Improved Aequorin for Ca Assay in Functioning Cells

改进的水母发光蛋白用于功能细胞中 Ca 的测定

• 批准号: 9403183
• 负责人: Osamu Shimomura
• 金额: $ 37.89万
• 依托单位: Marine Biological Laboratory
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9403183&HistoricalAwards=false
• 关键词: Improved; Aequorin; Ca; Assay; Functioning

9403183 Shimomura The photoprotein aequorin first isolated by the P.I. in 1962, has been widely used as an indicator for cellular calcium for more than 25 years. In the last several years, three improved forms of aequorin were made available: 1) various iso-forms of aequorin isolated from natural aequorin, with properties considerably different from each other; 2) recombinant aequorin prepared from the apoaequorin genetically produced in E. coli, which can be mass produced, and 3) more than 30 kinds of semi-synthetic aequorin have been obtained by replacing the coelenterazine moiety of aequorin with various synthetic analogues of coelenterazine. Semi-synthetic aequorins showed various properties that are highly advantageous in measuring calcium, including the widely different ranges of sensitivity to calcium. Recently, a new trend in the use of aequorin began. In the new method, apoaequorin is genetically produced in cells utilizing aequorin cDNA, then the apoaequorin is intracellularly regenerated into aequorin by the addition of coelenterazine and finally the recombinant aequorin produced is used to study calcium in the cells. The next objectives of this research are: 1) Further improvement of semi-synthetic aequorin, based on the data of various semi-synthetic aequorins that contain various coelenterazine analogues modified at position 2 of the imidazopyrazine ring. 2) In the new method that involves the production of semi-synthetic aequorins in cells, the regeneration of aequorins from apoaequorin and coelenterazine analogues is a crucial step that is strongly influenced by the characteristics of the analogue used. Thus, the characteristics of all coelenterazine analogues will be investigated, and the analogues that are suitable for intracellular aequorin regeneration will be identified. 3) Various preparations of aequorin, including isoaequorins and semi synthetic aequorins, will be produced for the purpose of distribution to cell biologists and p hysiologists for their use in research. In addition, coelenterazine analogues suitable for the intracellular regeneration of aequorins will also be supplied. 4) On the basis of the recent discovery that some of the coelenterazine analogues are extremely sensitive and highly specific to superoxide anion, the measurement and monitoring of superoxide anion in biological systems with those analogues will be explored. %%% One of the most important components of cellular signal transduction is the calcium ion. Levels of intracellular calcium are closely regulated and changes of calcium are commonly involved as steps in the cascade of responses to extracellular signals. The mechanisms by which cells control internal calcium and how calcium changes are involved in cellular signaling are major current themes in cell research. Much current research in this field has been made possible by the availability of indicators that measure intracellular free calcium and determine its location within cells over the time course of a physiological response. The subject of this research is aequorin, a naturally occurring complex of a protein (apoaequorin) with an associated smaller organic molecule (coelenterazine). When one aequorin molecule binds a calcium ion it gives off a pulse of light. Aequorin is thus an invaluable tool for measuring and localizing intracellular calcium. This laboratory first isolated aequorin from a marine jellyfish in 1962 and has since, primarily with NSF support, developed a wide range of important variants of the aequorin molecules for various applications in cell biology research. The next goals of this project are to produce further specialized calcium indicators and, if possible to develop variants to extend the use of the components of aequorin to measure another class of important cellular signal molecules, superoxide anion. These indicators will be made available for use in research on a wide range of problems in biology. ***

小行星9403183 发光蛋白水母发光蛋白首先由P.I.在1962年，已被广泛用作细胞钙的指标超过25年。 在过去的几年中，水母发光蛋白的三种改进形式是可用的：1）从天然水母发光蛋白分离的各种水母发光蛋白的异构体，其性质彼此相当不同; 2）从E.大肠杆菌，其可以大量生产; 3）通过用各种合成的腔肠素类似物代替水母发光蛋白的腔肠素部分，已经获得了30多种半合成水母发光蛋白。 半合成水母发光蛋白显示出在测量钙方面非常有利的各种性质，包括对钙的广泛不同的灵敏度范围。 最近，水母发光蛋白的使用开始出现新的趋势。 在新方法中，利用水母发光蛋白cDNA在细胞中遗传产生脱辅基水母发光蛋白，然后通过加入腔肠素使脱辅基水母发光蛋白在细胞内再生为水母发光蛋白，最后将产生的重组水母发光蛋白用于研究细胞中的钙。 本研究的下一个目标是：1）基于含有在咪唑并吡嗪环的2位修饰的各种腔肠素类似物的各种半合成水母发光蛋白的数据，进一步改进半合成水母发光蛋白。 2)在涉及在细胞中生产半合成水母发光蛋白的新方法中，从脱辅基水母发光蛋白和腔肠素类似物再生水母发光蛋白是一个关键步骤，受到所用类似物特性的强烈影响。 因此，将研究所有腔肠素类似物的特征，并鉴定适合于细胞内水母发光蛋白再生的类似物。 第三章 将生产各种水母发光蛋白制剂，包括异水母发光蛋白和半合成水母发光蛋白，以分发给细胞生物学家和生理学家用于研究。 此外，还将提供适用于水母发光蛋白细胞内再生的腔肠素类似物。 四、 基于最近发现的一些腔肠素类似物对超氧阴离子极其敏感和高度特异性，将探索在具有这些类似物的生物系统中测量和监测超氧阴离子。 细胞信号转导的最重要的组成部分之一是钙离子。 细胞内钙的水平受到密切调节，钙的变化通常涉及对细胞外信号的级联反应的步骤。 细胞控制内部钙的机制以及钙的变化如何参与细胞信号传导是当前细胞研究的主要主题。 目前在这一领域的许多研究已经成为可能的指标，测量细胞内游离钙，并确定其在细胞内的生理反应的时间过程中的位置的可用性。 这项研究的主题是水母发光蛋白，一种天然存在的蛋白质（脱辅基水母发光蛋白）与相关的较小有机分子（腔肠素）的复合物。 当一个水母发光蛋白分子与钙离子结合时，它会发出光脉冲。 因此，水母发光蛋白是测量和定位细胞内钙的宝贵工具。 该实验室于1962年首次从海洋水母中分离出水母发光蛋白，此后，主要在NSF的支持下，开发了水母发光蛋白分子的各种重要变体，用于细胞生物学研究的各种应用。 该项目的下一个目标是生产进一步专门的钙指标，如果可能的话，开发变体，以扩展水母发光蛋白组分的使用，以测量另一类重要的细胞信号分子，超氧阴离子。 这些指标将用于研究生物学中的广泛问题。 ***