MECHANISMS OF ENTEROCYTE CELL SURFACE POLARITY

这项提案的长期目标是界定 负责细胞蛋白质分选的细胞机制 注定在不同的域上表达 肠细胞质膜。具体目标是：(1)明确 微绒毛和基侧膜蛋白的生物合成 在肠道细胞中使用体内生物合成的组合 研究和免疫电子显微镜。双标签 将使用免疫金技术同时进行 氨基寡肽酶(微绒毛膜)的定位 蛋白质)和分泌成分(基侧膜 蛋白质)；(2)确定是否增溶或磷酸化 分泌成分在引导其从 基侧膜至微绒毛膜。反SC 免疫印迹将用于检测膜的转化 以可溶形式的酶。增溶的位置 酶将通过孵育亚细胞部分来确定。 检测增溶作用的标准方法。在识别出 抑制剂的体外增溶试验，尝试将 在离体灌流鼠体内抑制增溶作用 肝脏。对SC增溶的抑制将通过以下方法进行评估 体内分泌成分释放和分泌失败 胆汁。SC的磷酸化依赖于配体结合意愿 在离体鼠肝脏灌流中作为一种间接手段进行评估 确定SC的磷酸化是否在 引导来自 基侧膜至微绒毛膜；(3)发育 高尔基体到质膜的体外研究系统 运输：这个运输系统将仿照体外运输系统。 交通系统的开发是为了研究 蛋白质在高尔基体堆叠之间的运输：十二烷基硫酸钠 将进行聚丙烯酰胺凝胶电泳法和放射荧光法 用于评估来自供体的放射性标记蛋白质的运输 高尔基膜至非放射性标记受体刷子边缘 薄膜。这些研究的目的是回答基本的 肠上皮细胞如何在形态上和 在功能上极性建立和维持它们的细胞表面 两极。

The long term objectives of this proposal are to define the cellular mechanisms responsible for sorting of cell proteins that are destined to be expressed on separate domains of the enterocyte plasma membrane. The specific aims are (1) to define the biosynthesis of microvillar and basolateral membrane proteins in the enterocyte using a combination of in vivo biosynthetic studies and immunoelectron microscopy. Dual-labeled immunogold techniques will be used for the simultaneous localization of amino-oligopeptidase (a microvillar membrane protein) and secretory component (a basolateral membrane protein); (2) to determine if solubilization or phosphorylation of secretory component is important in directing its transport from the basolateral membrane to the microvillar membrane. Anti-SC immunoblots will be used to detect conversion of the membrane to soluble form of the enzyme. The location of the solubilizing enzyme will be determined by incubating subcellular fractions in the standard assay to detect solubilization. Upon identifying inhibitors of the in vitro solubilization assay, attempts will be made to inhibit solubilization in vivo in the isolated perfused rat liver. Inhibition of SC solubilization will be assessed by the failure of release and secretion of secretory component in the bile. The dependence of SC phosphorylation on ligand binding will be assessed in the isolated perfused rat liver as an indirect means of determining whether SC phosphorylation is important in directing the transport of secretory component from the basolateral membrane to the microvillar membrane; (3) to develop an in vitro system for the study of Golgi to plasma membrane transport: this transport system will be modeled after the in vitro transport systems which have been developed to study the transport of proteins between the Golgi stacks: SDS polyacrylamide gel electrophoresis and radiofluorography will be used to assess transport of radiolabeled proteins from the donor Golgi membrane to the non-radiolabeled acceptor brush border membrane. These studies are aimed at answering the basic question of how enterocytes which are morphologically and functionally polar establish and maintain their cell surface polarity.