Improved Aequorin for Ca Assay in Functioning Cells

光蛋白Aequorin于1962年由P.I.首次分离，已被广泛用作活细胞中的钙指示剂。过去所用的马兜铃素制剂是许多异马豆素的未定义混合物。最近，本实验室制备了三类改进形式的马钱子素：1)从异质马钱子素中分离得到均一异马钱子素，2)分子克隆制备重组马钱子素，3)用各种合成类似物取代各种马钱子素的官能团而得到半合成马钱子素。半合成的水通道蛋白在测定细胞内钙离子方面具有非常有利的性质，但还没有得到充分的利用。该项目的主要目标是提高新型马蹄铁的可用性。计划的研究包括：1)详细研究半合成马钱子素的钙触发发光，以提高这些马钱子素的有效利用；2)开发一种新的比率测钙法，使用半合成马钱子素；3)开发一种更便于运输和使用的稳定的马钱子素制剂。此外，还将生产足够数量的不同类型的水飞蓟素，供细胞生物学家和生理学家在研究中使用。这项研究的结果将是各种改进的发光探针，用于测量活细胞内的游离钙。这些探针与计算机辅助光学显微镜成像的现代技术相结合，最终将使可视化和量化钙在细胞对生理信号的反应期间进出细胞以及细胞内隔室之间的运动成为可能。

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.