MECHANISMS OF ENTEROCYTE SURFACE POLARITY IN VIVO

小肠上皮细胞表面由两种不同的 微绒毛膜（MVM）和侧基底膜 （LBM）。 正常的肠道功能依赖于这种维持。 严格的表面极性。 本项目的目标是定义 糖蛋白的生物合成途径，这些糖蛋白注定要在 体内肠上皮细胞质膜的独立结构域。 具体 本项目的目的是（1）确定MVM蛋白是否被转运 直接从高尔基体到MVM或最初插入LBM 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转运到细胞表面， 运输囊泡的子集。 第一个目标将在 在大鼠体内进行体内（3 H）-岩藻糖脉冲追踪实验， 小肠上皮细胞的细胞分级分离以制备 高度纯化的MVM、LBM和高尔基体膜。 特异性MVM（蔗糖酶-γ 糊精酶，氨基寡肽酶）和LBM（分泌成分，转铁蛋白 受体）糖蛋白将从这些膜免疫沉淀， 脉冲后的间隔（5'至180'）。 新合成糖蛋白 将通过闪烁计数定量，并通过SDS表征 聚丙烯酰胺电泳和放射荧光照相。 第二个目标将 通过纯化肠上皮细胞转运囊泡 通过密度梯度离心和 逆流分配分离。 双标免疫电子 将使用具有两个单独尺寸的胶体金颗粒的显微镜来 确定是否在相同或不同的组织中发现特定的MVM和LBM糖蛋白， 运输囊泡的不同子集。

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.