Thesis (M.S.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains vii, 72 p. : ill. (some col). Vita. Includes abstract. Includes bibliographical references (p. 63-70).

A novel molecular mechanism of autophosphorylation-dependent activation of the ser/thr S6/H4 kinase isolated from human placenta is described. Phosphopeptide mapping of the enzyme was used to determine the rate and extent of site-specific autophosphorylation. These data were correlated to phosphotransferase activity of the protein kinase. The results indicated that a sequential phosphorylation of two sites in the catalytic domain is required for maximum activation. Kinetic analysis determined that site 1 is modified by an intramolecular phosphorylation, and site 2 is modified by an intermolecular phosphorylation. On the basis of these data a model is proposed in which autophosphorylation of the pseudosubstrate domain and on a serine residue in subdomain VIII are both required for maximum activation of the S6/H4 kinase.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

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A Dissertation Submitted to the Faculty of SCience University of the Witwatersrand, Johannesburg In fulfilment of the requirements for the Degree of Doctor of Philosophy / Living cells are highly auto-dynamic entities which means that the underlying biochemistry is equally dynamic, a reality which is ignored by most researchers. Theoretical studies indicate that such a state must be due to the existence of oscillatory variations in the levels and activities of key components in the cell. In this study, the dynamic behaviour of four major, interrelated areas of cell biochemistry (phosphorylation, dephosphorylation, the terminal reaction of glycolysis and the amount of soluble protein) were examined and all systems found to oscillate in murine erythroleukaemic cells (MEL) and, where examined, also in the human HL-60 leukaemic cell line. (Abbreviation abstract) / AC 2018

A Dissertation Submitted to the Faculty of Science University of the Witwatersrand, Johannesburg In fulfilment of the requirements for the Degree of Doctor of Philosophy / Living cells are highly auto-dynamic entities which means that the underlying biochemistry is equally dynamic, a reality which is ignored by most researchers. Theoretical studies indicate that such a state must be due to the existence of oscillatory variations in the levels and activities of key components in the cell. In this study, the dynamic behaviour of four major, interrelated areas of cell biochemistry (phosphorylation, dephosphorylation, the terminal reaction of glycolysis and the amount of soluble protein) were examined and all systems found to oscillate in murine erythroleukaemic cells (MEL) and, where examined, also in the human HL-6Q leukaemic cell line. certain processes have been shown to be oscillatory for the first time ( phosphorylation potential, the lactate dehydrogenase active isozyme level and aspects of the regulation thereof). While others have been shown to occur at a higher frequency than previously reported (phosphotyrosine phosphatase activity, the activity and apparent isozyme pattern of lactate dehydrogenase, the amount of extractable protein). All rhythms are shown (for the first time) to be complex and to involve several contributing periodicities, some modulating the period and amplitude of the observed oscillation. The frequencies are very high (periods of 1-20 minutes and probably Less) and the amplitudes are equally high (variations in magnitude of as much as a hundred fold). Phosphorylation processes, currently of particular interest with regard to the nature and control of cell proliferation are thus found to be more highly dynamic than previously believed, a fact which throws some doubt on the current ideas on cell proliferation. The actual lactate dehydrogenase (LDH) active isozyme pattern is shown not to be constant (as generally believed) but to vary at high frequency (possibly due to phosphorylation of the the enzyme) while the kinetics and specificity of the lone isozyme in murine erythroleukaemic cells appear to be varying at equally high frequency due to the action of regulators (perhaps arising elsewhere within the glycolytic pathway). Similar results were obtained with HL-60 leukaemic cells with at least two of the isozymes varying in level, to some extent independently. The hormone, insulin, and the inducer of cell differentiation, HMBA (hexamethylenebisacetamide), have been found to affect the dynamics of the four systems although, because of the complexity of the rhythms the actual effects on the dynamics are not easily defined. Insulin has a marked effect on the mean level of the activity of the LDH isozyme. The fact that all oscillations are seen despite no attempt being made to synchronise the cell population suggests the existence of communication between cells but how this can occur when the rhythms are of such high frequency is intriguing. All the results add further support for the long standing view of my supervisor, that the properties and behaviour of cells reflect the internal dynamics and that differentiation, cancer and intracellular signalling occur through changes in the pattern of temporal organisation of cellular oscillations. / AC2018

Malaria is caused by Plasmodium parasites and is the world’s most devastating tropical infectious disease. The need for identifying novel drug targets is fuelled by an increased resistance of these parasites against available drugs. The human host red cell membrane plays an important role during invasion and subsequent development of the parasite within the red cell and undergoes several structural, functional and biochemical changes triggered by various protein-protein interactions between the parasite and the host cells. These interactions form a fundamental part of malaria research, since the parasite spends the pathogenic stage of its life cycle in the human erythrocyte. The Plasmodium kinome is complex and the exact role of protein phosphorylation in malaria parasites is not yet fully understood. This study aims to characterise the kinase domain of Plasmodium falciparum (3D7) Protein Kinase 8 (PfPK8), described as a putative protein on the Plasmodium falciparum database. PfPK8 is encoded by the PfB0150c gene (recently renamed as PF3D7_0203100) situated on chromosome 2 of the parasite genome. A 1 507bp section of the PfB0150c gene, containing a 822bp centrally located kinase domain was cloned into a pTriEx-3 expression vector. A soluble recombinant octa-histidine-tagged PfPK8 was expressed in Escherichia coli Rosetta 2 (DE3) cells, but with relatively low yield and purity.To improve the expression, a recombinant PfB0150c-baculovirus infected Spodoptera frugiperda (Sf9) insect cell system was attempted, but without success. A different tag was employed and glutathione-S-transferase-PfPK8 was successfully expressed in Escherichia coli Rosetta 2 (DE3) cells, with a higher yield and purity. Recombinant GST-PfPK8 was used in non-radioactive coupled spectrophotometric kinase assays in the presence of known kinase substrates casein, MBP and H1 to determine kinetic parameters of the enzyme. It phosphorylated all three substrates at a temperature of 37ºC and pH of 7.4. Recombinant GST-PfPK8 was inactive at a pH below 6 and most active at pH 7.4. The relative activity of the enzyme was highest at a temperature synonymous to a fever spike in a Plasmodium falciparum infected individual. Secondary structural analysis of PfPK8 revealed the position of a conserved substrate binding domain containing an ATP-binding site and binding loop within the kinase domain. The kinase domain of rPfPK8 was modelled using available crystal structures of its identified homologues. The gene is expressed throughout the intraerythrocytic stages of the parasite life cycle, as well as in gametocytes. Protein-protein binding studies revealed that host-parasite protein-protein interactions exist between rPfPK8 and erythrocyte membrane protein, band 3. Plasmodium falciparum PK8 could therefore play a role during invasion of host erythrocytes and during the intraerythrocytic development of the parasite, by phosphorylating red blood cell membrane proteins. This study provides the groundwork for future X-ray crystallographic studies to elucidate the structure of the enzyme, and for additional gene manipulation experiments to ascertain whether it is essential for parasite survival in all the intraerythrocytic stages and therefore a potential new drug target candidate.

A Dissertation Submitted to the Faculty of Science University of the Witwatersrand, Johannesburg In fulfilment of the requirements for the Degree of Doctor of Philosophy Johannesburg 1994 / Living cells are highly auto-dynamic entities which means that the underlying biochemistry is equally dynamic, a reality which is ignored by most researchers. Theoretical studies indicate that such a state must be due to the existence of oscillatory variations in the levels and activities of key components in the cell. In this study, the dynamic behaviour of four major, interrelated areas of cell biochemistry (phosphorylation, dephosphorylation, the terminal reaction of glycolysis and the amount of soluble protein) were examined and all systems found to oscillate in murine erythroleukaemic cells (MEL) and, where examined, also in the human HL-6Q leukaemic cell line. [Abbreviated Abstract. Open document to view full version] / MT2017