High throughput fluorescence-based assays for cyclic ADP-ribose, NAADP, and their metabolic enzymes

Abstract

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two novel Ca2+ messengers derived respectively from NAD and NADP. Since their discovery in sea urchin eggs, both have now been shown to serve messenger functions in a wide range of cells from plant to human. In this article, a series of fluorimetric assays for cADPR, NAADP and their metabolic enzymes is compiled. The enzyme assay makes use of an analog of NAD, nicotinamide guanine dinucleotide, which is non-fluorescent but is cyclized by the enzymes to a fluorescent analog of cADPR, cyclic GDP-ribose. Other NAD utilizing enzymes are not capable of catalyzing the cyclization and thus produce no interference. The fluorimetric assays for cADPR and NAADP make use of coupled-enzyme amplification and can readily detect nanomolar concentrations of either messenger. All the assays described can be performed in multi-well format, allowing ready automation and use in high throughput screening. An added advantage of these assays is that all the required reagents are commercially available, facilitating general adoption of the techniques by all those who are interested in the physiology and enzymology of the novel Ca2+ signaling pathways mediated by cADPR and NAADP.