CALCIUM REGULATION SYSTEMS IN SEA URCHIN EGGS

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Mobilization of intracellular Ca+2 plays an important role in many cellular processes. It is generally accepted that inositol trisphosphate is a second messenger for transducing surface receptor activation to mobilization of internal Ca+2. That inositol trisphosphate may not be the only Ca+2 messenger as suggested by several recent studies. We have identified a Ca+2 mobilization system in sea urchin eggs which is totally independent of the inositol trisphosphate pathway. This system is activated by a metabolite of NAD+ we named cyclic ADP-ribose (cADPR). Evidence we have suggests that cADPR may be an endogenous regulator of the Ca+2-induced-Ca+2 release mechanism in cells. Detailed characterization of the cADPR system should provide useful information toward understanding the basic mechanisms involved in mobilization of cellular Ca+2. Research is proposed to elucidate the physiological role of the cADPR system by, first, determining if cADPR increases the Ca+2 sensitivity of the Ca+2-induced-Ca+2 release system in intact eggs and homogenates, second, measuring the endogenous cADPR levels in eggs from fertilization to cleavage and, third, developing specific inhibitors to block the cADPR system and assessing the resultant changes in cell functions. We will devise methods to purify both the soluble and membrane bound forms of ADP- ribosyl cyclase, the cADPR synthesizing enzyme, and generate specific antibodies against them. Structures of cADPR and the soluble ADP-ribosyl cyclase from Aplysia ovotestis will be determined by X-ray crystallography. cADPR will be chemically modified to produce agonists, antagonists and immunogens of the metabolite. The degradation enzyme of cADPR will be characterized. We will develop methods to radioactively label the microsomal receptor for cADPR. Generally applicable procedures for introducing cADPR into cells will be devised to facilitate the assessment of how widespread the cADPR system among cells is. We will characterize yet another independent Ca+2 release system in egg homogenates that is activated by a derivative of NADP.