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

NA,K-ATP酶——结构、生物合成和调节

• 批准号: 3283965
• 负责人: LOWELL E HOKIN
• 金额: $ 18.39万
• 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 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283965
• 关键词: DNA footprinting; Malacostraca; adenosinetriphosphatase; cell free system; chimeric proteins; complementary DNA; enzyme substrate; genetic manipulation; genetic regulation; genetic transcription; genetic translation; genome; laboratory rabbit; messenger RNA; molecular cloning; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; polysomes; protein biosynthesis; protein structure function; ribonucleoproteins; saltwater environment; sodium potassium exchanging ATPase

The Na+ and K+-activated adenosine triphosphatase (Na+K- ATPase) is an intrinsic membrane protein which transports Na+ and K+ across the plasma membrane against electrochemical gradients. This is essential for maintenance of low (Na+) and high (K+) inside the cell. The resulting gradients underly many important physiological processes, including conduction of electrical stimuli in nerve, excitation of muscle, co-transport of sugars and amino acids, counter-transport of ions, maintenance of osmotic pressure, and regulation of cell volume. Our laboratory will address several important, but poorly understood aspects of the (Na+K)-ATPase: (1) structure-function relationships, (2) genomic structure, and (3) molecular mechanism of the regulation of biosynthesis in a developmental system. These studies will take advantage of unique properties of the (Na+K)-ATPase of brine shrimp. Brine shrimp contain two forms of alpha-subunits of the (Na+K)-ATPase which have different activities. Structure- function relationships will be examined by determining the properties of chimeric alpha-subunits in which key domains of one form of alpha-subunit have been substituted by those from the other. The modification of primary structures will be accomplished by recombinant DNA technology. Recombinant DNA technology will also be used to prepare partial-length subunits in order to identify those domains which are required for protein translocation. The ability of these partial length and/or chimeric proteins to effect insertion into the membrane will be examined in cell-free translation systems. This system will also be used to test for involvement of signal recognition particle. A brine shrimp genomic library will be constructed in order to examine the genomic structure of the genes encoding the subunits. The genomic clones will provide tools for future experiments concerning the regulation of gene expression and answer questions regarding the evolution and linkage of the genes encoding the subunits. Portions of the genomic sequence which may be involved in regulation will be identified by gel retardation assays and DNase footprinting. The sequences of these areas will also be determined. The levels of mRNA alpha and mRNA beta increase dramatically during the first 24 hr of development of the brine shrimp. Molecular mechanisms of regulation of the levels of these mRNAs will be examined, including alterations in transcription, processing, or recruitment from RNP.

Na+ 和 K+ 激活的三磷酸腺苷酶 (Na+K- ATPase）是一种内在膜蛋白，可转运 Na+ 和 K+ 穿过质膜对抗电化学 梯度。 这对于维持低 (Na+) 和高浓度至关重要 (K+)在细胞内。 由此产生的梯度在许多 重要的生理过程，包括传导 神经电刺激、肌肉兴奋、协同运输 糖和氨基酸、离子的反向运输、维持 渗透压和细胞体积的调节。 我们的实验室 将解决几个重要但知之甚少的方面 (Na+K)-ATP酶：(1) 结构-功能关系，(2) 基因组结构，以及（3）调控的分子机制 发育系统中的生物合成。 这些研究将 利用 (Na+K)-ATPase 的独特特性 盐水虾。 丰年虾含有两种形式的α亚基 (Na+K)-ATP酶具有不同的活性。 结构- 将通过确定来检查函数关系 嵌合α亚基的特性，其中一个关键域 α-亚基的形式已被来自 其他。 主要结构的修改将是 通过重组DNA技术完成。 重组 DNA技术也将用于制备部分长度 子单元，以便识别所需的域 蛋白质易位。 这些部分长度和/或的能力 实现插入膜的嵌合蛋白将是 在无细胞翻译系统中进行检查。 该系统还将 用于测试信号识别粒子的参与。 一个 将构建丰年虾基因组文库 检查编码基因的基因组结构 亚单位。 基因组克隆将为未来提供工具 有关基因表达调控的实验 回答有关基因进化和连锁的问题 对子单元进行编码。 基因组序列的部分 可能参与调节将通过凝胶阻滞来识别 分析和 DNase 足迹分析。 这些区域的顺序将 也可以确定。 mRNA α 和 mRNA β 的水平 在发育的前 24 小时内急剧增加 盐水虾。 调节水平的分子机制 这些 mRNA 将被检查，包括 RNP 的转录、处理或募集。