Specific binding of cyclic ADP-ribose to calcium-storing microsomes from sea urchin eggs.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ which is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that it may be a general messenger for Ca2+ mobilization in cells. In this study I address questions of whether an intracellular receptor for cADPR exists and, if so, whether it is different from the IP3 receptor. A procedure employing nitrogen decompression was used to homogenize sea urchin eggs, and the Ca2(+)-storing microsomes were separated from mitochondria and other organelles by Percoll density centrifugation. Radioactive cADPR with high specific activity was produced by incubating [32P]NAD+ with the synthesizing enzyme and the product purified by high pressure liquid chromatography. The enzyme was membrane bound and was isolated from dog brain extracts by sucrose density gradient centrifugation. Partial purification of the enzyme was achieved by DEAE ion-exchange chromatography after solubilization with 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate. Specific binding of 32P-labeled cADPR to a saturable site on the Ca2(+)-storing microsomes was detected by a filtration assay. Scatchard analysis indicated a binding affinity of about 17 nM and a capacity of about 25 fmol/mg protein. The binding was not affected by either NAD+ (the precursor) or ADP-ribose (the hydrolysis product) at 0.5 microM but was eliminated by 0.3 microM nonlabeled cADPR. The receptor for cADPR appeared to be different from that of IP3 since IP3 was not an effective competitor at a concentration as high as 3 microM. Similarly, heparin at a concentration that inhibits most of the IP3-induced calcium release from the microsomes did not affect the binding. The binding showed a prominent pH optimum at about 6.7. Calcium at 40 microM decreased the binding by about 50%. These dependencies of the binding on pH and Ca2+ are different from those reported for the IP3 receptor and provide further support that the intracellular receptors for cADPR and IP3 are different.