High Throughput Fluorescence-Based Assays for Cyclic ADP-Ribose, NAADP, and Their Metabolic Enzymes

Authors: Graeff R.M.; Lee H.Cheung

Source: Combinatorial Chemistry & High Throughput Screening, Volume 6, Number 4, June 2003 , pp. 367-379(13)

Publisher: Bentham Science Publishers

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.

Keywords: cyclic adp-ribose; naadp; cd38; adp-ribosyl cyclase; pyridine nucleotides; calcium messengers; calcium stores; calcium signaling

Language: English

Document Type: Review article

DOI: http://dx.doi.org/10.2174/138620703106298464

Publication date: 2003-06-01

• Combinatorial Chemistry & High Throughput Screening publishes full length original research articles and reviews describing various topics in combinatorial chemistry (e.g. small molecules, peptide, nucleic acid or phage display libraries) and/or high throughput screening (e.g. developmental, practical or theoretical). Ancillary subjects of key importance, such as robotics and informatics, will also be covered by the journal. In these respective subject areas, Combinatorial Chemistry & High Throughput Screening is intended to function as the most comprehensive and up-to-date medium available. The journal should be of value to individuals engaged in the process of drug discoveryand development, in the settings of industry, academia or government.

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