MECHANISMS OF ENTEROCYTE SURFACE POLARITY IN VIVO

小肠上皮细胞表面由两个截然不同的 微绒毛膜(MVM)和基底侧膜 (LBM)。正常的肠道功能有赖于这种功能的维持 严格的表面极性。此项目的目标是定义 注定要表达的糖蛋白的生物合成途径 活体内肠细胞质膜的分离结构域。具体的 这个项目的目的是(1)确定MVM蛋白是否被运输 直接从高尔基体到MVM或最初插入到LBM中 并随后重新分配给MVM，以及(2)确定MVM和 LBM糖蛋白以共同或不同的方式运输到细胞表面。 运输囊泡的亚群。第一个目标将通过在 大鼠体内岩藻糖脉冲追逐实验 小肠上皮细胞的细胞分级法制备 高纯度的MVM、LBM和高尔基膜。特异性MVM(蔗糖酶-伽马 糊精酶，氨基寡肽酶)和LBM(分泌成分，转铁蛋白 受体)糖蛋白将从这些膜上免疫沉淀 脉冲后的间隔(5‘到180’)。新合成的糖蛋白 将用闪烁计数法定量，并用十二烷基硫酸钠表征 聚丙烯酰胺凝胶电泳法和放射荧光法。第二个目标是 是通过纯化肠细胞运输囊泡来实现的 密度梯度离心法和密度梯度离心法相结合 逆流分布分离。双标记免疫电子 带有两个不同大小的胶体金颗粒的显微镜将用于 确定是否在相同或相同的 运输囊泡的不同亚群。

The small intestinal epithelial cell surface consists of two distinct domains, the microvillaus membrane (MVM) and the laterobasal membrane (LBM). Normal intestinal function is dependent on the maintenance of this strict surface polarity. The goal of this project is to define the biosynthetic pathways of glycoproteins that are destined to be expressed on separate domains of the enterocyte plasma membranes in vivo. The specific aims of this project are (1) to determine if MVM proteins are transported directly from the Golgi to the MVM or are initially inserted into the LBM and subsequently redistributed to the MVM, and (2) to determine if MVM and LBM glycoproteins are transported to the cell surface in common or distinct subsets of transport vesicles. The first aim will be accomplished by in vivo (3H)-Fucose pulse chase experiments in vivo in the rat followed by cell fractionation of the small intestinal epithelial cells to prepare highly purified MVM, LBM and Golgi membranes. Specific MVM (sucrase-Gamma dextrinase, aminooligopeptidase) and LBM (secretory component, transferrin receptor) glycoproteins will be immunoprecipitated from these membranes at intervals (5' to 180') after the pulse. Newly synthesized glycoproteins will be quantitated by scintillation counting and characterized by SDS polyacrylamide electrophoresis and radioflurography. The second aim will be accomplished by purification of the enterocyte transport vesicle population by a combination of density gradient centrifugation and counter-current distribution separation. Double-labeled immunoelectron microscopy with two separate sized colloidal gold particles will be used to determine if specific MVM and LBM glycoproteins are found in the same or different subsets of transport vesicles.