BIOCHEMICAL STUDIES OF ERYTHROCYTE TRANSPORT PROTEINS

这个项目的长期目标是了解分子 蛋白质介导的生物膜转运机制。 该提案的重点是三种红细胞膜蛋白： 1-HCO3-交换体(带3或AE1)；43 kDa乳酸转运体； 和水通道蛋白，芯片28。这三种蛋白质都是 丰富到足以使研究结构-功能关系成为可能 在天然的细胞膜上。 第一个目标是使用生化技术来改善地形 带3多肽在膜和膜中的排列模型 定义精氨酸、赖氨酸和天冬氨酸或谷氨酸残基 功能重要性。最近的数据表明，红细胞带3 由于交替剪接的RNA，多肽可能是异质的； 这种异质性的性质将被详细描述，使用 包括生化和分子生物学方法。进一步的进展 在对波段3的理解上将需要更高分辨率的结构。 拟议工作的目标之一是准备二维 天然膜中带3的晶体；这些晶体将提供 用于其他实验室结构研究的材料。 乳酸转运在几种病理生理学中具有重要意义 情况，包括糖尿病、缺血性心脏病和肿瘤。 然而，对运输蛋白本身几乎一无所知。这个 该蛋白的cdna将从一个红血球中克隆和测序。 文库，使用从分离蛋白序列衍生的探针。这个 然后，cDNA序列将被用作进一步研究的指南 这种蛋白质中的结构-功能关系。的功能 蛋白质也将在处于稳定状态前的完整细胞中进行研究。 条件，以测试催化循环的动力学模型 运输。 水通道(芯片28)将相对于 使用最近提出的一种单克隆抗体对该蛋白质进行地形分析 在这个实验室里。这些研究的目标是测试一种简单的 膜中蛋白质折叠的结构模型。这个 具有重要功能的氨基酸残基的位置也将 使用为频带3开发的方法确定。最后， 质子或氢离子通过水通道的传输速率 将会被量化。

The long-term goal of this project is to understand the molecular mechanisms of protein-mediated transport across biological membranes. The proposal is focused on three red blood cell membrane proteins: the C1--HCO3- exchanger (band 3 or AE1); a 43 kDa lactic acid transporter; and the water channel protein, CHIP 28. All three of these proteins are abundant enough to make it possible to study structure-function relations in the native membrane. The first aim is to use biochemical techniques to refine the topographic model of the arrangement of the band 3 polypeptide in the membrane and to define arginine, lysine and aspartate or glutamate residues of functional importance. Recent data indicate that red cell band 3 polypeptide may be heterogeneous as a result of alternately spliced RNA; the nature of this heterogeneity will be characterized in detail, using both biochemical and molecular biological approaches. Further advances in the understanding of band 3 will require higher resolution structure. One of the goals of the proposed work is to prepare two-dimensional crystals of band 3 in native membranes; these crystals will provide material for structural studies in other laboratories. Lactate transport is of significance in several pathophysiological situations, including diabetes, ischemic heart disease, and neoplasia. However, almost nothing is known about the transport protein itself. The cDNA for the protein will be cloned and sequenced from an erythroid library, using probes derived from the sequence of isolated protein. The cDNA sequence will then be used as a guide for further studies of structure-function relations in this protein. The function of the protein will also be studied in intact cells under pre-steady state conditions, to test a kinetic model for the catalytic cycle for transport. The water channel (CHIP 28) will be characterized with respect to the topography of the protein, using a monoclonal antibody recently raised in this laboratory. The goal of the studies is to test a simple structural model for the folding of the protein in the membrane. The locations of functionally important amino acid residues will also be determined, using methods that have been developed for band 3. Finally, the rate of proton or hydroxyl ion transport through the water channel will be quantified.