Browsing by Author "Muiyser, S. M."
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- ItemThe role of PFK in the oscillatory yeast strain X2180(Stellenbosch : Stellenbosch University, 2019-04) Muiyser, S. M.; Van Niekerk, D. D.; Snoep, Jacky L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: The elucidation of mechanisms involved in glycolytic oscillations in yeast can lead to a deeper understanding of the metabolic organisation in living systems. These oscillations, detected in the concentrations of the glycolytic intermediates of yeast, are a well-studied topic in the context of cell populations and extracts. In a recent study sustained oscillations were detected in individually placed single cells of the X2180 yeast strain in a microfluidic flow-chamber. Literature states that the specific regulation of the phosphofructokinase (PFK) enzyme by the metabolites AMP and ATP are a necessary feature in the glycolytic pathway that enables these oscillations. This regulation was also observed in a previous study where a mathematical model for the oscillatory system was created by numerical optimisation of enzyme kinetic parameters in an experimentally validated detailed model of steady state glycolysis. One of the results of the optimisation was an increased inhibition of PFK by its substrate, ATP. The aim of the current study was to perform an experimental investigation into the enzyme kinetic characteristics of phosphofructokinase in the X2180 oscillatory strain, as well as in the VIN13 strain, which has not been tested for oscillations. To address this, we used wide-field microscopy to detect oscillations in the X2180 strain and to test for oscillations in VIN13, when they were both cultured under conditions that have been shown to elicit oscillations in X2180. We optimised a protocol for inducing oscillations in our local microscope setup, and subsequently detected oscillations in X2180, and not in VIN13. We were able to optically resolve oscillations on the single cell level which will allow for the investigation of synchronization between cells (a feature that is observed in oscillating populations) in future studies. We then performed a kinetic characterisation of the phosphofructokinase enzyme in both yeast strains to test whether differences in its regulatory characteristics could be the reason for oscillations in one strain and not the other. We found that there are differences in PFK kinetic parameters of these two strains. Finally, by using our experimentally determined PFK kinetic parameters in a non-oscillating mathematical model, and following the previously published optimisation steps for other enzymes, we were able to simulate oscillations for the X2180 strain but could not do so for VIN13. From this we conclude that VIN13 requires further kinetic characterisation to be able to determine if it exhibits oscillatory behaviour.