BIOCHEMICAL STUDIES OF ERYTHROCYTE TRANSPORT PROTEINS

该项目的长期目标是了解分子 蛋白质介导的跨生物膜转运机制。 该提案集中在三种红细胞膜蛋白： C1-HCO 3-交换器（带3或AE 1）; 43 kDa乳酸转运蛋白; 和水通道蛋白CHIP 28。 这三种蛋白质都是 丰富到足以研究结构-功能关系 在原生膜中。 第一个目标是使用生物化学技术来改善地形 带3多肽在膜中的排列模型， 为了定义精氨酸、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RNA的交替剪接，多肽可以是异质的; 这种异质性的性质将详细描述，使用 生物化学和分子生物学方法。 思进 在理解频带3时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 其他实验室的结构研究材料。 乳酸转运在几种病理生理学中具有重要意义， 这些疾病包括糖尿病、缺血性心脏病和肿瘤。 然而，对转运蛋白本身几乎一无所知。 的 该蛋白的cDNA将从红细胞中克隆并测序。 文库，使用来自分离的蛋白质序列的探针。 的 cDNA序列将被用作进一步研究的指导。 这种蛋白质的结构-功能关系。 的功能 蛋白质也将在前稳态下的完整细胞中进行研究 条件下，测试催化循环的动力学模型， 运输 水通道（CHIP 28）的特征将关于 蛋白质的拓扑结构，使用最近提出的单克隆抗体 在这个实验室里。 研究的目的是测试一个简单的 蛋白质在膜中折叠的结构模型。 的 功能上重要的氨基酸残基的位置也将被 确定，使用已开发的频带3的方法。 最后， 质子或氢氧根离子通过水通道的速率 将被量化。

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.