STRUCTURAL STUDIES OF CRYSTALLINE (NA++K+)-ATPASE

结晶 (NA K )-ATP酶的结构研究

基本信息

批准号：
3280229
负责人：
GUIDO A ZAMPIGHI
金额：
$ 8.52万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-09-01 至 1987-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3280229
关键词：
X ray crystallography adenosinetriphosphatase cell membrane chemical structure function crosslink crystallization electron microscopy enzyme structure freezing image enhancement

项目摘要

Sodium and potassium ion-activated adenosine triphosphatase [(Na++K+)-ATPase] is inserted into the plasma membranes of animal cells and is responsible for the high concentration of potassium and low concentration of sodium in the cytoplasm. The consequent differences in the steady-state concentrations of these cations between the cytoplasm and the extracellular environment provide potential energy employed to maintain cellular volume, to drive the uptake of nutrients, to move water, and to create the resting potential of cells. Conventional electron microscopy, x-ray diffraction and computer-assisted, three-dimensional reconstruction of low-dose micrographs will be employed to investigate the structural organization of (Na++K+)-ATPase and the conformational changes that this molecular assembly undergoes in order to mediate active transport. The structural data will be collected from crystalline specimens of native (Na++K+)-ATPase molecules poised in defined conformational states. We will produce the crystalline specimens both from membrane-bound preparations and from fully active enzyme dissolved in solutions of detergent. Preliminary studies have shown that crystalline patches of (Na++K+)-ATPase molecules can be produced by changing the concentrations of ionic ligands in the medium. In these crystalline specimens, we will focus our attention on the subunit composition of each molecular assembly as well as its exact position relative to the plane of the membrane. Also, the assignment of the locations of the Alpha and Beta subunits in the unit cell will be confirmed by specifically labeling the arrays with antibodies against the Alpha subunit of the enzyme or lectins that bind to the carbohydrate moiety of the Beta subunit and processing the images by Fourier methods. One of the most exciting aspects of these investigations will be our ability to study the molecules of (Na++K+)-ATPase in the frozen hydrated state at liquid nitrogen temperature. These experiments will provide a low-resolution, three-dimensional map of the molecular structure of this enzyme.

钠和钾离子激活的腺苷三磷酸酶 [（Na ++ K+） - ATPase]插入动物细胞的质膜和负责高浓度的钾和低浓度细胞质中钠的浓度。随之而来的差异这些阳离子的稳态浓度在细胞质和细胞外环境提供维护的潜在能量蜂窝体积，以驱动养分的吸收，移动水，然后移动到创建细胞的静息潜力。常规的电子显微镜，X射线衍射和计算机辅助，将采用低剂量显微照片的三维重建调查（Na ++ K+）ATPase和ATPase的结构组织和该分子组装发生的构象变化是为了中介主动运输。结构数据将从天然（Na ++ K+）的结晶标本 - ATPase分子已定义构象状态。我们将从膜结合的制剂，以及溶解在完全活性的酶中洗涤剂的溶液。初步研究表明结晶（Na ++ K+） - ATPase分子的斑块可以通过更改来产生培养基中离子配体的浓度。在这些结晶中标本，我们将注意力集中在每个的亚基组成上分子组件以及相对于平面的确切位置膜。另外，Alpha和Beta的位置分配单位单元中的亚基将通过特异性标记来确认抗体抗体针对酶或凝集素的α亚基的阵列与β亚基的碳水化合物部分结合并处理通过傅立叶方法的图像。这些最令人兴奋的方面之一调查将是我们研究分子的能力（Na ++ K+） - 在液氮处冷冻水合状态的ATPase 温度。这些实验将提供低分辨率该酶的分子结构的三维图。