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The analysis of glycogen phosphate and glucose-1,6-bisphosphate metabolism in escherichia coli

dc.contributor.advisorLloyd, Jamesen_ZA
dc.contributor.authorJewell, Jonathan Fredericken_ZA
dc.contributor.otherStellenbosch University. Faculty of Agrisciences. Dept. of Genetics.en_ZA
dc.date.accessioned2015-05-20T09:12:54Z
dc.date.available2015-05-20T09:12:54Z
dc.date.issued2015-03
dc.identifier.urihttp://hdl.handle.net/10019.1/96612
dc.descriptionThesis (MScAgric)--Stellenbosch University, 2015.en_ZA
dc.description.abstractENGLISH ABSTRACT: This thesis examined two aspects of E. coli carbon metabolism, the incorporation of covalently bound phosphate into glycogen as well as the manufacture of glucose-1,6-bisphosphate (GBP). In vitro analysis using recombinant maltodextrin phosphorylase (MalP) incubated together with maltodextrin, glucose-1-phosphate (Glc-1-P) and GBP resulted in the incorporation of phosphate into manufactured polymer at levels of 15 nmol Glc-6-P/mg polymer. No phosphate could be detected in the same incubation lacking only GBP. Moreover, higher amounts of polymer were also present in incubations where GBP was present with Glc-1-P, compared with Glc-1-P alone. Attempts were made to purify glycogen phosphorylase (GlgP), but these were unsuccessful. To examine if MalP and/or GlgP carry out this reaction in vivo, strains lacking them were produced. However, analysis revealed no significant difference in the phosphate content of glycogen extracted from wild type, single and double mutants lacking glgP and malP. A protein responsible for the synthesis of a phosphoglucomutase (PGM) stimulatory compound was purified to apparent homogeneity. This was identified, through tryptic fingerprinting, as the acid glucose-1-phosphate phosphatase (AGP) protein. Using recombinant AGP protein it was demonstrated that it was able to produce GBP from Glc-1-P in a phosphotransferase reaction, where one phosphate from Glc-1-P phosphorylates the C6 position of another. However, agp mutant cells were unchanged in the amounts of GBP they accumulate and crude protein extracts from them were still capable of synthesizing GBP from Glc-1-P. A mutant strain lacking both agp and pgm could no longer produce a PGM stimulatory compound, indicating that PGM most likely also synthesises GBP.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Hierdie tesis het twee aspekte van E. coli koolstof metabolisme, naamlik die inkorporasie van kovalent gebonde fosfaat in glikogeen en die vervaardiging van glukose-1,6-bisfosfaat (GBP), ondersoek. In vitro analise met behulp van rekombinante maltodekstrien-fosforilase (MalP), geïnkubeer met maltodekstrien, glukose-1-fosfaat (Glc-1-P) en GBP het gelei tot die inkorporasie van fosfaat teen vlakke van 15 nmol glukose-6-fosfaat (Glc-6-P) per milligram vervaardigde polimeer. Fosfaat was nie teenwoordig in die inkubasie waarin GBP uitgelaat was nie. Verder was hoër vlakke van polimeer vervaardig tydens die ko inkubasie van GBP en Glc-1-P as wat opgemerk was toe net Glc-1-P as substraat gedien het. Pogings wat aangewend was om glikogeen-fosforilase (GlgP) te suiwer was onsuksesvol. Om vas te stel of MalP en/of GlgP hierdie reaksie in vivo kan uitvoer, was mutante wat die gene ontbreek geproduseer. Daar was egter geen beduidende verskil in die fosfaat inhoud van glikogeen tussen wilde-tipe en enkel en dubbel-mutante van die glgP en malP gene opgemerk nie. 'n Proteïen wat verantwoordelik is vir die sintese van 'n fosfoglukomutase (PGM) stimulant, is gesuiwer tot oënskynlike homogeniteit. Dit was geïdentifiseer met proteïen vertering-vingerdrukking as die suurglukose-1-fosfaat fosfatase (AGP) proteïen. Deur gebruik te maak van rekombinante AGP proteïen was daar gedemonstreer dat die proteïen in staat is om GBP te vervaardig deur gebruik te maak van Glc-1-P as substraat in ʼn fosfotransferase reaksie. Die reaksie behels die oordrag van 'n fosfaat van een Glc-1-P eenheid na die C6 posisie van 'n ander Glc-1-P eenheid. Die vlakke van GBP was egter onveranderd in die selle van die agp mutant en boonop was ru-proteïen uittreksels nog steeds in staat om GBP uit Glc-1-P te sintetiseer. „n Dubbele mutant, ontbreek in beide agp en pgm, was nie in staat om 'n PGM-stimulerende verbinding te vervaardig nie, wat daarop dui dat PGM ook waarskynlik verantwoordelik is vir die sintese van GBP.af_ZA
dc.format.extent77 pages : illustrationsen_ZA
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.subjectGlycogen phosphate -- Analysisen_ZA
dc.subjectGlucose-1,6-bisphosphate -- Analysisen_ZA
dc.subjectAcid glucose-1-phosphateen_ZA
dc.subjectPhosphorylaseen_ZA
dc.subjectEscherichia coli carbon metabolismen_ZA
dc.subjectUCTDen_ZA
dc.titleThe analysis of glycogen phosphate and glucose-1,6-bisphosphate metabolism in escherichia colien_ZA
dc.typeThesisen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.embargo.terms2016-12-31


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