Improved Aequorin for Ca Assay in Functioning Cells

光蛋白aequorin于1962年由P.I.首次分离，作为细胞钙的指示物已被广泛应用了25年以上。在过去的几年中，有三种改进形式的水芹素可用：1)从天然水芹素中分离出各种同型水芹素，它们的性质彼此有很大的不同；2)从大肠杆菌中遗传产生的水芹蛋白制备重组水芹蛋白，可大规模生产；3)用各种人工合成的水芹蛋白类似物取代水芹蛋白的铜肠嗪部分，获得了30多种半合成水芹蛋白。半合成相思素显示出各种特性，在测量钙方面非常有利，包括对钙的敏感性范围大不相同。最近，一种新的使用趋势开始出现。在新方法中，利用水龙蛋白cDNA在细胞中产生水龙蛋白，然后在细胞内通过添加异戊肠嗪将其再生为水龙蛋白，最后将重组水龙蛋白用于研究细胞内的钙。本研究的下一个目标是：1)基于在咪唑吡嗪环2位修饰含有各种异肠enterazine类似物的各种半合成金针叶苷的数据，进一步改进半合成金针叶苷。2)在新方法中，涉及在细胞中生产半合成水仙素，从水仙素和复方肠enterazine类似物中再生水仙素是一个关键步骤，这受到所用类似物特性的强烈影响。因此，我们将研究所有coelenterazine类似物的特性，并确定适合于细胞内aequorin再生的类似物。3)将生产各种黄蜡素制剂，包括异黄蜡素和半合成黄蜡素，以分发给细胞生物学家和生理学家用于研究。此外，还将提供适合于青龙蛋白细胞内再生的复方肠enterazine类似物。4)基于最近发现的一些异肠菌嗪类似物对超氧阴离子非常敏感和高度特异性的研究，将探索这些类似物在生物系统中对超氧阴离子的测量和监测。细胞信号转导中最重要的成分之一是钙离子。细胞内钙的水平受到密切调节，钙的变化通常作为响应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。由于有了测量细胞内游离钙并确定其在生理反应的时间过程中在细胞内的位置的指标，目前在这一领域的许多研究已经成为可能。本研究的主题是一种天然存在的蛋白质复合物（apoaequorin）和相关的较小有机分子（coelenterazine）。当一个钙蛋白分子与钙离子结合时，它会发出光脉冲。因此，Aequorin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麒麟，此后主要在美国国家科学基金会的支持下，开发了大量水麒麟分子的重要变体，用于细胞生物学研究的各种应用。该项目的下一个目标是进一步生产专门的钙指示剂，如果可能的话，开发变体来扩展aequorin成分的使用，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广泛的问题。* * *

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. ***