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Article: Structural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity

TitleStructural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activity
Authors
Issue Date1989
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 1989, v. 264 n. 3, p. 1608-1615 How to Cite?
AbstractIncubation of NAD+ with extracts from sea urchin eggs resulted in production of a metabolite which could mobilize intracellular Ca2+ stores of the eggs. In this study we present structural evidence indicating that the metabolite is a cyclized ADP-ribose having an N-glycosyl linkage between the anomeric carbon of the terminal ribose unit and the N6-amino group of the adenine moiety. In view of this structure we propose cyclic ADP-ribose as the common name for the metabolite. The purification procedure for the metabolite consisted of deproteinizing the incubated egg extracts and sequentially chromatographing the extracts through three different high pressure liquid chromatography (HPLC) columns. The homogeneity of the purified metabolite was further verified by HPLC on a Partisil 5 SAX column. Using radioactive precursor NAD+ with label at various positions it was demonstrated that the metabolite was indeed derived from NAD+ and that the adenine ring as well as the adenylate α-phosphate were retained in the metabolite whereas the nicotinamide group was removed. This was confirmed by 1H NMR and two-dimensional COSY experiments, which also allowed the identification of all 12 protons on the two ribosyl units as well as the two protons on the adenine ring. From the chemical shifts of the two anomeric protons it was concluded that the C-1 carbons of both ribosyl units were still bonded to nitrogen. The positive and negative ion fast atom bombardment mass spectra showed (M + Na)+, (M - H + 2Na)+, (M - H)-, and (M - 2H + Na)- peaks at m/z 564, 586, 540, and 562, respectively. Exact mass measurements indicated a molecular weight of 540.0526 for (M - H)-. This together with the constraints imposed by the results from NMR, radioactive labeling, and total phosphate determination uniquely specified a molecular composition of C15H21N5O13P2. Analysis by 1H NMR and mass spectroscopy of the only major breakdown product of the metabolite after prolonged incubation at room temperature established that it was ADP-ribose, thus providing strong support for the cyclic structure.
Persistent Identifierhttp://hdl.handle.net/10722/171534
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorLee, HCen_US
dc.contributor.authorWalseth, TFen_US
dc.contributor.authorBratt, GTen_US
dc.contributor.authorHayes, RNen_US
dc.contributor.authorClapper, DLen_US
dc.date.accessioned2012-10-30T06:15:34Z-
dc.date.available2012-10-30T06:15:34Z-
dc.date.issued1989en_US
dc.identifier.citationJournal Of Biological Chemistry, 1989, v. 264 n. 3, p. 1608-1615en_US
dc.identifier.issn0021-9258en_US
dc.identifier.urihttp://hdl.handle.net/10722/171534-
dc.description.abstractIncubation of NAD+ with extracts from sea urchin eggs resulted in production of a metabolite which could mobilize intracellular Ca2+ stores of the eggs. In this study we present structural evidence indicating that the metabolite is a cyclized ADP-ribose having an N-glycosyl linkage between the anomeric carbon of the terminal ribose unit and the N6-amino group of the adenine moiety. In view of this structure we propose cyclic ADP-ribose as the common name for the metabolite. The purification procedure for the metabolite consisted of deproteinizing the incubated egg extracts and sequentially chromatographing the extracts through three different high pressure liquid chromatography (HPLC) columns. The homogeneity of the purified metabolite was further verified by HPLC on a Partisil 5 SAX column. Using radioactive precursor NAD+ with label at various positions it was demonstrated that the metabolite was indeed derived from NAD+ and that the adenine ring as well as the adenylate α-phosphate were retained in the metabolite whereas the nicotinamide group was removed. This was confirmed by 1H NMR and two-dimensional COSY experiments, which also allowed the identification of all 12 protons on the two ribosyl units as well as the two protons on the adenine ring. From the chemical shifts of the two anomeric protons it was concluded that the C-1 carbons of both ribosyl units were still bonded to nitrogen. The positive and negative ion fast atom bombardment mass spectra showed (M + Na)+, (M - H + 2Na)+, (M - H)-, and (M - 2H + Na)- peaks at m/z 564, 586, 540, and 562, respectively. Exact mass measurements indicated a molecular weight of 540.0526 for (M - H)-. This together with the constraints imposed by the results from NMR, radioactive labeling, and total phosphate determination uniquely specified a molecular composition of C15H21N5O13P2. Analysis by 1H NMR and mass spectroscopy of the only major breakdown product of the metabolite after prolonged incubation at room temperature established that it was ADP-ribose, thus providing strong support for the cyclic structure.en_US
dc.languageengen_US
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_US
dc.relation.ispartofJournal of Biological Chemistryen_US
dc.subject.meshAdenosine Diphosphate Ribose - Analogs & Derivatives - Analysis - Metabolismen_US
dc.subject.meshAnimalsen_US
dc.subject.meshCalcium - Metabolismen_US
dc.subject.meshChromatography, High Pressure Liquiden_US
dc.subject.meshCyclic Adp-Riboseen_US
dc.subject.meshMagnetic Resonance Spectroscopyen_US
dc.subject.meshMass Spectrometryen_US
dc.subject.meshModels, Molecularen_US
dc.subject.meshNad - Metabolismen_US
dc.subject.meshOocytes - Drug Effects - Metabolismen_US
dc.subject.meshSea Urchinsen_US
dc.titleStructural determination of a cyclic metabolite of NAD+ with intracellular Ca2+-mobilizing activityen_US
dc.typeArticleen_US
dc.identifier.emailLee, HC:leehc@hku.hken_US
dc.identifier.authorityLee, HC=rp00545en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid2912976-
dc.identifier.scopuseid_2-s2.0-0024496833en_US
dc.identifier.volume264en_US
dc.identifier.issue3en_US
dc.identifier.spage1608en_US
dc.identifier.epage1615en_US
dc.identifier.isiWOS:A1989R890300040-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridLee, HC=26642959100en_US
dc.identifier.scopusauthoridWalseth, TF=7005424273en_US
dc.identifier.scopusauthoridBratt, GT=7006386681en_US
dc.identifier.scopusauthoridHayes, RN=7402082400en_US
dc.identifier.scopusauthoridClapper, DL=6701733043en_US

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