9722982 Shimomura The photoprotein aequorin has been used in monitoring intracellular calcium for many years, and the functional group of this protein, coelenterazine, and its analogues are sensitive probes for measuring superoxide anion. The main objective of this project is to design and prepare optimally improved forms of aequorin and coelenterazine for studying various biological events that involve free calcium and superoxide anion. Aequorin was discovered in a jellyfish by the PI in 1962. It emits blue light in the presence of ionized calcium by an intramolecular reaction, decomposing itself into apoaequorin, coelenteramide and carbon dioxide. Apoaequorin can be regenerated into the original aequorin by incubation with coelenterazine in the presence of oxygen. In the past several years, about 50 kinds of coelenterazine analogue were synthesized and incorporated into apoaequorin. Many of the products, which are called semisynthetic aequorins, showed excellent properties for measuring cellular calcium. Some had a very high sensitivity, others had a very low sensitivity, and some had a very fast response, thus making possible the monitoring of rapid, transient changes of calcium ions in a wide concentration range (1 nM - 1 mM). A recent trend is to express recombinant apoaequorin in cells, which is followed by the in situ reconstitution of aequorin by the addition of a coelenterazine. In this procedure, the choice of coelenterazine analogue is critical for the success of the reconstitution of an aequorin. The significance of superoxide radical generation in biological systems has long been recognized, but the technique of measuring it lags far behind that of measuring calcium. This is largely due to the unavailability of the superoxide probes that are sufficiently sensitive for the purpose. In the past two years, more than 20 kinds of coelenterazine analogues were prepared and tested at the PI's lab. Some of them showed significantly improved responses to superoxide, and the structure- sensitivity relationships of the analogues provided useful information for further improvement of superoxide probes. The research plan for the next three years includes: (1) Preparation of the semisynthetic aequorins that emit red light (550-650 nm), by extending the conjugation system of coelenterazine at the position 8 of its imidazopyrazinone ring. The products will be useful in monitoring calcium in colored and opaque cells and tissues, and also in certain two-wavelength techniques. (2) Identification of suitable coelenterazine analogues for the reconstitution of the recombinant apoaequorin that is expressed in cells. This information should significantly contribute to the work in progress at many laboratories. (3) New, improved superoxide probes will be designed and prepared based on the information now available; the probes will have at least 100 times higher sensitivity than MCLA, the most sensitive probe currently available, with a very low background luminescence. (4) The PI's laboratory will continue to supply various aequorin preparations and coelenterazine analogues to cell biologists to aid their studies. The supersensitive superoxide-probes, when prepared, will also be provided. The perception of extrinsic environmental effects such as light, temperature and chemicals influences cells and organisms to exert a multitude of overt changes in physiology and development. The initial perception of environmental changes is detected by specific proteins that transmit the signal downstream in the cell through mechanisms that often involve rapid changes or fluxes of intracellular calcium. The PI discovered a protein, apoaequorin with coelenterazine, is used for bioluminescence in jelly fish is highly sensitive to calcium. The gene for apoaequarin can be expressed in cells to provide an intrinsic visual probe for calcium with the addition of coelenterazine. To be able to visualize the changes a nd fluxes of calcium within cells in response to signals is extremely useful in the study of cellular reactions to environmental and developmental changes. With this award, Dr. Shimomura will continue to refine the use of aequorin by producing new analogs of coelenterazine that will emit light in the red instead of blue and to be sensitive to oxidative burst that accompanies many stress reactions. Attaining these goals will provide the scientific community with extremely valuable tools to explore many types of reactions to changes in the physiology of animal, plant and fungal cells. ***
