STRUCTURE DETERMINATION OF THE NA/K-ATPASE

NA/K-ATP酶的结构测定

• 批准号: 3288067
• 负责人: MANIJEH MOHRAZ
• 金额: $ 16.37万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-06 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288067
• 关键词: X ray crystallography; adenosinetriphosphatase; cell membrane; chemical structure function; circular dichroism; conformation; crystallization; electron microscopy; electron optics; enzyme model; enzyme reconstitution; enzyme structure; image processing; kidney; lipids; lyophilization; protein engineering; sodium potassium exchanging ATPase; swine

The focus of this project is the structure determination of Na, K- ATPase, an integral membrane protein that constitutes the Na, K pump in the plasma membrane of animal cells. Three-dimensional (3-D) reconstruction from tilted views of negatively stained sheets has revealed the structure of the cytoplasmic component of Na, K-ATPase At 2.5nm resolution. The domains corresponding to the two subunits of the enzyme have been identified in the structure. This information will be extended by two new approaches: electron microscopy and image processing of frozen hydrated specimens of the sheets will reveal structural organization of both the intra- and the extra-membrane regions of the enzyme; freeze-drying and high resolution metal shadowing of the crystalline sheets and surface reconstruction will provide information about the exoplasmic surface that is not available in the current map. Furthermore, it will provide a baseline for interpreting subsequent images of the crystalline enzyme after biochemical modification. Attempts will be made to extend the structural studies to higher resolution by forming better-ordered and larger crystalline sheets. The carbohydrate moiety of the beta subunit will be removed. Crystallization of the latter should result in the formation of sheets with greater degree of order. Additionally, comparison of the modified structure with that of the intact enzyme will give further evidence for the location of the beta subunit. Solubilization of the enzyme in nonionic detergents and reconstitution with controlled amounts of phospholipids would also lead to the formation of better-ordered and larger crystalline sheets. An ultimate goal for the electron microscopy work is obtaining a 3-D structure of the enzyme at approximately 1.0nm resolution. This would be sufficient to resolve structures such as helices and channels. By correlating these results with the information from the location of the two subunits, and the sequence the structure of various domains can be related to their role in the transport process. In parallel with the above studies, the structure of H, K-ATPase will be determined by method similar to those used for Na, K- ATPase. H, K-ATPase constitutes the gastric pump that is responsible for the secretion of acid into the stomach. It is closely related to Na, K-ATPase and the resulting structural similarities and differences could shed light on the mechanism of ion transport by these important biological systems.

本项目的重点是Na，K-的结构测定。 ATPase，构成Na，K的一种完整的膜蛋白 泵存在于动物细胞的质膜中。 倾斜视图的三维(3-D)重建 负染的薄片揭示了 在2.5 nm分辨率下，Na，K-ATPase的细胞质成分。这个 与该酶的两个亚基相对应的结构域已被 在结构中被识别。此信息将通过以下方式扩展 两种新方法：电子显微镜和图像处理 冰冻水合的薄片样本将揭示出 膜内区和膜外区的组织 酶；冷冻干燥和高分辨率金属阴影 水晶片和表面重建将提供 中未提供的有关外质表面的信息 当前地图。此外，它还将为 解释晶体酶的后续图像后 生化修饰。将尝试将 通过形成更有序的结构来实现更高分辨率的结构研究 和更大的水晶片。贝塔的碳水化合物部分 子单元将被删除。后者的结晶应该是 导致形成具有更大有序度的片材。 此外，还对改进后的结构与原结构进行了比较。 完整的酶将为定位提供进一步的证据 β亚基。酶在非离子介质中的增溶作用 洗涤剂和含有受控数量的洗涤剂 磷脂也将导致形成更有序的 和更大的水晶片。 电子显微镜工作的最终目标是获得一种 酶的三维结构，分辨率约为1.0 nm。 这将足以解析螺旋和 频道。通过将这些结果与来自 两个亚基的位置，以及结构的序列 不同领域的关系可以与它们在运输中的作用有关 进程。 在上述研究的同时，H，K-ATPase的结构 将通过与用于Na、K-的方法类似的方法测定 ATPase。H，K-ATPase构成胃泵，即 负责将酸分泌到胃里。它是 与Na，K-ATPase及由此产生的结构密切相关 相似之处和不同之处可能有助于揭示 通过这些重要的生物系统进行离子运输。