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.

钠钾离子激活三磷酸腺苷酶 [(Na++K+)-ATP酶]插入动物细胞的质膜并是造成钾浓度高和钾浓度低的原因细胞质中钠的浓度。随之而来的差异这些阳离子在细胞质和细胞质之间的稳态浓度细胞外环境提供用于维持的潜在能量细胞体积，驱动营养物质的吸收，移动水分，并创造细胞的静息电位。传统电子显微镜、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研究 (Na++K+)-ATPase 的结构组织和该分子组装体经历构象变化，以便介导主动运输。结构数据将收集自天然 (Na++K+)-ATP 酶分子的晶体样本处于规定的状态构象状态。我们将生产结晶标本膜结合制剂和完全活性的酶溶解在洗涤剂溶液。初步研究表明，结晶 (Na++K+)-ATPase 分子的补丁可以通过改变介质中离子配体的浓度。在这些结晶体中样本，我们将把注意力集中在每个样本的亚基组成上分子组装及其相对于平面的精确位置膜。另外，Alpha 和 Beta 的位置分配晶胞中的亚基将通过专门标记来确认带有针对酶或凝集素的 Alpha 亚基的抗体的阵列与β亚基的碳水化合物部分结合并加工傅里叶方法的图像。其中最令人兴奋的方面之一研究将是我们研究分子的能力 (Na++K+)-ATP酶在液氮中处于冷冻水合状态温度。这些实验将提供低分辨率、该酶分子结构的三维图。