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

NA,K-ATP酶——结构、生物合成和调节

• 批准号: 3283966
• 负责人: LOWELL E HOKIN
• 金额: $ 18.9万
• 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/3283966
• 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+激活的腺苷三磷酸酶（Na+K- ATP酶）是一种转运Na+的膜内蛋白 和K+穿过质膜对抗电化学 梯度。 这对于维持低（Na+）和高（Na+）浓度至关重要。 （K+）在细胞内。 由此产生的梯度是许多 重要的生理过程，包括传导 神经电刺激，肌肉兴奋， 糖和氨基酸，离子的反向转运， 渗透压和调节细胞体积。 本实验室 将解决几个重要的，但了解不多的方面， （Na+K）-ATP酶：（1）结构-功能关系，（2） 基因组结构;（3）调控的分子机制 在发育系统中的生物合成。 这些研究将 利用（Na+K）-ATP酶的独特性质， 盐水虾 盐水虾含有两种形式的α亚基， （Na+K）-ATP酶活性不同。 结构- 功能关系将通过确定 嵌合α亚单位的特性，其中一个亚单位的关键结构域 α-亚基的形式已经被来自 其他. 一级结构的修改将是 通过重组DNA技术完成。 重组 DNA技术也将用于制备部分长度的 子单元，以确定这些领域所需的 蛋白质易位 这些部分长度和/或 将嵌合蛋白插入膜中， 在无细胞翻译系统中检测。 该系统还将 用于检测信号识别颗粒的参与。 一 将构建卤虫基因组文库， 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 有关基因表达调节的实验， 回答有关基因进化和连锁的问题 编码子单元。 部分基因组序列， 可能参与调节将确定凝胶阻滞 测定和DNA酶足迹法。 这些区域的序列将 也要坚决。 mRNA α和mRNA β的水平 在发育的前24小时内急剧增加， 盐水虾 调节蛋白质水平的分子机制 将检查这些mRNA，包括 转录、加工或从RNP招募。