form a new membrane. This mixing reduces the surface density of the energy acceptor resulting in a decreased efficiency of resonance energy transfer which is measured experimentally. These changes in transfer efficiency allow kinetic and quantitative measurements of the fusion process. Using this system, we have studied the ability of phosphatidylcholine, phosphatidylserine, and phosphatidylcholine--phosphatidylserine (1:1) vesicles to fuse with cultured fibroblasts. Under the conditions employed, the majority of the cellular uptake of vesicle lipid could be attributed to the adsorption of intact vesicles to the cell surface regardless of the composition of the vesicle bilayer.

4-Chloro-7-nitrobenzofurazan↗
PubMed1981

Cell-induced leakage of liposome contents.

Using the principle of relief of self-quenching of carboxyfluorescein [Weinstein, J. N., Yoshikami, S., Henkart, P., Blumenthal, R., & Hagins, W. A. (1977) Science 195, 489-492] upon leakage of the dye from the interior of lipid vesicles, we investigated the integrity of sonicated small unilamellar vesicles in the presence of isolated hepatocytes, Zajdela ascites hepatoma cells, and plasma membranes of either cell type. We observed that cells as well as plasma membranes induce leakage of carboxyfluorescein from vesicles. Two parameters (initial rate and maximal level of induced leakage) were determined to quantitate the leakage events and were found to depend on cell density, vesicle concentration, and vesicle lipid composition. The magnitude of both parameters is shown to increase with cell density and to decrease with increasing vesicle lipid concentration and seems to be proportional to the number of vesicles found in close contact with the cell. For vesicles made of phosphatidylcholine and cholesterol, the degree of induced leakage increases steeply with cholesterol contents increasing from 30 to 40 mol %. In the case of simultaneous presence of 10 mol % phosphatidylserine, induced leakage can be observed at cholesterol contents exceeding 20 mol %. We show that leak-inducing activity resides in the plasma membrane and that it can be considerably reduced by treatment of the plasma membranes with neuraminidase or trypsin, suggesting the involvement of cell-surface glycoprotein(s). Release of activity from intact cells and isolated plasma membranes into the medium occurs spontaneously (at a slow rate) but can be facilitated by freezing and thawing; the activity can subsequently be recovered in a soluble form from the medium.

Animals↗
PubMed1980

Interactions of phospholipid vesicles with rat hepatocytes in vitro. Influence of vesicle-incorporated glycolipids.

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either N-lignoceroyldihydrolactocerebroside or the monosialoganglioside, GM1, enhanced liposomal lipid uptake 4-5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin. In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [14C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%). The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryloleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicle is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.

Animals↗
PubMed1979

Effect of fetal calf serum and serum protein fractions on the uptake of liposomal phosphatidylcholine by rat hepatocytes in primary monolayer culture.

We studied the effect of fetal calf serum and serum proteins fractions on the interaction of phospholipid vesicles consisting of phosphatidylcholine, cholesterol and dicetylphosphate (molar ratio 7 : 2 : 1), with rat liver parenchymal cells in a primary monolayer culture. During incubation of such vesicles with fetal calf serum part of the labeled phosphatidylcholine is transferred to a lipoprotein particle similar to the one we identified previously as a derivative of high density lipoprotein (Scherphof, G., Roerdink, F.H., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296--307). When the particle thus formed is incubated with the cells a transfer of the phospholipid label to the cells is observed. When vesicles are incubated with the cells in presence of serum such lipoprotein-mediated lipid transfer may conceivably contribute to the total lipid uptake observed. However, we found that the presence of fetal calf serum in the culture medium greatly diminished rather than increased the total transfer of liposomal lipid to the cells. Also bovine serum albumin and bovine beta-globulins reduced this transfer, although to a lesser extent than whole serum. alpha-Globulins, on the other hand, were as effective as complete serum in reducing the uptake of liposomal phospholipid. A gamma-globulin fraction failed to exhibit any effect on the uptake of [14C]phosphatidylcholine by the cells. All protein fractions which were able to inhibit cellular uptake of liposomal phospholipid were shown to bind to the phospholipid vesicles. Furthermore, lipid vesicles reincubated with fetal calf serum and then separated from it showed reduced transfer of labeled phosphatidylcholine ot parenchymal cells. These observation were taken to suggest that the diminished uptake of liposomal lipid may be caused by a modification of tm proteins. On the other hand, we cannot rule out that plasma membrane modifications are involved in the mechanism of inhibition as well.

Animals↗
PubMed1978

Interaction of phospholipid vesicles with rat hepatocytes in primary monolayer culture.

We studied the interaction of positively and negatively charged unilamellar and multilamellar phospholipid vesicles (liposomes) with rat-liver parenchymal cells in primary monolayer culture. Radioactive liposomal phosphatidylcholine was taken up more rapidly and to a larger extent from unilamellar than from multilamellar vesicles. No significant difference in uptake characteristics was observed between vesicles of different charge. The presence of serum greatly reduced uptake of liposomal phosphatidylcholine of both unilamellar and multilamellar vesicles. This serum effect was independent of surface charge of the vesicles. When cells were allowed to take up radioactive liposomal phospholipid and then incubated further in absence of vesicles, part of the radioactivity associated with the cells was released into the medium, most of it as water soluble degradation products. When cells were preincubated with vesicles containing horseradish peroxidase and then, after removal of the vesicles, further incubated, peroxidase activity could be demonstrated in the culture medium, part of it only after addition of Triton X-100. These observations were taken to indicate that part of the phospholipid taken up the cells represented vesicles binding to the cell surface rather than having been internalized. Vesicle-entrapped [125I]albumin was taken up by the cells and rapidly hydrolyzed as indicated by the appearance of radioactivity soluble in trichloroacetic acid within minutes after starting the incubation. No uptake of free albumin could be demonstrated. The kinetics of albumin uptake and release of trichloroacetic acid-soluble radioactivity from the cells suggest that, initially, liposomes are internalized predominantly by endocytosis, while during prolonged incubation fusion of the liposomal membrane with the plasma membrane gradually contributes more substantially to the overall uptake process. The significance of these findings is emphasized with special reference to the use of liposomes as intravenous carriers of enzymes or drugs.

Blood↗
PubMed1976

Release of outer membrane fragments from normally growing Escherichia coli.

A complex containing lipopolysaccharides, phospholipids and proteine separated from the medium by gelfiltration on Sephadex G-200 or by centrifugation. Electron microscopy revealed that this material is released as vesicles and membrane fragements. To determine the origin of these fragments, they were compared to outer and cytoplasmic membranes with respect to keto-deoxyoctulosonic acid, phospholipid, and protein content, phospholipid composition, fatty acid composition, protein distribution on sodium dodecyl sulfate-polyacrylamide gels, buoyant density, and content of several membrane marker enzymes. The results of this comparison indicate that the membrane fragments found in the culture supernatant of normally growing Escherichia coli consist of practically unmodified outer membrane. Possible mechanisms as to the cause of the release of outer membrane fragments, and its relationship to cell-division, are discussed.

Bacterial Proteins↗