CHARACTERIZATION AND SORTING OF ZYMOGEN GRANULE PROTEINS

描述(改编自调查员的摘要和/或目标)： 申请人指出，上皮极性的建立是必不可少的 外分泌胰腺的功能。尽管心尖部的血浆 胰腺的膜约占总数的5%。 质膜表面积、酶原颗粒为靶标 特别是这一区域导致消化酶的释放。 胰腺外分泌中上皮极性的生物发生将是 通过询问酵素原颗粒是否代表唯一途径进行检查 至顶膜。为了实现这一目标，外分泌细胞系， AR42J将编码血凝素的基因稳定地导入 流感蛋白，一种顶膜蛋白，通常不存在于 分泌颗粒。利用放射性脉冲追逐技术和 亚细胞分级，血凝素的细胞内途径 质膜与内生膜进行比较。 存在于酶原颗粒中的蛋白质。内源性膜蛋白将是 通过使用产生的单抗进行鉴定和表征 对纯化的酵母菌颗粒有抑制作用。申请人希望这些 实验将提供对蛋白质分选机制的洞察 极化的分泌上皮。除了它们在定义 细胞内途径，单抗也将被用来 确定在调节分泌过程中具有重要功能的蛋白质。 将对单抗进行测试，以确定其抑制能力 通过两种检测方法测量的调节分泌。一种化验方法测量 酶原颗粒与质膜的体外融合。第二 利用红细胞将抗体输送到AR42J的检测 细胞。那些表现出抑制作用的抗体将被描述为 再远一点。最后，还将努力发展 培养中的极化外分泌胰腺细胞系。申请人 相信这样的细胞系将提供更多的研究机会 用分子和细胞生物学技术研究胰腺功能。

DESCRIPTION (adapted from investigator's abstract and/or aims): The applicant notes that the establishment of epithelial polarity is essential to the function of the exocrine pancreas. Although the apical plasma membrane of the pancreas represents approximately 5 percent of the total surface area of the plasma membrane, zymogen granules are targeted specifically to this area resulting in the release of digestive enzymes. The biogenesis of epithelial polarity in the exocrine pancreas will be examined by asking whether the zymogen granule represents the sole pathway to the apical membrane. To achieve this goal, the exocrine cell line, AR42J, will be stably transfected with cDNA encoding the hemagglutinin protein of influenza, an apical membrane protein normally not found in secretory granules. Utilizing radioactive pulse-chase techniques and subcellular fractionation, the intracellular pathway of hemagglutinin to the plasma membrane will be compared to that of endogenous membrane proteins present in zymogen granules. Endogenous membrane proteins will be identified and characterized by the use of monoclonal antibodies generated against purified zymogen granules. The applicant hopes that these experiments will offer insight into mechanisms of protein sorting in polarized secretory epithelia. In addition to their utility in defining intracellular pathways, the monoclonal antibodies will also be used to identify proteins that are functionally important in regulated secretion. The monoclonal antibodies will be tested for their ability to inhibit regulated secretion as measured by two assays. One assay measures the fusion of zymogen granules with the plasma membrane in-vitro. The second assay utilizes red blood cells for the delivery of antibodies into AR42J cells. Those antibodies that display inhibition will be characterized further. Finally, efforts will also be directed towards the development of a polarized exocrine pancreatic cell line in culture. The applicant believes that such a cell line will offer greater opportunities to study pancreatic function with molecular and cell biological techniques.