COMPONENTS AND KINETICS IN EXOCYTOSIS

我们继续对膜融合的研究，这是最基本的一步 在分泌物、病毒感染、受精和神经传递方面。大海 海胆卵皮质颗粒胞吐，钙触发的一例 膜融合，已经成为一种模型系统。我们一直在 研究某些蛋白质如何(NSF等人)可能会规范 卵子皮质颗粒的胞吐作用。我们已经确定 胞浆不能恢复与孤立的胞吐机械的融合 已被NEM停用。我们用免疫印迹分析发现SEA 海胆卵的胞浆中含有NSF，但没有这种蛋白质 来自体外制剂，该制剂在供应的基础上进行融合 钙离子的含量。然而，NSF和美国国家科学基金会之间稳定而具体的互动 在适当的条件下，可以发生孤立的胞吐机械。 这些结果表明，NSF的作用可能是在 指融合能力强的排列，而不是膜融合本身： 一种新的、不依赖于NSF的融合机制。 评估了失活和异质性的相对作用。 进一步在从海胆卵分离的平面皮质中使用 多种溶液交换方案和不同速率的灌流。 没有发现任何证据支持这一假设 与速率相关或与速率无关的失活是导致 在产生亚最大值的钙浓度时停止融合 皮质脱颗粒的反应。钙触发的胞吐作用是 异质性：某些钙离子的胞吐反应次之。 浓度，因为所有的颗粒都能在 钙的浓度已经融化了。秘密模特具有 钙依赖失活不适合用来描述大脑皮质 海胆卵内脱颗粒。钙离子的最小数量 通过考虑行为来估计参与融合过程的 一个单一的，阶梯式的转变，在一个门槛钙浓度。 这一分析表明，超过四个钙离子参与了 引发胞吐。我们的模型，它描述了动力学和 海胆皮质颗粒胞吐的稳态特征， 已通过添加第三个参数进行扩展，表示 融合复合体的激活时间。模型之间的差异 而最初的胞吐过程已经用这个消除了 修改。

We have continued our research on membrane fusion, the fundamental step in secretion, viral infection, fertilization and neuro-transmission. Sea urchin egg cortical granule exocytosis, an example of calcium triggered membrane fusion, has served as a model system. We have been investigating how certain proteins (NSF et al.) might regulate the exocytosis of the egg's cortical granules. We have established that cytosol is unable to restore fusion to the isolated exocytotic machinery inactivated by NEM. We have found using immunoblot analysis that sea urchin eggs contain NSF in their cytosol but that this protein is absent from an in vitro preparation which undergoes fusion upon the provision of Ca2+. However, a stable and specific interaction between NSF and the isolated exocytotic machinery can occur under the appropriate conditions. These results suggest that the role of NSF might be in the establishment of a fusion-competent arrangement rather than in membrane fusion per se: a novel, NSF-independent mechanism of fusion. The relative roles of inactivation and heterogeneity were evaluated further in the isolated planar cortex from the sea urchin eggs using multiple solution exchange protocols and perfusion at different rates. No evidence was found to support the hypothesis that either rate-dependent or rate-independent inactivation is responsible for the cessation of fusion at calcium concentrations which produced sub-maximal responses in cortical degranulation. Calcium-triggered exocytosis was heterogeneous: exocytotic responses were sub-maximal at certain calcium concentrations because all the granules capable of fusing at those concentrations of calcium had fused. Secretory models featuring calcium-dependent inactivation are inappropriate for describing cortical degranulation in the sea urchin egg. The minimum number of calcium ions involved in the fusion process was estimated by considering the behavior of a single, step-like transition at a threshold calcium concentration. This analysis suggests that more than four calcium ions are involved in triggering exocytosis. Our model, which describes both the kinetic and steady-state features observed in sea urchin cortical granule exocytosis, has been extended with the addition of a third parameter representing the activation time for fusion complexes. Discrepancies between the model and the initial time course of exocytosis have been eliminated with this modification.