Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase

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dc.contributor.advisorMcIntosh, David Ben_ZA
dc.contributor.authorSeebregts, Christopher Jen_ZA
dc.date.accessioned2018-01-31T13:44:38Z
dc.date.available2018-01-31T13:44:38Z
dc.date.issued1989en_ZA
dc.description.abstractWe have synthesized a new class of photoaffinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP, -ADP and -AMP (TNP- 8N₃ATP, -ADP and -AMP), and their radiolabeled derivatives, and characterized their interaction with the sarcoplasmic reticulum Ca²⁺-ATPase. The TNP-8N₃-nucleotides were synthesized from ATP in three steps involving bromination in the 8-position of the adenine ring followed by displacement with an azido group and then trinitrophenylation of the resulting 8N₃-nucleotide with TNBS. Inclusion of the oxidizing agent, DTNB, in the final reaction was found to be necessary to prevent reduction of the azido group by the released sulfite anion and also elevated the yield of trinitrophenylation to about 80%. Purity was determined spectrophotometrically, as well as by anion exchange TLC and reversed phase HPLC. In the dark, the compounds were found to display most of the features of the parent TNP-nucleotides and interacted with the Ca²⁺-ATPase in a similar way. When activated by illumination, the probes were specifically incorporated into SR vesicles with high efficiency at alkaline pH. The site of labeling was identified as being on the A₁ tryptic fragment.en_ZA
dc.identifier.apacitationSeebregts, C. J. (1989). <i>Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology. Retrieved from http://hdl.handle.net/11427/27167en_ZA
dc.identifier.chicagocitationSeebregts, Christopher J. <i>"Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology, 1989. http://hdl.handle.net/11427/27167en_ZA
dc.identifier.citationSeebregts, C. 1989. Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Seebregts, Christopher J AB - We have synthesized a new class of photoaffinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP, -ADP and -AMP (TNP- 8N₃ATP, -ADP and -AMP), and their radiolabeled derivatives, and characterized their interaction with the sarcoplasmic reticulum Ca²⁺-ATPase. The TNP-8N₃-nucleotides were synthesized from ATP in three steps involving bromination in the 8-position of the adenine ring followed by displacement with an azido group and then trinitrophenylation of the resulting 8N₃-nucleotide with TNBS. Inclusion of the oxidizing agent, DTNB, in the final reaction was found to be necessary to prevent reduction of the azido group by the released sulfite anion and also elevated the yield of trinitrophenylation to about 80%. Purity was determined spectrophotometrically, as well as by anion exchange TLC and reversed phase HPLC. In the dark, the compounds were found to display most of the features of the parent TNP-nucleotides and interacted with the Ca²⁺-ATPase in a similar way. When activated by illumination, the probes were specifically incorporated into SR vesicles with high efficiency at alkaline pH. The site of labeling was identified as being on the A₁ tryptic fragment. DA - 1989 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1989 T1 - Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase TI - Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase UR - http://hdl.handle.net/11427/27167 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/27167
dc.identifier.vancouvercitationSeebregts CJ. Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology, 1989 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/27167en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Chemical Pathologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherSarcoplasmic reticulumen_ZA
dc.subject.otherAdenosinetriphosphataseen_ZA
dc.subject.otherAffinity labelingen_ZA
dc.subject.otherBinding sites (Biochemistry)en_ZA
dc.subject.otherNucleotidesen_ZA
dc.subject.otherCa²⁺-Transporting Atpaseen_ZA
dc.subject.otherAffinity Labelsen_ZA
dc.subject.otherBinding sitesen_ZA
dc.subject.otherNucleotidesen_ZA
dc.subject.otherSarcoplasmic reticulumen_ZA
dc.titlePhotoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPaseen_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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