BIOCHEMICAL STUDIES OF ERYTHROCYTE TRANSPORT PROTEINS

红细胞转运蛋白的生物化学研究

• 批准号: 2174824
• 负责人: MICHAEL L JENNINGS
• 金额: $ 14.42万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174824
• 关键词: animal tissue; arginine; aspartate; band 3 protein; cell water; crystallization; dogs; erythrocyte membrane; fatty acid transport; genetic library; glutamates; human subject; hydrogen transport; ion transport; laboratory rabbit; lactates; lysine; membrane transport proteins; molecular cloning; monoclonal antibody; physical model; protein folding; protein sequence; protein structure function

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.

该项目的长期目标是了解分子 蛋白质介导的跨生物膜运输机制。 该提案重点关注三种红细胞膜蛋白： C1--HCO3-交换剂（带3或AE1）； 43 kDa 乳酸转运蛋白； 和水通道蛋白 CHIP 28。这三种蛋白都是 足够丰富，可以研究结构-功能关系 在天然膜中。 第一个目标是利用生化技术来完善地形 带3多肽在膜中的排列模型和 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交替剪接，多肽可能是异质的； 这种异质性的本质将被详细描述，使用 生物化学和分子生物学方法。 进一步的进展 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 其他实验室结构研究的材料。 乳酸转运在多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，人们对转运蛋白本身几乎一无所知。 这 将从红系细胞中克隆该蛋白质的 cDNA 并进行测序 文库，使用源自分离蛋白质序列的探针。 这 cDNA序列将作为进一步研究的指南 该蛋白质的结构-功能关系。 的功能 还将在预稳态下的完整细胞中研究蛋白质 条件，测试催化循环的动力学模型 运输。 水通道 (CHIP 28) 将根据以下方面进行表征 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 膜中蛋白质折叠的结构模型。 这 功能上重要的氨基酸残基的位置也将被 使用为频段 3 开发的方法确定。最后， 质子或氢氧根离子通过水通道的传输速率 将被量化。