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.
