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STRUCTURE DETERMINATION OF THE NA/K-ATPASE

NA/K-ATP酶的结构测定

基本信息

批准号：
2177885
负责人：
MANIJEH MOHRAZ
金额：
$ 17.81万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-09-06 至 1995-08-31
项目状态：
已结题

项目摘要

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.

本课题的重点是Na，K- ATP酶，一种组成Na，K 泵入动物细胞的质膜。三维（3-D）重建从倾斜的意见，负染色的床单揭示了 Na，K-ATP酶的胞质组分。的对应于酶的两个亚基的结构域已经被在结构中识别。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，冰冻的水化标本将揭示组织内和膜外区域的酶;冷冻干燥和高分辨率金属阴影晶体片和表面重构将提供关于外质表面的信息，当前地图此外，它还将提供一个基线，解释晶体酶的后续图像，生化改造将努力延长通过形成更好的有序结构，和更大的结晶片。 β的碳水化合物部分子单元将被删除。后者的结晶应导致形成具有更大有序度的片材。此外，还比较了修饰结构与完整的酶将进一步证明 β亚基酶在非离子液体中的溶解洗涤剂和用受控量的磷脂也会导致形成更好的有序和更大的结晶片。电子显微镜工作的最终目标是获得酶的三维结构，分辨率约为1.0nm。这将足以解决结构，如螺旋和渠道通过将这些结果与来自两个亚基的位置和结构的顺序可以与它们在运输中的作用有关过程在上述研究的同时，H，K-ATP酶的结构将通过与Na，K- ATP酶 H，K-ATP酶构成胃泵，负责将酸分泌到胃中。是与Na，K-ATPase密切相关，相似性和差异性可以揭示这些重要的生物系统的离子运输。