PHYSIOLOGICAL SIGNIFICANCE OF NA, K-PUMP ALPHA ISOFORMS

描述：Na，K ATP酶将细胞内的Na交换为 细胞外钾，水解ATP提供能量泵送这些 离子对它们的浓度梯度。该酶由α 和β亚基，其中亚基含有ATP的结合位点 和强心苷。 该提案的目标是利用网站 定向诱变，然后在cos-1或Ltk（- ）细胞系中，以检查细胞的差异加工的影响。 氨基末端序列对泵功能和同种型特异性 ATP结合位点附近的区域对 同种型。 有三个具体目标。 第一个是测试是否同种型- 泵功能需要特殊处理。 为了实现这一 目的是产生针对前八个氨基酸的抗体 以及沿着特异于 三种同种型的未处理区域，将用于检查 四个氨基末端嵌合体的加工。 第二个目的是使用α 1氨基末端的缺失诱变 区域（但保留前八个氨基酸）以分离该位点 蛋白水解裂解测定功能表达， 免疫反应性（以测量裂解）。 最终目的是构建嵌合体，其中11个氨基酸 位于ATP结合位点附近的亚型特异性区域是 互换。 嵌合体和W.T. 亚型将包括V（max），通过后门泵丰度 磷酸化，K，Na和ATP的表观亲和力和阳离子 主动运输的浓度依赖性。 对应于 嵌合体的动力学参数相对于 两个W.T.将检查ATP酶以检测非特异性结构 特定突变的影响。 如果可以从 嵌合体，点突变体将被构建，以尝试识别单个 导致观察到的性质差异的氨基酸。 如果构建体是无功能的，则突变、组装 和靶向将使用概述的免疫学方法进行检查 以上如果这个区域在催化作用中没有作用， Na，K ATP酶和H，K或Ca同源区的嵌合体 将建造泵以证实这一结论。

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.