Improved Aequorin for Ca Assay in Functioning Cells

光蛋白aequorin于1962年首次被P.I.分离出来，作为活细胞中的钙指示物被广泛应用。过去使用的水曲霉素制剂是许多异水曲霉素的未定义混合物。近年来，本实验室制备了三种改进形式的水仙素：1)从异质水仙素中分离得到的均质异水仙素，2)通过分子克隆制备的重组水仙素，以及3)用各种合成类似物取代各种水仙素的官能团获得的半合成水仙素。半合成相思素在测定细胞内钙含量方面具有非常有利的性能，但尚未得到充分利用。该项目的主要目标是提高新型蜂鸟素的可用性。拟开展的研究包括：1)深入研究半合成水仙素的钙激发发光，提高水仙素的有效利用；2)开发了一种新的钙含量比测定方法，采用半合成萘醌；3)开发一种生产更便于运输和使用的稳定制剂的方法。此外，还将生产足够数量的各种黄酮，分发给细胞生物学家和生理学家用于研究。这项研究的结果将是用于测量活细胞内游离钙的各种改进的发光探针。这些探针与现代计算机辅助光学显微镜成像技术相结合，最终将有可能可视化和量化在细胞对生理信号的反应过程中钙进出细胞以及细胞内隔室之间的运动。

The photoprotein aequorin, first isolated by the P.I. in 1962, has been widely used as a calcium indicator in living cells. The preparations of aequorin used in the past were undefined mixtures of many isoaequorins. Recently, three categories of improved forms of aequorin were prepared in this laboratory: 1) homogeneous isoaequorins isolated from heterogeneous aequorin, 2) recombinant aequorin prepared by molecular cloning, and 3) semi-synthetic aequorins obtained by replacing the functional group of various aequorins with various synthetic analogs. Semi-synthetic aequorins have highly advantageous properties in measuring intracellular calcium but are not yet fully utilized. The main objective of this project is to improve the usability of the new types of aequorin. Studies planned include: 1) detailed investigation concerning the calcium triggered luminescence of semi-synthetic aequorins, to improve the effective utilization of these aequorins; 2) development of a new ratio method of measuring calcium, employing semi-synthetic aequorins; 3) development of a method to produce stabilized preparations of aequorin that are more convenient to transport and use. In addition, sufficient amounts of various kinds of aequorin will be produced for distribution to cell biologists and physiologists for their use in research. The results of this research will be a variety of improved luminescent probes for measurement of free calcium within living cells. These probes, in combination with modern techniques of computer assisted light microscope imaging, will ultimately make it possible to visualize and quantitate the movement of calcium into and out of cells and between intracellular compartments during the response of a cell to a physiological signal.