STUDIES ON INTESTINAL BRUSH BORDER MEMBRANES

这项提案的总体目标是界定机制 参与钴胺(CBL)的细胞内运动 在它的细胞吸收之后，通过研究两者的生物学 CBL结合蛋白的主要受体。我们将使用现有的 内源性因子(IF)-CBL受体抗血清及提高抗血清水平 转钴胺II(TC II)受体。这些研究将会进行 使用组织外植体或极化肾(MDCK)和肠 (Caco-2)细胞。细胞内转运、分布与碳水化合物 处理过程将通过使用3H的脉冲追逐实验进行评估 氨基酸和糖。受体的贩运将是 用标记物的免疫沉淀物在SDS PAGE上监测 在转运过程中从各种细胞器中提取的受体 牢房。我们将确定受体是否经历了 专门化的翻译后修饰，如脂肪酸 酰化反应。如果受体确实是酰化的，我们将表征 被酰化的结构域和其性质(酯与酰胺) 与脂肪酸的联系。脂蛋白的本质 对锚定和受体活性很重要的相互作用 将使用人工双层系统和纯净的 受体。膜插入的性质和位置(NH2或 COOH终点站)将被检查。为了进一步探讨 IF-CBL受体的结构及其可能的同源性 对于配基(IF)，我们将对选定的片段进行测序 蛋白水解法获得受体，制备寡核苷酸 探测器。我们将筛选一个大小不同的lambda gtll大鼠 含有抗体和寡核苷酸探针的肾脏文库 目的：分离编码IF-CBL受体的cDNA克隆。克隆人 将用于通过DNA测序来阐明受体结构 和生物合成，通过组织和细胞系的斑点印迹分析。 利用培养的正常细胞和转化细胞，我们将研究 游离和蛋白质结合的CBL的摄取和运动 研究a)IF的细胞内命运，b)TC II-CBL的形成，c) TC II受体在CBL胞吐作用中的作用这些研究是 旨在了解调节细胞内事件的细胞内事件 参与CBL摄取的蛋白质的合成，并可以提供 关于理解CBL缺乏机制的线索 家族性CBL吸收不良和大脑中动脉畸形患者 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.