EXOCYTOSIS MODELING--KINETICS OF MEMBRANE AGGREGATION AND FUSION

神经递质和神经调节剂的释放通过胞吐作用发生； 钙离子流入细胞或神经末梢触发 细胞质膜上的贮藏颗粒。膜聚变 可以通过研究人工或生物的融合来模拟事件 相互连接的膜。一种新型的多通道计算机控制系统 建立了停流快速混合光谱仪，并对其进行了研究。 这些反应的动力学。我们之前的停流混合研究已经 结果表明，小泡的聚集和融合的动力学 结构(人造脂泡、神经递质储存颗粒、 等)聚合度为聚集率的二级动力学 有限的。 我们独创的基于共振的膜融合停流试验 荧光磷脂之间的能量转移受到伪影的影响 NBD标记的磷脂酰乙醇胺与 钙离子等融合催化剂与NBD探针的直接相互作用 并改变它的量子产额。我们已经开发了一种新的分析方法，基于 芘标记磷脂酰胆碱的荧光变化。这 检测方法检测了芘准分子/单体荧光的比率 混合后的时间函数。这一比例与钙无关 浓度，并与芘之间的距离成线性关系 荧光团，从而解决了这些问题，并另外允许容易 计算当小泡融合在一起时预期的幅度变化。 该方法已应用于镁促进的人工囊泡融合。 以及蛋白质催化的融合。

Neurotransmitter and neuromodulator release takes place by exocytosis; the influx of calcium into the cell or nerve terminal triggers the fusion of the storage granule with the cell plasma membrane. The membrane fusion events can be modeled by studying the fusion of artifical or biological membranes with each other. A new multichannel, computer controlled stopped-flow rapid mixing spectrometer has been constructed to study the kinetics of these reactions. Our previous stopped-flow mixing studies have shown that the kinetics of both aggregation and fusion of small vesicular structures (artifical lipid vesicles, neurotransmitter storage granules, etc.) follow second order kinetics with fusion being aggregation rate limited. Our original stopped-flow assay for membrane fusion, based on resonance energy transfer between fluorescent phospholipids was subject to artifacts arising from the interactions of NBD-labelled phosphatidylethanolamine with the fusion catalysts such as Ca2+ interacting directly with the NBD probe and changing its quantum yield. We have developed a new assay, based on changes in the fluorescence of pyrene-labelled phosphatidylcholine. This assay examines the ratio of pyrene excimer/monomer fluorescence as a function of time after mixing. This ratio is independent of calcium concentration and is linearly dependent upon the distance between pyrene fluorophores, thus solving these problems and additionally allowing easy calculation of expected amplitude changes as the vesicles fuse together. This assay has been applied to the Mg-promoted fusion of artifical vesicles as well as protein-catalyzed fusion.