CYCLIC GMP-DEPENDENT AND GMP-INDEPENDENT EFFECTS ON THE SYNTHESIS OF THE CALCIUM MESSENGERS CYCLIC ADP-RIBOSE AND NICOTINIC-ACID ADENINE-DINUCLEOTIDE PHOSPHATE

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phos phate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate, Although cADPR and NAAD P are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity, In this study , three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+ rel ease assay, and a thin layer chromatographic assay, Soluble and membra ne-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR, The soluble cyclase was half-maximally stimulated by 5 .3 mu M cGMP, but not by cAMP, while the membrane-bound form was indep endent of cGMP, The two forms of the cyclase were also different in th e pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separat e enzymes, The stimulatory effect of cGMP required ATP or ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) and a cGMP-dependent kinase acti vity was shown to be present in the soluble fraction, The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was fou nd to be predominantly associated with membranes. Similar to the membr ane-bound cyclase, the cADPR hydrolase activity was also independent o f cGMP. Both the soluble and membrane fractions also catalyzed the syn thesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA), The base-exchange activity was independent of cG MP and the half-maximal concentrations of NADP and NA needed were abou t 0.2 mM and 10 mM, respectively. The exchange reaction showed a prefe rence for acidic pH, contrasting with the neutral pH optimum of the cy clase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.