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

(NA,K)-ATP酶--结构、生物合成和调节

• 批准号: 3283964
• 负责人: LOWELL E HOKIN
• 金额: $ 13.14万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1987-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283964
• 关键词: adenosinetriphosphatase; cell free system; complementary DNA; enzyme substrate; genetic manipulation; genetic regulation; genetic translation; messenger RNA; microsomes; molecular cloning; nucleic acid sequence; polysomes; saltwater environment

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.

（Na+，K+）激活的腺苷三磷酸酶[（Na，K）-ATP酶]是 分子机器，影响Na+和K+的耦合运输， 动物细胞 为了了解这台机器的分子机制， 必须阐明三维结构。 这尤其 需要了解亚基（α和β）的氨基酸序列， Beta）。 由于大量的疏水性氨基酸导致 部分水解产物的聚集，尝试对 蛋白质在五年以上的时间内通过经典氨基酸 测序失败了。 拟议研究的主要目标是 通过制备cDNA确定亚基的氨基酸序列， 克隆，确定cDNA插入片段的核苷酸序列，以及 从所述核苷酸序列衍生所述氨基酸序列。 另一 目的是研究合成和翻译后的位点 亚基的修饰。 合成位点将通过以下方式确定： 从游离和膜结合的多核糖体翻译mRNA。 的 翻译后修饰（切割和糖基化）将被 在含有犬微粒体的无细胞翻译系统中检查 胰腺 我们还将研究发育调节的合成 卤虫（Artemia salina）的（Na，K）-ATPase亚基 检测蛋白质合成和特定mRNA水平。 mRNA水平 将通过杂交分析和定量特异性 无细胞翻译产物在整个卤虫的不同时间 发展 加工（蛋白水解切割和糖基化）也将 研究，以及插入膜的机制。