Fusion of phospholipid vesicles containing a trypsin‐sensitive fluorogenic substrate and trypsin

Hoekstra, Dick; Yaron, Avraham; Carmel, Amos; Scherphof, Gerrit L.

doi:10.1016/0014-5793(79)80722-x

Cited by 36 publications

(13 citation statements)

References 25 publications

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“…If Ca2+ and Mg 2 + are added to the liposomes simultaneously, the effect of both ions is additive (Table II). These characteristics are very similar to properties found with divalent cation-induced liposome fusion in other model systems [37][38][39][40][41][42][43][44].…”

Section: Fusion 01 Liposomessupporting

confidence: 83%

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Ekerdt

Dahl²,

Gratzl³

1981

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Secretory vesieles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca 2 +. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesieies in freeze-fractured suspensions of vesieles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10-7 M and 10-4 M Ca 2 +, was specific for ea 2 +, was inhibited by other divalent cations and was abolished by pretreatment of vesieles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca 2 + in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesieles: ea 2 + could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type 11 of intact secretory vesieies as weil as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca 2 + concentrations we have not found a major influence of Ca 2 + asymmetry on fusion. Incorporation of the ganglioside GM 3 , which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca 2 +-selective electrode we have identified in secretory vesiele membranes both high affinity binding sites for Ca 2 + (/(} = 1.6 . 10-6 M) and low affinity sites (/(} = 1.2· 10-4 M).

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Section: Fusion 01 Liposomessupporting

confidence: 83%

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Ekerdt

Dahl²,

Gratzl³

1981

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…Comparison of these data with our results is hampered by differences in the type and lipid composition of the vesicles used. Recently, an elegant and simple fusion assay has been described by Hoekstra et al (1979) which involves the fluorescence development due to the degradation by trypsin within vesicles of an intramolecularly quenched fluorogenic substrate in the presence of a trypsin inhibitor in the external medium. Fusion can be continuously monitored and accurately quantitated; however, the assay may not be particularly suitable for following very fast kinetics of fusion since an enzyme reaction is involved.…”

Section: Discussionmentioning

confidence: 99%

“…In any case, the new assay would meet a more rigorous criterion for fusion than the criteria that have been considered most frequently before, such as vesicle aggregation (Kremer & Wiersema, 1977), mixing of membrane components (Maeda & Ohnishi, 1974;; Papahadjopoulos et al, 1974Papahadjopoulos et al, , 1976, release of vesicle contents (Portis et al, 1979), or increase of vesicle size and/or morphological changes (Lau & Chan, 1975;Lawaczeck et al, 1976; Kantor & Prestegard, 1978;Liao & Prestegard, 1979;Verkleij et al, 1974;Koter et al, 1978;Stollery & Vail, 1977;Papahadjopoulos et al, 1975;Day et al, 1977). Previous attempts to monitor mixing of vesicle contents have been limited mainly to methods based on enzyme reactions (Ingolia & Koshland, 1978;Holz & Stratford, 1979;Hoekstra et al, 1979). However, enzyme reactions in general may not be fast enough to adequately follow rapid vesicle fusion.…”

mentioning

confidence: 99%

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

Wilschut¹,

Düzgüneş²,

Fraley³

et al. 1980

Biochemistry

493

378

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“…Pure PC vesicles will not aggregate or fuse in the presence of 10 mM Ca 2 § and 100 mM NaC1 (Papahadjopoulos etal., 1974;Dfizgiine~ & Ohki, 1977). Increasing the PS fraction to 50% enables the vesicles to undergo fusion in this electrolyte, but at a very slow rate (Hoekstra, Yaron, Carmel & Scherphof, 1979;N. Dfizgfine~, unpublished observations).…”

mentioning

confidence: 96%

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

et al. 1981

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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.

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Fusion of phospholipid vesicles containing a trypsin‐sensitive fluorogenic substrate and trypsin

Cited by 36 publications

References 25 publications

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

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

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

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