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Article: A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity

TitleA novel cycling assay for cellular cADP-ribose with nanomolar sensitivity
Authors
KeywordsADP-ribosyl cyclase
Ca2+ signallin
CD38
NAD+
NADH
Issue Date2002
PublisherPortland Press Ltd. The Journal's web site is located at http://www.biochemj.org
Citation
Biochemical Journal, 2002, v. 361 n. 2, p. 379-384 How to Cite?
AbstractcADP-ribose (cADPR) is a novel cyclic nucleotide derived from NAD that has now been established as a general Ca2+ messenger in a wide variety of cells. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, its measurement has been technically difficult and requires specialized reagents. In this study a widely applicable high-sensitivity assay for cADPR is described. ADP-ribosyl cyclase normally catalyses the synthesis of cADPR from NAD+, but the reaction can be reversed in the presence of high concentrations of nicotinamide, producing NAD+ from cADPR stoichiometrically. The resultant NAD+ can then be coupled to a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD+ cycles through these coupled reactions, a molecule of highly fluorescent resorufin is generated. The reaction can be conducted for hours, resulting in more than a thousand-fold amplification of cADPR. Concentrations of cADPR in the nanomolar range can be measured routinely. The unique ability of ADP-ribosyl cyclase to catalyse the reverse reaction provides the required specificity. Using this assay, it is demonstrated that cADPR is present in all tissues tested and that the levels measured are directly comparable with those obtained using a radioimmunoassay. All the necessary reagents are widely available and the assay can be performed using a multiwell fluorescence plate reader, providing a high-throughput method for monitoring cADPR levels. This assay should be valuable in elucidating the messenger role of cADPR in cells.
Persistent Identifierhttp://hdl.handle.net/10722/132565
ISSN
2015 Impact Factor: 3.562
2015 SCImago Journal Rankings: 2.582
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorGraeff, Ren_HK
dc.contributor.authorLee, HCen_HK
dc.date.accessioned2011-03-28T09:26:21Z-
dc.date.available2011-03-28T09:26:21Z-
dc.date.issued2002en_HK
dc.identifier.citationBiochemical Journal, 2002, v. 361 n. 2, p. 379-384en_HK
dc.identifier.issn0264-6021en_HK
dc.identifier.urihttp://hdl.handle.net/10722/132565-
dc.description.abstractcADP-ribose (cADPR) is a novel cyclic nucleotide derived from NAD that has now been established as a general Ca2+ messenger in a wide variety of cells. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, its measurement has been technically difficult and requires specialized reagents. In this study a widely applicable high-sensitivity assay for cADPR is described. ADP-ribosyl cyclase normally catalyses the synthesis of cADPR from NAD+, but the reaction can be reversed in the presence of high concentrations of nicotinamide, producing NAD+ from cADPR stoichiometrically. The resultant NAD+ can then be coupled to a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD+ cycles through these coupled reactions, a molecule of highly fluorescent resorufin is generated. The reaction can be conducted for hours, resulting in more than a thousand-fold amplification of cADPR. Concentrations of cADPR in the nanomolar range can be measured routinely. The unique ability of ADP-ribosyl cyclase to catalyse the reverse reaction provides the required specificity. Using this assay, it is demonstrated that cADPR is present in all tissues tested and that the levels measured are directly comparable with those obtained using a radioimmunoassay. All the necessary reagents are widely available and the assay can be performed using a multiwell fluorescence plate reader, providing a high-throughput method for monitoring cADPR levels. This assay should be valuable in elucidating the messenger role of cADPR in cells.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.subjectADP-ribosyl cyclaseen_HK
dc.subjectCa2+ signallinen_HK
dc.subjectCD38en_HK
dc.subjectNAD+en_HK
dc.subjectNADHen_HK
dc.titleA novel cycling assay for cellular cADP-ribose 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.doi10.1042/0264-6021:3610379en_HK
dc.identifier.pmid11772410-
dc.identifier.scopuseid_2-s2.0-0037081860en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0037081860&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume361en_HK
dc.identifier.issue2en_HK
dc.identifier.spage379en_HK
dc.identifier.epage384en_HK
dc.identifier.isiWOS:000173982600021-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridGraeff, R=7003614053en_HK
dc.identifier.scopusauthoridLee, HC=26642959100en_HK

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