Understanding yeast glycolysis dynamics using HPLC analysis and mathematical modelling.

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dc.contributor.advisorVan Niekerk, David Douglasen_ZA
dc.contributor.advisorSnoep, Jacob Leendert en_ZA
dc.contributor.authorVan Schalkwyk, Clara Elizabethen_ZA
dc.contributor.otherStellenbosch University. Faculty of Science. Dept. of Biochemistry.en_ZA
dc.date.accessioned2023-03-03T12:38:33Z
dc.date.accessioned2023-05-18T07:17:37Z
dc.date.available2023-03-03T12:38:33Z
dc.date.available2023-05-18T07:17:37Z
dc.date.issued2023-03
dc.descriptionThesis (MSc)--Stellenbosch University, 2023. en_ZA
dc.description.abstractENGLISH ABSTRACT: Glycolysis is a universally conserved metabolic pathway that forms the backbone of carbon metabolism and energy generation. A comprehensive understanding of this pathway may lead to a more complete understanding of energy metabolism. A novel HPLC-based method was recently developed for the detection of metabolite, cofactor and coenzyme concentrations during metabolic studies. The purpose of the current study is to implement this novel method to generate data which can be used to eluci- date dynamics of glycolytic intermediates and cofactors in conjunction with a existing enzyme kinetic model of yeast glycolysis in Saccharomyces cerevisiae, a model organism for eukariotic biology. The HPLC method was used to study the dynamic behaviour of three key glycolytic intermediates in response to a glucose pulse in cell free extracts of S. cerevisiae. It was found that in response to a glucose pulse, glucose is consumed, fructose-1,6- bisphosphate accumulates prior to glucose depletion and ethanol accumulates. Sub- sequently, the dynamic behaviour of the adenine nucleotides in response to a glucose pulse was investigated. It was found that while glycolysis is active, AMP accumulates and ATP depletes while ADP remains at a constant low concentration. Once glucose is depleted ATP increases and AMP decreases. This is the result of the combined action of adenylate kinase, glycolytic enzymes and ATPases. Finally, the effect of phosphate on glycolytic intermediate dynamics and specific enzyme activity was investigated. It was found that phosphate influences the rate of glucose consumption and degree of F16BP accumulation. The inhibitory effect of phosphate on pyruvate kinase activity was char- acterised and incorporated into the model, however, the adapted model could not yet describe the observed phenomena and further investigation is required.en_ZA
dc.description.abstractAFRIKAANSE OPSOMMING: Glikolise is ’n universeel-bewaarde metaboliese pad wat die ruggraat van kool- stofmetabolisme en energieopwekking vorm. ’n Omvattende begrip van hierdie pad kan lei tot ’n meer volledige begrip van energiemetabolisme. ’n Nuwe HPLC- gebaseerde metode is onlangs ontwikkel vir die opsporing van metaboliet-, kofaktor- en koënsiemkonsentrasies tydens metaboliese studies. Die doel van die huidige studie is om hierdie nuwe metode te implementeer om data te genereer wat gebruik kan word om die dinamika van glikolitiese tussenprodukte en kofaktore te verduidelik in samew- erking met ’n bestaande ensiemkinetiese model van gisglikolise in Saccharomyces cere- visiae, ’n model-organisme vir eukariotiese biologie. Die HPLC-metode is gebruik om die dinamiese gedrag van drie sleutel glikolitiese tussenprodukte te bestudeer in reaksie op ’n glukosepuls in selvrye ekstrakte van S. cerevisiae. Daar is gevind dat in reaksie op ’n glukose puls glukose verbruik word en fruktose-1,6-bisfosfaat ophoop voordat glukose opgebruik word en etanol akku- muleer. Vervolgens is die dinamiese gedrag van die adeniennukleotiede in reaksie op ’n glukosepuls ondersoek. Daar is gevind dat terwyl glikolise aktief is, AMP ophoop en ATP uitgeput word terwyl ADP by ’n konstante lae konsentrasie bly. Sodra glukose uitgeput is, neem ATP toe en AMP neem af. Dit is die gevolg van die gekombineerde werking van adenilaatkinase, glikolitiese ensieme en ATPases. Laastens is die effek van fosfaat op glikolitiese intermediêre dinamika en spesifieke ensiemaktiwiteit onder- soek. Daar is gevind dat fosfaat die tempo van glukoseverbruik en graad van F16BP- akkumulasie beïnvloed. Die inhiberende effek van fosfaat op piruvaatkinase aktiwiteit is gekarakteriseer en in die model geïnkorporeer, maar die aangepaste model kon egter nog nie die waargenome verskynsels beskryf nie en verdere ondersoek is nodig.af_ZA
dc.description.versionMastersen_ZA
dc.format.extentxii, 72 pagesen_ZA
dc.identifier.urihttp://hdl.handle.net/10019.1/127351
dc.language.isoen_ZAen_ZA
dc.publisherStellenbosch : Stellenbosch Universityen_ZA
dc.rights.holderStellenbosch Universityen_ZA
dc.subject.lcshGlycolysisen_ZA
dc.subject.lcshSaccharomyces cerevisiae -- Biotechnologyen_ZA
dc.subject.lcshHigh performance liquid chromatography -- Methodsen_ZA
dc.subject.lcshAdenine nucleotidesen_ZA
dc.subject.nameUCTDen_ZA
dc.titleUnderstanding yeast glycolysis dynamics using HPLC analysis and mathematical modelling.en_ZA
dc.typeThesisen_ZA
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