STUDIES ON INTESTINAL BRUSH BORDER MEMBRANES

本提案的总体目标是确定 参与钴胺素（cbl）的细胞内运动 在其细胞摄取后，通过检查两种细胞的生物学特性， CBL结合蛋白的主要受体。 我们将利用现有的 内因子（IF）-cbl受体抗血清和升高抗血清 转钴胺素II（TC II）受体。 这些研究将在 使用组织外植体或极化肾（MDCK）和肠 （Caco-2）细胞。 细胞内转运、分配和碳水化合物 将通过使用3 H的脉冲追踪实验来评估处理 氨基酸和糖。 受体的运输将是 使用标记的免疫沉淀物在SDS PAGE上监测 受体在转运过程中从各种细胞器中提取， 牢房 我们将确定受体是否经历 专门翻译后修饰，如脂肪酸 酰化反应。 如果受体确实被酰化，我们将表征 酰化的结构域和酰化结构域的性质（酯对酰胺）。 与脂肪酸连接。 脂蛋白质的性质 相互作用对锚定和受体的活性很重要 将使用人工双层系统和纯 受体的 膜插入的性质和位点（NH 2或 COOH末端）进行检查。 为了进一步探讨 IF-cbl受体的结构及其可能的同源性 与配体（IF），我们将测序选择的片段， 通过蛋白水解获得受体，并制备寡核苷酸 probes. 我们将筛选大小分级的λ gtll cDNA大鼠， 用抗体和寡核苷酸探针构建肾文库， 分离编码IF-cbl受体的cDNA克隆。 克隆 将用于通过DNA测序阐明受体结构 和生物合成，通过组织和细胞系的斑点印迹分析。 使用培养正常和转化细胞，我们将研究 游离和蛋白结合的CBL的摄取和移动， 研究a）IF的细胞内命运，B）TC II-cbl的形成，c） TC Ⅱ受体在cbl胞吐中的作用 这些研究 旨在了解细胞内的事件，调节 参与CBL摄取的蛋白质的合成，并且可以提供 了解cbl缺乏机制的线索， 患有家族性CBL吸收不良和 CBL的细胞内路由。

The overall objective of this proposal is to define the mechanisms involved in the intracellular movement of cobalamin (cbl) following its cellular uptake, by examining the biology of the two major receptors for cbl binding proteins. We will use existing intrinsic factor (IF)-cbl receptor antiserum and raise antiserum to Transcobalamin II (TC II) receptor. These studies will be carried out using tissue explants or polarized kidney (MDCK) and intestine (Caco-2) cells. Intracellular transit, distribution and carbohydrate processing will be assessed by pulse chase experiments using 3H amino acids and sugars. The trafficking of the receptor will be monitored on SDS PAGE using immunoprecipitates of the labeled receptor extracted from various organelles during its transit in the cell. We will determine whether the receptor undergoes specialized post translational modification such as fatty acid acylation. If receptor is indeed acylated we will characterize the domains which are acylated and the nature (ester vs amide) of the linkage with the fatty acid. The nature of lipid-protein interaction important for anchoring, and activity of the receptor will be examined using artificial bilayer systems and the pure receptor. The nature and site of membrane insertion (NH2 or COOH terminus) will be examined. In order to probe further into the structure of the IF-cbl receptor and its possible homology with the ligand (IF), we will sequence selected fragments of the receptor obtained by proteolysis, and prepare oligonucleotide probes. We will screen a size fractionated lambda gtll cDNA rat kidney library with antibody and oligonucleotide probes, in order to isolate a cDNA clone encoding the IF-cbl receptor. The clone will be used to elucidate receptor structure by DNA sequencing and biosynthesis, by dot blot analysis of tissues and cell lines. Using culture normal and transformed cells, we will study the uptake and movement of free and protein bound cbl in order to study a) intracellular fate of IF, b) formation of TC II-cbl, c) the role of TC II-receptor in the exocytosis of cbl. These studies are designed to understand the intracellular events which regulate the synthesis of proteins involved in cbl uptake, and could provide clues in understanding the mechanism of cbl deficiency noted in patients with familial cbl malabsorption and defects in the intracellular routing of cbl.