[26] Bioassay for determining endogenous levels of cyclic ADP-ribose

This chapter describes a bioassay for cyclic ADP-ribose (ADPR) on the basis of its Ca2+-releasing activity in homogenates prepared from sea urchin eggs. Substances that could potentially interfere with the Ca2+ release bioassay, such as Ca2+, inositol 1,4,5-triphosphate, and NAD+, must be removed from the sample. Cyclic ADP-ribose in the extracts is purified by a two-step system consisting of phenylboronate chromatography followed by anion-exchange chromatography on AG MP-1. Strongylocentrotus purpuratus eggs are obtained by stimulating ovulation of a female sea urchin with a 1-ml injection of 0.5 M KCI and washed once in artificial seawater, twice in Ca2+-free seawater containing 1 mM ethylene glycol tetraacetic acid (EGTA), twice in Ca2+-free seawater without EGTA, once with the homogenization buffer, and resuspended with the same medium to 25%. The sensitivity of the bioassay can be increased by pretreating the homogenate with caffeine, which is known to potentiate cADPR-induced Ca2+ release.