Multiple intracellular Ca+2 transients occur during fertilization and early development of sea urchin eggs. Inositol trisphosphate (IP3) has been shown to be the second messenger transducing sperm-egg interactions at the surface to the mobilization of internal ca+2 stores during fertilization. The messenger molecule(s) responsible for later transients, whether it is IP3 or not, has not been identified. In view of the possibility that other messenger molecules may exist we have developed a cell free system using egg homogenates for screening a large number of potential Ca+2 release activators. With this assay system, we have identified a novel metabolite of NAD with Ca+2 mobilizing activity as potent as IP3. Structural determination established that the metabolite is a cyclized ADP - ribose and we have proposed cyclic ADP- ribose (cADPR) as a descriptive common name for it. cADPR is not only active in vitro but can also induce cortical reaction when microinjected into the egg. The enzyme responsible for synthesizing cADPR appears to be ubiquitous since it is found not only in sea urchin egg extracts but also in mammalian tissue extracts. I propose to investigate the physiological role of cADPR in the next grant period by measuring and comparing the time course of in vivo production of cADPR and IP3 from fertilization to first cleavage and correlating the time course with intracellular Ca+2 changes. An IP 3 - receptor blocker heparin, will be microinjected into fertilized eggs to determine if any of the Ca+2 transients are not mediated by IP3. We will characterize the cADPR system by purifying the enzymes responsible for synthesizing and degrading it and antibodies will be raised against them. The Ca+2 stores in the egg homogenates which are sensitive to cADPR as well as those that are sensitive to IP3 and NADP will be separated by density gradient centrifugation and characterized by marker enzyme distributions and receptor binding assays. These studies will be extended to other cell systems to determine if cADPR is a general second messenger for intracellular Ca+2 mobilization similar to IP3.
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