Inaugural Addresses (Biochemistry)
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Browsing Inaugural Addresses (Biochemistry) by browse.metadata.type "Thesis"
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- ItemComparative characterization and mutational analysis of type III pantothenate kinases(Stellenbosch : University of Stellenbosch, 2006-03) Brand, Leisl Anne; Strauss, Erick; Swart, Pieter; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.This thesis reports the cloning, overexpression and characterization of the coaX gene product from Bacillus subtilis and its homologue from Helicobacter pylori. It demonstrates that these proteins have pantothenate kinase activity. Compared to the two pantothenate kinase analogues classified to date, these two enzymes exhibit distinctly different characteristics, suggesting that they are the first characterized examples of a third pantothenate kinase analogue. In addition, mutational studies are presented that probe the importance of conserved aspartate residues within the active sites of these newly characterized analogues. The results show that these residues are important for the activity of the protein.
- ItemDevelopment of a synthetic affinity membrane for the purification of recombinant maltose binding proteins(Stellenbosch : Stellenbosch University, 2008-12) Asongwe, Lionel Ateh Tantoh; Swart, P.; Jacobs, E. P.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.The aim of this project was to fabricate a new affinity membrane-based system that is biospecific and biocompatible, and which could be used as an adsorption matrix for the immobilization of the recombinant protein maltose binding protein human estrogen receptor alpha ligand binding domain (MBP-hER LBD). The viability of the affinity membrane system (AMS) for the detection of estrogenic compounds (ECs) in drinking water, using affinity principles was determined. This affinity separation was based on the interaction between the analyte 17 -estradiol (E2) and the recombinant protein MBP-hER LBD. The MBP-hER LBD was immobilized on a solid matrix support. The alpha human estrogen hormone receptor (hER ) was used to test for the binding affinity of the fusion protein to a ligand, radiolabelled E2. Each component of this bioaffinity system, from the membrane matrix to the expression/purification of the bioligand, and raising of antibodies against the purified bioligand, was studied with the aim of producing a well-characterized system with the following advantages: robust in nature, cost effective and high loading capacity. Specifically, this study describes: 1. Expression of the bioligand maltose-binding protein (MBP) to be used as an affinity ligand for immobilization onto a solid membrane matrix. 2. Expression of MBP as a fusion protein to the human estrogen receptor alpha ligand binding domain (hER LBD). 3. The affinity purification of biospecific bioligands (MBP and MBP-hER LBD) using a one-step affinity purification system with amylose forming the solid phase of the affinity chromatographic column. 4. Generation of anti MBP-hER LBD antibodies to be used for the characterization of the bioligands by means of western blotting. 5. The fabrication and characterization of a flat-sheet membrane as a model affinity-matrix. 6. Developing an affinity immobilization protocol for the immobilization of the bioligand onto the affinity membrane (AM) matrix. 7. Quantitative analysis of the immobilized bioligand present on the surface of the membrane matrix using tritiated E2. The recombinant protein (MBP-hER LBD) was successfully expressed and purified to form a bio-specific ligand for its immobilization onto a cellulose acetate (CA)/amylose functionalized affinity membrane. Polyclonal antibodies were successfully raised against the purified recombinant protein. The anti-MBP-hER LBD antibodies were subsequently used as a potential ‘marker’ to confirm the immobilization of the recombinant protein onto the CA/amylose functionalized membrane. Attempts to utilize the protein-coated membrane for the selective recovery of E2 were, however, unsuccessful.
- ItemGeneric kinetic equations for modelling multisubstrate reactions in computational systems biology(Stellenbosch : University of Stellenbosch, 2006-03) Hanekom, Arno J.; Rohwer, J. M.; Hofmeyr, J-H. S.; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.Systems biology is a rapidly developing field, studying biological systems by methodically perturbing them either chemically, genetically or biologically. The system response is observed and incorporated into mathematical models. These computational models describe the system structure, predicting its behaviour in response to individual perturbations. Metabolic networks are examples of such systems and are modelled in silico as kinetic models. These kinetic models consist of the constituent enzyme reactions that make up the different pathways of a metabolic network. Each enzyme reaction is represented as a mathematical equation. The main focus of a kinetic model is to portray as realistically as possible a view in silico of physiological behaviour. The equations used to describe model reactions therefore need to make accurate predictions of enzyme behaviour. Numerous enzymes in metabolic networks are cooperative enzymes and many equations have been put forward to describe these reactions. Examples of equations used to model cooperative enzymes are the Adair equation, the uni-reactant Monod, Wyman and Changeux model, Hill equation, and the recently derived reversible Hill equation. Hill equations fit the majority of experimental data very well and have many advantages over their uni-substrate counterparts. In contrast to the abovementioned equations, the majority of enzyme reactions in metabolism are of a multisubstrate nature. Moreover, these multisubstrate reactions should be modelled as reversible reactions, as the contribution of the reverse reaction rate on the net conversion rate can not be ignored [1]. To date, only the bi-substrate reversible MWC equation has been formulated to describe cooperativity for a reversible reaction of more than one substrate. It is, however, difficult to use as a result of numerous parameters, not all of which have clear operational meaning. Moreover, MWC equations do not predict realistic allosteric modifier behaviour [2, 3]. Hofmeyr & Cornish-Bowden [3] showed how the uni-reactant reversible Hill equation succeeds in predicting realistic allosteric inhibitor behaviour, compared to the uni-reactant MWC equation, which does not. The aim of this study was to therefore derive a reversible Hill equation that can describe multisubstrate cooperative reactions and predicts realistic allosteric modifier behaviour. In this work, we present a generalised multisubstrate reversible Hill (GRH) equation. The bi-substrate and three substrate cases of this equation were also extended to incorporate any number of independently binding allosteric modifiers. The derived GRH equation is evaluated against the above mentioned cooperative models and shows good correlation. Moreover, the predicted behaviour of the bi-substrate reversible Hill equation with one allosteric inhibitor is compared to the MWC equation with one allosteric inhibitor in silico. This showed how the bi-substrate reversible Hill equation is able to account for substrate-modifier saturation, unlike the MWC equation, which does not. Additionally, the bi-substrate reversible Hill equation behaviour was evaluated against in vitro data from a cooperative bi-substrate enzyme which was allosterically inhibited. The experimental data confirm the validity of the behaviour predicted by the bi-substrate reversible Hill equation. Furthermore, we also present here reversible Hill equations for two substrates to one product and one substrate to two products reactions. Reactions of this nature are often found in metabolism and the need to accurately describe their behaviour is as important as reactions with equal substrates and products. The proposed reversible Hill equations are all independent of underlying enzyme mechanism, they contain parameters that have clear operational meaning and all of the newly derived equations can be transformed to non-cooperative equations by setting the Hill coefficient equal to one. These equations are of great use in computational models, enabling the modeller to accurately describe the behaviour of a vast number of cooperative and non-cooperative enzyme reactions with only a few equations.
- ItemSelective extraction of Cyclopia for enhanced in vitro phytoestrogenicity(Stellenbosch : University of Stellenbosch, 2008-03) Mfenyana, Ciko; Louw, Ann; Joubert, Lizette; University of Stellenbosch. Faculty of Science. Dept. of Biochemistry.Phytoestrogens are plant compounds whose ability to mimic the action of estrogens has resulted in their usage for the treatment of menopausal symptoms. Despite uncertainties about the safety and effectiveness of phytoestrogens in humans, the use of market phytoestrogenic nutraceuticals and botanicals is on the increase. Positive epidemiological study findings coupled to an entrenched belief in many societies about the superiority of what they view as “natural” remedies, as well as the reluctance of women to use the traditional hormone replacement therapy due to its association with detrimental health effects as reported by studies such as the World Health Initiative, the Million Women and the Kronos Early Estrogen Prevention studies, are thought to be instrumental in the growth of the phytoestrogen market. As the subject of the current thesis, we investigated the candidacy of extracts of the honeybush plant (genus Cyclopia), which is used for the manufacture of popular tea beverages, for use in the formulation of a high quality phytoestrogenic nutraceutical with a competitive market edge. We evaluated four harvestings of Cyclopia (M6-9) available in bulk and selected 2 harvestings (M6 and M7) for further extraction using solvents of differing polarity and also mimicking the preparation of a cup of tea. Our findings clearly demonstrate that of the resultant 22 extracts the SM6Met and SM6EAc extracts had the highest in vitro potency and efficacy, respectively. Another exciting finding from our study is the unequivocal demonstration of phytoestrogenic activity by extracts prepared in the same manner as the traditional cup of honeybush tea. Additionally, our study has highlighted the importance and the influence of experimental variables such as the specific harvesting evaluated and the characteristics of the extraction solvent (e.g. polarity and temperature) on the yield and the estrogenic activity of the extracts. In addition, the advantage of certain in vitro assays over others for discriminating between estrogenic substances based on their efficacies and potencies was demonstrated with the alkaline phosphatase assay being most suitable for discriminating efficacy and the E-screen most suitable for discriminating potency. Furthermore, our study has imparted a valuable lesson about the pharmacological behavior of estrogenic substances by presenting a conundrum in the form of the two desirable pharmacological parameters (potency and efficacy) occurring in different extracts, an outcome that complicates the central aim of our study, which is the preparation of an extract that embodies both parameters. Additionally, the low quantity of known putative phytoestrogens and the presence of unidentified polyphenols in M6, the source of our choice extracts (SM6Met and SM6EAc), makes the high estrogenic potency and efficacy of the choice extracts that much more intriguing. Nonetheless, benchmarking against four market phytoestrogen extracts indicate that the Cyclopia extracts have comparable estrogenicity suggesting potential as marketable phytoestrogenic preparations. The combination of the achievement of aims and the birth of new questions from that very achievement, which are the hallmark of scientific endeavors, have made this study a rewarding experience and we hope to share the feeling in its entirety with the reader.