(NA,K)-ATPASE--STRUCTURE, BIOSYNTHESIS, AND REGULATION

(Na+，K+)激活的三磷酸腺苷酶[(Na，K)-ATPase]是 影响急性淋巴细胞Na~+、K~+耦合转运的分子机制 动物细胞。为了了解这台机器的分子机制，它的 必须阐明其三维结构。在其他方面，这一点 需要了解亚基的氨基酸序列(阿尔法和 测试版)。因为大量的疏水氨基酸会引起 部分水解物的聚集，试图对 在五年以上的时间内由经典氨基酸测定蛋白质 测序都失败了。拟议研究的主要目标是 用制备cDNA方法测定亚基的氨基酸序列 克隆，测定cDNA插入物的核苷酸序列，以及 从所述核苷酸序列导出所述氨基酸序列。另一个 目的是研究合成和翻译后的位置 亚基的修饰。合成的地点将通过以下方式确定 游离型和膜结合型多聚体的mRNA翻译。这个 翻译后修饰(切割和糖基化)将是 在含有狗微粒体的无细胞翻译系统中进行检测 胰腺。我们还将研究合成的发育规律。 卤虫(Artemia Salina)的(Na，K)-ATPase亚基 检测蛋白质合成和特定的mRNA水平。基因的表达水平 将通过杂交分析和特定基因的定量来估计 卤水虾不同时间的无细胞翻译产物 发展。加工(蛋白质分解和糖基化)也将 以及插入到膜中的机制。

The (Na+,K+)-activated adenosine triphosphatase [(Na,K)-ATPase] is the molecular machine which effects the coupled transports of Na+ and K+ in all animal cells. To understand the molecular mechanism of this machine, its three-dimensional structure must be elucidated. Among other things, this requires a knowledge of the amino acid sequences of the subunits (Alpha and Beta). Because of large stretches of hydrophobic amino acids which cause aggregation of partial hydrolytic products, attempts to sequence the protein over a period of more than five years by classical amino acid sequencing have failed. The primary objective of the proposed research is to determine the amino acid sequence of the subunits by preparation of cDNA clones, determination of the nucleotide sequences of the cDNA inserts, and deriving the amino acid sequences from the nucleotide sequences. Another objective is to study the site of synthesis and post-translational modifications of the subunits. The site of synthesis will be determined by translation of mRNA from free and membrane-bound polysomes. The post-translational modifications (cleavage and glycosylation) will be examined in a cell-free translation system containing microsomes from dog pancreas. We will also study developmental regulation of the synthesis of the (Na,K)-ATPase subunits in the brine shrimp, Artemia salina, by examining protein synthesis and specific mRNA levels. The levels of mRNA will be estimated by hybridization analysis and quantitation of specific cell-free translation products at various times throughout brine shrimp development. Processing (proteolytic cleavage and glycosylation) will also be studied, as well as the mechanism of insertion into the membrane.