PANCREATIC ACINAR SECRETION: RECONSTITUTED EXOCYTOSIS

胰腺腺泡细胞通过胞吐作用合成和分泌，多种 在所有主要食物的消化过程中起重要作用的酶。 胞吐作用是一种普遍存在的细胞过程，只有在 最笼统的术语。在胰腺腺泡细胞中，胞吐作用包括 酶原颗粒膜与心尖血浆的结合与融合 膜结构域，将消化酶释放到腺泡腔内。这个 长期目标是在分子水平上理解胞吐作用 开发一种由亚细胞部分重组的模型系统 胰腺腺泡细胞。 具体目的是：1)产生单抗(MAb) 血浆心尖区和基底外侧区的特异性标志物 胰腺腺泡细胞膜和酶原颗粒膜；2) 用结构域特异性单抗纯化顶端质膜结构域 无论是在免疫亲和技术中还是作为免疫化学标志物 密度梯度分级中不同的膜结构域；3)至 确定颗粒顶端质膜形成的必要条件 结合(例如，钙离子、镁-三磷酸腺苷和非水解性类似物、GTP和 类似物、电解质或胞浆成分)，使用免疫化学 跟踪互动的技术；4)确定进一步的因素 结合的顶端质膜和酶原颗粒的融合所必需的 (例如，游离脂肪酸加上那些将被测试对结合的影响) 通过释放颗粒内容物、电子显微镜和 免疫化学上作为两个膜之间不可逆的相互作用； 和5)使用mAbs来识别作为受体的分子 胰腺中的酶原颗粒膜和顶端质膜 腺泡细胞。

Pancreatic acinar cells synthesize and secrete by exocytosis, a variety of enzymes important in the digestion of all major classes of food. Exocytosis is a ubiquitous cellular process which is understood only in the most general terms. In the pancreatic acinar cell, exocytosis involves binding and fusion of the zymogen granule membrane with the apical plasma membrane domain, releasing digestive enzymes into the acinar lumen. The long term goal is to understand exocytosis at a molecular level by developing a model system reconstituted from subcellular fractions of the pancreatic acinar cell. The specific aims are: 1) to generate monoclonal antibodies (mAb) as specific markers for the apical and basolateral domains of the plasma membrane and the zymogen granule membrane of the pancreatic acinar cell; 2) to purify the apical plasma membrane domain by using domain specific mAbs either in immuno-affinity techniques or as immunochemical markers for the different membrane domains in density gradient fractionation; 3) to determine the necessary conditions for granule-apical plasma membrane binding (e.g., Ca2+, Mg-ATP and nonhydrolyzable analogues, GTP and analogues, electrolytes, or cytosolic components), using immunochemical techniques to follow the interaction; 4) to determine further factors necessary for fusion of bound apical plasma membranes and zymogen granules (e.g., free fatty acids plus those to be tested for effects on binding) monitored by release of granule content, electron microscopy, and immunochemically as an irreversible interaction between the two membranes; and 5) to use mAbs to identify the molecules that act as receptors between zymogen granule membranes and apical plasma membranes in the pancreatic acinar cell.