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Article: Specific binding of cyclic ADP-ribose to calcium-storing microsomes from sea urchin eggs

TitleSpecific binding of cyclic ADP-ribose to calcium-storing microsomes from sea urchin eggs
Authors
Issue Date1991
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, 1991, v. 266 n. 4, p. 2276-2281 How to Cite?
AbstractCyclic ADP-ribose (cADPR) is a metabolite of NAD+ which is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that it may be a general messenger for Ca2+ mobilization in cells. In this study I address questions of whether an intracellular receptor for cADPR exists and, if so, whether it is different from the IP3 receptor. A procedure employing nitrogen decompression was used to homogenize sea urchin eggs, and the Ca2+-storing microsomes were separated from mitochondria and other organelles by Percoll density centrifugation. Radioactive cADPR with high specific activity was produced by incubating [32P]NAD+ with the synthesizing enzyme and the product purified by high pressure liquid chromatography. The enzyme was membrane bound and was isolated from dog brain extracts by sucrose density gradient centrifugation. Partial purification of the enzyme was achieved by DEAE ion-exchange chromatography after solubilization with 3-[(cholamidopropyl)dimethylammonio]-1- propanesulfonate. Specific binding of 32P-labeled cADPR to a saturable site on the Ca2+-storing microsomes was detected by a filtration assay. Scatchard analysis indicated a binding affinity of about 17 nM and a capacity of about 25 fmol/mg protein. The binding was not affected by either NAD+ (the precursor) or ADP-ribose (the hydrolysis product) at 0.5 μM but was eliminated by 0.3 μM nonlabeled cADPR. The receptor for cADPR appeared to be different from that of IP3 since IP3 was not an effective competitor at a concentration as high as 3 μM. Similarly, heparin at a concentration that inhibits most of the IP3-induced calcium release from the microsomes did not affect the binding. The binding showed a prominent pH optimum at about 6.7. Calcium at 40 μM decreased the binding by about 50%. These dependencies of the binding on pH and Ca2+ are different from those reported for the IP3 receptor and provide further support that the intracellular receptors for cADPR and IP3 are different.
Persistent Identifierhttp://hdl.handle.net/10722/171547
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorLee, HCen_US
dc.date.accessioned2012-10-30T06:15:37Z-
dc.date.available2012-10-30T06:15:37Z-
dc.date.issued1991en_US
dc.identifier.citationJournal Of Biological Chemistry, 1991, v. 266 n. 4, p. 2276-2281en_US
dc.identifier.issn0021-9258en_US
dc.identifier.urihttp://hdl.handle.net/10722/171547-
dc.description.abstractCyclic ADP-ribose (cADPR) is a metabolite of NAD+ which is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that it may be a general messenger for Ca2+ mobilization in cells. In this study I address questions of whether an intracellular receptor for cADPR exists and, if so, whether it is different from the IP3 receptor. A procedure employing nitrogen decompression was used to homogenize sea urchin eggs, and the Ca2+-storing microsomes were separated from mitochondria and other organelles by Percoll density centrifugation. Radioactive cADPR with high specific activity was produced by incubating [32P]NAD+ with the synthesizing enzyme and the product purified by high pressure liquid chromatography. The enzyme was membrane bound and was isolated from dog brain extracts by sucrose density gradient centrifugation. Partial purification of the enzyme was achieved by DEAE ion-exchange chromatography after solubilization with 3-[(cholamidopropyl)dimethylammonio]-1- propanesulfonate. Specific binding of 32P-labeled cADPR to a saturable site on the Ca2+-storing microsomes was detected by a filtration assay. Scatchard analysis indicated a binding affinity of about 17 nM and a capacity of about 25 fmol/mg protein. The binding was not affected by either NAD+ (the precursor) or ADP-ribose (the hydrolysis product) at 0.5 μM but was eliminated by 0.3 μM nonlabeled cADPR. The receptor for cADPR appeared to be different from that of IP3 since IP3 was not an effective competitor at a concentration as high as 3 μM. Similarly, heparin at a concentration that inhibits most of the IP3-induced calcium release from the microsomes did not affect the binding. The binding showed a prominent pH optimum at about 6.7. Calcium at 40 μM decreased the binding by about 50%. These dependencies of the binding on pH and Ca2+ are different from those reported for the IP3 receptor and provide further support that the intracellular receptors for cADPR and IP3 are different.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 - Metabolismen_US
dc.subject.meshAnimalsen_US
dc.subject.meshBinding, Competitiveen_US
dc.subject.meshCalcium - Metabolismen_US
dc.subject.meshCalcium Channelsen_US
dc.subject.meshCentrifugation, Density Gradienten_US
dc.subject.meshChromatography, High Pressure Liquiden_US
dc.subject.meshChromatography, Ion Exchangeen_US
dc.subject.meshCyclic Adp-Riboseen_US
dc.subject.meshDogsen_US
dc.subject.meshHydrogen-Ion Concentrationen_US
dc.subject.meshInositol 1,4,5-Trisphosphate Receptorsen_US
dc.subject.meshInositol Phosphates - Metabolismen_US
dc.subject.meshMicrosomes - Metabolismen_US
dc.subject.meshOocytesen_US
dc.subject.meshReceptors, Cell Surface - Metabolismen_US
dc.subject.meshReceptors, Cytoplasmic And Nuclearen_US
dc.subject.meshSea Urchinsen_US
dc.subject.meshSolubilityen_US
dc.titleSpecific binding of cyclic ADP-ribose to calcium-storing microsomes from sea urchin eggsen_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.pmid1846622-
dc.identifier.scopuseid_2-s2.0-0025766614en_US
dc.identifier.volume266en_US
dc.identifier.issue4en_US
dc.identifier.spage2276en_US
dc.identifier.epage2281en_US
dc.identifier.isiWOS:A1991EV51500042-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridLee, HC=26642959100en_US

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