CHARACTERIZATION AND SORTING OF ZYMOGEN GRANULE PROTEINS

描述（改编自研究者的摘要和/或目的）： 申请人注意到上皮极性的建立是必要的 对胰腺外分泌功能的影响 虽然顶端的血浆 胰腺的膜约占总量的5%， 质膜的表面积，酶原颗粒是靶向的 特别是这个区域，导致消化酶的释放。 胰腺外分泌上皮极性的生物起源将是 通过询问酶原颗粒是否代表唯一的途径来检查 到顶端膜。 为了实现这一目标，外分泌细胞系， AR 42 J将用编码血凝素的cDNA稳定转染 流感病毒蛋白，一种顶端膜蛋白，通常在 分泌颗粒 利用放射性脉冲追踪技术， 亚细胞分离，血凝素的细胞内途径， 将质膜与内源性膜进行比较 存在于酶原颗粒中的蛋白质。 内源性膜蛋白将 通过使用产生的单克隆抗体鉴定和表征 针对纯化的酶原颗粒。 申请人希望这些 实验将提供深入了解蛋白质分选的机制， 极化分泌上皮 除了它们在定义 细胞内途径，单克隆抗体也将用于 鉴定在调节分泌中功能重要的蛋白质。 将测试单克隆抗体抑制 调节分泌，如通过两种测定所测量的。 一种测定方法测量 酶原颗粒与质膜的体外融合。 第二 该测定利用红细胞将抗体递送到AR 42 J中 细胞 将表征显示抑制的那些抗体 进一步. 最后，还将努力发展 培养的极化外分泌胰腺细胞系。 申请人 相信这样的细胞系将为研究 胰腺功能的分子和细胞生物学技术。

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.