PHYSIOLOGICAL SIGNIFICANCE OF NA, K-PUMP ALPHA ISOFORMS

描述：Na，K-ATPase在细胞内交换Na 胞外钾，水解三磷酸腺苷，提供能量来泵送这些 离子对其浓度梯度的影响。该酶由α 和β亚基，其中亚基包含与ATP的结合位点 和强心苷。该提案的目标是使用Site 定向诱变后突变体在cos-1或Ltk(-)中的表达 )细胞系，以研究差异处理对 泵功能上的氨基末端序列及其异构体特异性 ATP结合位点附近区域对酶动力学性质的影响 异构体。 有三个具体目标。第一个是测试异构体是否- 为保证泵的正常工作，必须进行特殊处理。要做到这一点 目的，将产生针对前八种氨基酸的抗体 每种异构体的同种异构体，并与针对 三个同种类型的未加工区域将被用来检查 四个氨基末端嵌合体的加工。 第二个目的是使用A1氨基末端的缺失突变 区域(但保留前八个氨基酸)来分离该位点 蛋白水解性切割检测功能表达和 免疫反应性(测量卵裂)。 最终目标是构建嵌合体，在嵌合体中11种氨基酸 位于ATP结合位点附近的亚型特定区域为 互换了。嵌合体和W.T.的动力学特性分析。 异构体将包括V(Max)，通过后门获得泵的丰度 磷酸化、钾、钠和三磷酸腺苷的表观亲和力及阳离子 主动转运的浓度依赖性。两国之间的通信 嵌合体相对于任何一个嵌合体的动力学参数 将对两个W.T.ATPase进行检测以检测非特异性结构 某一特定突变的影响。如果可以从 嵌合体，点突变将被构建来试图识别单个 造成观察到的性质差异的氨基酸(S)。 如果构建体无功能，则可能是突变、组装中的缺陷 并将使用概述的免疫学方法检查靶向 上面。如果这个区域似乎在催化中没有作用， Na，K-ATPase嵌合体及其H、K、Ca同源区 将建造水泵来证实这一结论。

DESCRIPTION: The Na, K ATPase exchanges intracellular Na for extracellular K, hydrolyzing ATP to provide the energy for pumping these ions against their concentration gradients.The enzyme consists of alpha and beta subunits where the subunit contains the binding sites for ATP and cardiac glycosides. The goal of the proposal is to use site directed mutagenesis followed by expression of mutants in cos-1 or Ltk(- ) cell lines to examine the effect of the differential processing of the amino terminal sequences on pump function and of the isoform specific region near the ATP binding site on the kinetic properties of the isoforms. There are three specific aims. The first is to test whether isoform- specific processing is necessary for pump function. To accomplish this aim, antibodies will be generated against the first eight amino acids of each isoform and, along with polyclonal antibodies specific for nonprocessed regions of the three isotypes, will be used to examine the processing of four amino terminal chimeras. The second aim is to use deletion mutagenesis of the a1 amino terminal region (but maintaining the first eight amino acids) to isolate the site of proteolytic cleavage assaying functional expression and immunoreactivity (to measure cleavage). The final aim is to construct chimeras in which the eleven amino acid subtype specific regions located near the ATP binding site are interchanged. Analysis of the kinetic properties of chimeras and W.T. isoforms will include V(max), pump abundance by backdoor phosphorylation, apparent affinities for K, Na and ATP and cation concentration dependence for active transport. Correspondence of the kinetic parameters of the chimera's with respect to those of either of the two W.T. ATPases will be examined to detect nonspecific structural effects of a given mutation. If an assignment can be made from the chimeras, point mutants will be constructed to try to identify single amino acids responsible for the observed difference(s) in properties. If constructs are nonfunctional, possible defects in mutation, assembly and targeting will be examined using the immunological methods outlined above. If there appears to be no role for this region in catalysis, chimeras of the Na, K ATPase and homologous regions of the H, K or Ca pumps will be constructed to confirm this conclusion.