MECHANISMS OF ENTEROCYTE CELL SURFACE POLARITY

肠细胞表面极性的机制

• 批准号: 3232084
• 负责人: DENNIS J AHNEN
• 金额: $ 11.18万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3232084
• 关键词: Golgi apparatus; apical membrane; basolateral membrane; bile; brush border membrane; electron microscopy; gel electrophoresis; intestinal mucosa; ion transport; laboratory rat; membrane activity; membrane permeability; membrane proteins; phosphorylation; protein biosynthesis; radiotracer; small intestines; surface property; transferrin; tritium

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.

该提案的长期目标是确定 负责分类细胞蛋白质的细胞机制 注定要在不同的域上表达 肠细胞质膜。 具体目标是 (1) 定义 微绒毛和基底外侧膜蛋白的生物合成 在肠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 免疫金技术将用于同时 氨基寡肽酶（微绒毛膜）的定位 蛋白质）和分泌成分（基底外侧膜 蛋白质）; (2) 确定是否溶解或磷酸化 分泌成分对于指导其运输很重要 基底外侧膜至微绒毛膜。 抗SC 免疫印迹将用于检测膜的转化 为酶的可溶形式。 增溶的位置 酶将通过在中孵育亚细胞级分来测定 检测溶解的标准测定。 识别后 体外增溶测定的抑制剂，将尝试 抑制离体灌注大鼠体内溶解 肝。 SC 溶解的抑制将由 分泌成分的释放和分泌失败 胆汁。 SC 磷酸化对配体结合的依赖性 作为间接手段在离体灌注大鼠肝脏中进行评估 确定 SC 磷酸化是否重要 指导分泌成分的运输 基底外侧膜至微绒毛膜； (3) 开发 用于研究高尔基体质膜的体外系统 运输：该运输系统将模仿体外 为研究交通运输系统而开发的 高尔基体堆栈之间蛋白质的运输：SDS 聚丙烯酰胺凝胶电泳和放射荧光检查 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状缘 膜。 这些研究旨在回答基本问题 肠上皮细胞的形态和结构如何变化的问题 功能极性建立并维持其细胞表面 极性。