A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as weil as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic PercoU™ gradient. In this way, secretory vesicles were separated from mitochondrial, mierosomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 ~mol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg 2 + and Ca 2 + concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonie media «400 mosmol/kg) or in Triton X-loO (0.2% final concentration) acetylcholinesterase activity was markedly higher. During hypotonie treatment or when secretory vesicles were specifically lyzed with 2 mM Mg 2 + and 2 mM A TP, adrenalin as weil as part of acetylcholinesterase was released from the vesicular conte nt. On polyacrylamide gel electrophoresis this soluble enzyme exhibited the same electrophoretie mobility as the enzyme released into the perfusate from adrenal glands upon stimulation. In addition to the soluble enzyme a membrane bound form of acetylcholinesterase exists within secretory vesicles, which sediments with the secretory vesicle membranes and exhibits a different electrophoretic mobility compared to the soluble enzyme. It is concluded, that the soluble enzyme found within isolated secretory vesicles is secreted via exocytosis, whilst the membrane-bound form is transported to the cell membrane during this process, contributing to the biogenesis of the cell membrane.

Binding of Ca2+ to liposomes composed of phosphatidylserine (PtdSer) was analyzed by potentiometric titrations. Ca2+ binding to large unilamellar PtdSer vesicles was saturable at a stoichiometry of 1:2 (Ca2+/PtdSer). At approximately 6 X 10(-4) M [Ca2+]free, the binding curve exhibited a discontinuity that can be attributed to the formation of a Ca2+/PtdSer complex with a higher affinity. When both Ca2+ and Mg2+ are present, depending on the relative concentrations, Mg2+ can either complete or can enhance Ca2+ binding. Concomitant to the enhanced binding, the vesicle suspension was found to aggregate, suggesting that close contact of membranes is a prerequisite for the abrupt change in affinity. This concept was tested by binding studies with liposomes of mixed composition. It was found that the incorporation of 50 mol% phosphatidylethanolamine (PtdEtn) into PtdSer liposomes produced a similar binding pattern to that of pure PtdSer with a saturable stoichiometry of 1:2 (Ca2+/PtdSer). However, incorporation of 50 mol% phosphatidylcholine (PtdCho) completely abolished the discontinuous shift in affinity and apparent saturation was reached at a stoichiometry of 1:4 (Ca2+/PtdSer). In addition, Ca2+ binding to PtdSer liposomes with 10 mol% galactosylcerebroside was not altered when compared to pure PtdSer, whereas 10 mol% of the glycolipid GL-4 abolished the increased binding. The results are closely correlated with recent findings on the role of the membrane composition in Ca2+-induced fusion of liposomes and argue in favor of a specific Ca2+/PtdSer complex (with 1:2 stoichiometry) forming only at points of close contact between membranes and serving as the trigger for membrane fusion.

The effect of synexin (an adrenal medullary protein) on the kinetics of Ca2+-and Mg2"-mediated membrane fusion was examined. Membrane fusion was studied by monitoring intermixing of-the aqueous contents of phospholipid vesicles. Synexin 'facilitated Ca2+-mediated, but not Mg2+-mediated, fusion of phosphatidate/phosphatidylethanolamine (1:3) and phosphatidate/phosphatidylserine/phosphatidylethanolamine/cholesterol (1: 2: 3:2) vesicles. The threshold concentration of Ca2+ for fusion was decreased to 10 jM in the presence of synexin at 6 jg/ ml and 1.5 mM Mg2+ in vesicle suspensions containing 50 ,IM lipid. This effect of synexin was drastically inhibited by including 25% phosphatidylcholine (mol/mol) in the vesicle membrane. It is proposed that the Ca2+-dependent lipid-specific enhancement of membrane fusion by synexin contributes to an increase in the sensitivity of specific intracellular membranes to Ca2`with respect to fusion.Membrane fusion, believed to be the key event in exocytosis, is poorly understood due to the complexity of the membranes involved. Thus, a variety of model systems have been introduced for elucidating the molecular mechanism of Ca2+-mediated membrane fusion. The most striking difference in the fusion behavior of secretory vesicles in vitro (1) compared with that of phospholipid vesicles (2-4) is the lower threshold concentration ofCa2+ needed to obtain measurable fusion (0.01-0.1 mM for the former compared with 1 mM for the latter). Indirect evidence suggests that proteins are involved in decreasing the Ca2+ concentration required for membrane fusion. To establish a specific role for proteins in relationship to Ca2+ concentration thresholds, we have studied the effects of several Ca2+-binding proteins on phospholipid vesicle fusion by using a fluorescence assay to monitor the intermixing of aqueous vesicle contents (2, 5). Calmodulin and parvalbumin weakly inhibit the fusion ofphospholipid vesicles (6, 7). Bovine prothrombin and its proteolytic fragment 1 have a strong inhibitory effect on fusion (7). Synexin, a Ca2+-binding protein originally isolated from bovine adrenal medulla (8), can cause aggregation of isolated chromaffin granules at Ca2O concentrations >6 ,AM. We have previously found that synexin increases appreciably the rate of fusion of phospholipid vesicles containing phosphatidylserine (PtdSer) and mixtures of PtdSer and phosphatidylethanolamine (PtdEtn) (6). To obtain a membrane model that has the same sensitivity to Ca2+ with respect to fusion as biological membranes, we have investigated further the effect of synexin on vesicles containing acidic phospholipids other than PtdSer. In this report, we demonstrate that synexin responds to Ca2+ but not to M-g2+ in promoting fusion ofphosphatidate (PtdO)-containing vesicles. The lowest threshold of Ca2+ is -10 uAM, which is in the range of intracellular Ca2+ levels necessary for exocytosis (9-12). In addition, we report that phosphatidylcholine (PtdCho) -inhibits Ca2"-induced fusion and discuss the possible role of synex...

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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