Improved Aequorin for Ca Assay in Functioning Cells

9403183下村是由P.I.于1962年首次分离到的光蛋白Aequorin，被广泛用作细胞钙的指示剂已有25年之久。在过去的几年里，人们已经推出了三种改进形式的马钱子素：1)从天然马钱子素中分离出各种性质迥异的异构体；2)从大肠杆菌中转基因产生的马钱子素制备出可批量生产的重组马钱子素；3)用不同的合成类似物取代马钱子中的马钱子素，得到了30多种半合成马钱子素。半合成的Aequorin显示了在测量钙方面非常有利的各种性质，包括对钙的敏感度范围广泛不同。最近，Aequorin的使用出现了一种新的趋势。在新的方法中，利用aequorin基因在细胞中产生apoaequorin，然后通过加入coelentazine将apoaequorin在细胞内再生为aequorin，最后将产生的重组aequorin用于细胞内钙的研究。本研究的下一个目标是：1)进一步改进半合成马钱子素，基于各种半合成马钱子素的数据，这些半合成马钱子素含有在咪唑并吡嗪环上2位修饰的各种coelenterazine类似物。2)在细胞内生产半合成水飞蓟素的新方法中，从水飞蓟素和乙胺嘧啶类似物中再生水飞蓟素是一个关键步骤，它受到所用类似物特性的强烈影响。因此，我们将研究所有腔肠净类似物的特性，并确定适合于细胞内木犀草素再生的类似物。3)将生产各种不同的Aequorin制剂，包括异Aequorin和半合成Aequorin，以便分发给细胞生物学家和生理医生用于研究。此外，还将供应适合于细胞内再生的辅酶Ⅱ类似物。4)基于最近发现的一些腔肠净类似物对超氧阴离子具有极高的敏感性和高度特异性，将探索利用这些类似物对生物体系中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。细胞内钙离子水平受到密切调控，钙离子的变化通常作为对细胞外信号的级联反应的步骤。细胞控制细胞内钙离子的机制以及钙离子变化如何参与细胞信号转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都是由于指示剂的存在，这些指示剂可以测量细胞内的游离钙并确定其在生理反应时间过程中的位置。这项研究的主题是aequorin，一种天然存在的蛋白质(Apoaequorin)与相关的较小有机分子(Coelenterazine)的复合体。当一个aequorin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无价工具。该实验室于1962年首次从海洋水母中分离出aequorin，此后主要在NSF的支持下，开发了一系列重要的aequorin分子变体，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更多专门的钙指示剂，如果可能的话，开发变种以扩大aequorin组分的使用范围，以测量另一类重要的细胞信号分子--超氧阴离子。这些指示器将用于对生物学中广泛问题的研究。***

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. ***