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

NA,K-ATP酶——结构、生物合成和调节

• 批准号: 3283968
• 负责人: LOWELL E HOKIN
• 金额: $ 19.76万
• 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/3283968
• 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; recombinant DNA; 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)是一种运输Na+的固有膜蛋白 和K+穿过质膜对抗电化学反应 渐变。这对于维持低(Na+)和高(Na+)是必不可少的 (K+)在细胞内。由此产生的渐变不足以 重要的生理过程，包括传导 神经中的电刺激，肌肉的兴奋，共运输 糖和氨基酸，离子的反向传输，维护 渗透压和细胞体积的调节。我们的实验室 将讨论以下几个重要但鲜为人知的方面 (Na+K)-ATPase：(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)-ATPase的独特性质 咸虾。卤水虾含有两种形式的α-亚基 具有不同活性的(Na+K)-ATPase。结构- 函数关系将通过确定 嵌合α-亚基的性质 阿尔法亚基的形式已经被来自 其他的。主要结构的修改将是 由重组DNA技术完成。重组 DNA技术也将用于制备部分长度的 子单元，以确定哪些结构域需要 蛋白质易位。这些部分长度和/或 影响插入膜的嵌合蛋白将是 在无单元翻译系统中进行了检查。该系统还将 用于检测信号识别粒子的介入情况。一个 将构建海水对虾基因组文库，以期 检查编码该基因的基因组结构 亚单位。基因组克隆将为未来提供工具 关于基因表达和基因调控的实验 回答有关基因进化和连锁的问题 对亚基进行编码。基因组序列的一部分 可能参与调节将被鉴定为凝胶迟滞 化验和DNA酶足迹。这些区域的顺序将 也要有决心。α、β信使核糖核酸的水平 在开发的前24小时内急剧增加 咸虾。人血清白蛋白水平调控的分子机制 这些mRNA将被检测，包括在 转录、加工或从RNP招募。

项目成果

In vitro biosynthesis of the beta-subunit of the Na+/K+-ATPase in developing brine shrimp: glycosylation and membrane insertion.

发育丰年虾中 Na /K -ATP 酶 β 亚基的体外生物合成：糖基化和膜插入。

• DOI: 10.1016/0005-2736(88)90566-4
• 发表时间: 1988
• 期刊: Biochimica et biophysica acta
• 作者: Baxter-Lowe,LA;Yohanan,JM;Hokin,LE
• 通讯作者: Hokin,LE

Differential expression of the alpha 2 and beta messenger RNAs of Na,K-ATPase in developing brine shrimp as measured by in situ hybridization.

通过原位杂交测量发育中的丰年虾中 Na,K-ATP 酶的 α2 和 β 信使 RNA 的差异表达。

• DOI: 10.1177/40.4.1313064
• 发表时间: 1992
• 期刊: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
• 作者: Sun,DY;Guo,JZ;Hartmann,HA;Uno,H;Hokin,LE
• 通讯作者: Hokin,LE

Molecular cloning of the beta-subunit of the Na,K-ATPase in the brine shrimp, Artemia. The cDNA-derived amino acid sequence shows low homology with the beta-subunits of vertebrates except in the single transmembrane and the carboxy-terminal domains.

丰年虾 Na,K-ATP 酶 β 亚基的分子克隆。

• DOI: 10.1016/0014-5793(90)81162-h
• 发表时间: 1990
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Bhattacharyya,KK;Bergstrom,EE;Hokin,LE
• 通讯作者: Hokin,LE

Na,K-ATPase expression in the developing brine shrimp Artemia. Immunochemical localization of the alpha- and beta-subunits.

Na,K-ATP酶在发育中的丰年虾中的表达。

• DOI: 10.1177/39.11.1655875
• 发表时间: 1991
• 期刊: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
• 作者: Sun,DY;Guo,JZ;Hartmann,HA;Uno,H;Hokin,LE
• 通讯作者: Hokin,LE

Regulation of Na,K-ATPase biosynthesis in developing Artemia salina.

卤虫发育过程中 Na,K-ATP 酶生物合成的调节。

• 发表时间: 1986
• 期刊: The Journal of biological chemistry
• 作者: Fisher,JA;Baxter-Lowe,LA;Hokin,LE
• 通讯作者: Hokin,LE