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Article: A novel cycling assay for nicotinic acid-adenine dinucleotide phosphate with nanomolar sensitivity

TitleA novel cycling assay for nicotinic acid-adenine dinucleotide phosphate with nanomolar sensitivity
Authors
KeywordsCa2+ signalling
NAD
NADP
Nicotinamideadenine dinucleotide
NMN-adenylyltransferase
Issue Date2002
PublisherPortland Press Ltd. The Journal's web site is located at http://www.biochemj.org
Citation
Biochemical Journal, 2002, v. 367 n. 1, p. 163-168 How to Cite?
AbstractNicotinic 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 10-20 nM 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.
Persistent Identifierhttp://hdl.handle.net/10722/132564
ISSN
2015 Impact Factor: 3.562
2015 SCImago Journal Rankings: 2.582
PubMed Central ID
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorGraeff, Ren_HK
dc.contributor.authorLee, HCen_HK
dc.date.accessioned2011-03-28T09:26:20Z-
dc.date.available2011-03-28T09:26:20Z-
dc.date.issued2002en_HK
dc.identifier.citationBiochemical Journal, 2002, v. 367 n. 1, p. 163-168en_HK
dc.identifier.issn0264-6021en_HK
dc.identifier.urihttp://hdl.handle.net/10722/132564-
dc.description.abstractNicotinic 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 10-20 nM 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.en_HK
dc.languageengen_US
dc.publisherPortland Press Ltd. The Journal's web site is located at http://www.biochemj.orgen_HK
dc.relation.ispartofBiochemical Journalen_HK
dc.subjectCa2+ signallingen_HK
dc.subjectNADen_HK
dc.subjectNADPen_HK
dc.subjectNicotinamideadenine dinucleotideen_HK
dc.subjectNMN-adenylyltransferaseen_HK
dc.titleA novel cycling assay for nicotinic acid-adenine dinucleotide phosphate with nanomolar sensitivityen_HK
dc.typeArticleen_HK
dc.identifier.emailGraeff, R: graeffr@hku.hken_HK
dc.identifier.emailLee, HC: leehc@hku.hken_HK
dc.identifier.authorityGraeff, R=rp01464en_HK
dc.identifier.authorityLee, HC=rp00545en_HK
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1042/BJ20020644en_HK
dc.identifier.pmid12117413-
dc.identifier.pmcidPMC1222877-
dc.identifier.scopuseid_2-s2.0-0036798917en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0036798917&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume367en_HK
dc.identifier.issue1en_HK
dc.identifier.spage163en_HK
dc.identifier.epage168en_HK
dc.identifier.isiWOS:000178571200019-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridGraeff, R=7003614053en_HK
dc.identifier.scopusauthoridLee, HC=26642959100en_HK

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