Cyclic ADP-ribose (cADPR) is a naturally occurring metabolite of NAD+ that is capable of mobilizing calcium from intracellular stores in a variety of biological systems. This chapter presents the procedures for the synthesis of three 8-substituted (8-amino-, 8-azido-, and 8-bromo-) analogs and a caged analog of cyclic ADP-ribose. The use of these analogs demonstrates that the occupation of the cADPR receptor site does not necessarily lead to Ca2+ release; appropriate interactions between the 8-position of the ligand and receptor are also required. All three compounds are competitive antagonists that bind to the same site as cADPR. The 8-azido-cADPR, with its photoactive azido group, is particularly useful as a photoaffinity probe for the identification and characterization of cADPR-binding proteins. The synthesis of high specific activity 8-azido-[32P]cADPR for use in photoaffinity labeling experiments is also described in the chapter. The use of photoactivable “caged” compounds represents a powerful tool for controlling the release of biologically active molecules in intact cells. Generally, the caging group incorporated into an active molecule is designed to render the molecule inactive. Removal of the caging group by photolysis results in liberation of the active substance, which can be controlled both spatially and temporally.

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CYCLIC ADP-ribose (cADPR) is a newly identified nucleotide(1,2) which can release calcium from a variety of cells(3-6), suggesting it is a messenger for mobilizing internal Ca2+ stores. Its cyclic structure has now been confirmed by X-ray crystallography(7). Available results are consistent with it being a modulator of Ca2+-induced Ca2+ releases(8-10). Here we report that sea urchin egg microsomes purified by Percoll gradients lose sensitivity to cADPR, but the response can be restored by a soluble protein in the supernatant. Purification and characterization of the protein indicate that it is calmodulin. It appears to be sensitizing the Ca2+ release mechanism because caffeine and strontium, agonists of Ca2+-induced Ca2+ release, can also mimic calmodulin in conferring cADPR-sensitivity. Although evidence indicates that cADPR may be an activator of the ryanodine receptor(8-10), present results point to the importance of accessory proteins such as calmodulin in modulating its activity.