Nicotinic acid—adenine dinucleotide phosphate (NAADP) is a novel nucleotide derived from NADP that has now been shown to be active in releasing Ca2+ from intracellular stores in a wide variety of cells ranging from plant to human. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, no assay has been reported for NAADP to date. In the present study, a widely applicable assay for NAADP with high sensitivity is described. NAADP was first dephosphorylated to nicotinic acid—adenine dinucleotide by treatment with alkaline phosphatase. The conversion was shown to be stoichiometric. NMN-adenylyltransferase was then used to convert nicotinic acid—adenine dinucleotide into NAD in the presence of high concentrations of NMN. The resultant NAD was amplified by a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD cycled through these coupled reactions, a molecule of highly fluorescent resorufin was generated. The reaction could be performed for hours, resulting in more than a 1000-fold amplification. Concentrations of NAADP over the 10—20nM range could be routinely measured. This novel cycling assay was combined with an enzymic treatment to provide the necessary specificity for the assay. NAADP was found to be resistant to NADase and apyrase. Pretreatment of samples with a combination of the hydrolytic enzymes completely eliminated the interference from common nucleotides. The versatility of the cycling assay can also be extended to measure nicotinic acid, which is a substrate in the synthesis of NAADP catalysed by ADP-ribosyl cyclase, over the micromolar range. All the necessary reagents for the cycling assay are widely available and it can be performed using a multi-well fluorescence plate reader, providing a high-throughput method. This is the first assay reported for NAADP and nicotinic acid, which should be valuable in elucidating the messenger functions of NAADP.
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October 2002
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Research Article|
October 01 2002
A novel cycling assay for nicotinic acid–adenine dinucleotide phosphate with nanomolar sensitivity Available to Purchase
Richard GRAEFF;
Richard GRAEFF
Department of Pharmacology, 321 Church Street SE, 4-145 Jackson Hall, University of Minnesota, Minneapolis, MN 55455, U.S.A.
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Hon Cheung LEE
Hon Cheung LEE
1
Department of Pharmacology, 321 Church Street SE, 4-145 Jackson Hall, University of Minnesota, Minneapolis, MN 55455, U.S.A.
1To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
April 23 2002
Revision Received:
June 21 2002
Accepted:
July 15 2002
Accepted Manuscript online:
July 15 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 367 (1): 163–168.
Article history
Received:
April 23 2002
Revision Received:
June 21 2002
Accepted:
July 15 2002
Accepted Manuscript online:
July 15 2002
Citation
Richard GRAEFF, Hon Cheung LEE; A novel cycling assay for nicotinic acid–adenine dinucleotide phosphate with nanomolar sensitivity. Biochem J 1 October 2002; 367 (1): 163–168. doi: https://doi.org/10.1042/bj20020644
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