Southeast Asian Ovalocytosis (SAO) is an autosomal dominantly inherited, classically asymptomatic condition, that is widespread in Southeast Asian populations of Malasia, Indonesia, Papua New Guinea and the Philippines. Some regions have close to 30% prevalence and this is thought to be due to SAO providing partial protection against malaria. SAO is characterized by rigid, spoonshaped, ovalocytic red blood cells. The underlying defect is a deletion of 27bp in the band 3 gene, resulting in the absence of 9 amino acids (400-408) at the boundary of the cytoplasmic and membrane domains of band 3, causing abnormal structure and function. SAO is tightly linked in all cases to the band 3 Memphis 1 polymorphism, which is a lysine 56 (AAG) –glutamic acid (GAG) substitution. This polymorphism can be inherited independently and the prevalence ranges from about 6-30% according to various populations studied. The presence of SAO in a Cape Coloured family is a recent finding. The purpose of this study was to further investigate the prevalence of SAO and the band 3 Memphis 1 polymorphism in the Cape Coloured population. 20 unrelated individuals with SAO morphology were identified. DNA analysis revealed the 27bp deletion of exon 11 of the band 3 gene in all 20 subjects, which is diagnostic of SAO. This indicates a high occurrence of the SAO mutation in the Cape Coloured population, which is speculated to be due to a founder effect. Some of the clinical features differed from classically described SAO as some individuals showed evidence of haemolysis. Protein analysis showed all 20 individuals to have a reduced band 3 mobility, indicating the band 3 Memphis 1 polymorphism. Detecting the band 3 Memphis 1 polymorphism on a protein level is time consuming and labour intensive, therefore a PCR assay, which utilizes DNA, was developed for the rapid screening of this polymorphism. The PCR assay was based on a nucleotide mismatch which created a Taq 1 restriction site when combined with the band 3 Memphis 1 allele, but not with the wild type allele. Digestion of the PCR product with Taq 1 allowed differentiation between the two alleles. It was established that the band 3 Memphis 1 polymorphism has a high prevalence in the Cape Coloured population as it was detected in 108/326 (33%) of the individuals studied. Analysis indicated the Memphis allele is in Hardy-Weinberg equilibrium. / Prof. T.L. Coetzer Dr. H. Abrahamse

M.Sc. / Please refer to full text to view abstract

A procedure is described for the purification of human erythrocyte aldolase (EC 4.1.2.13). The process involves a specific substrate elution of the enzyme from phosphocellulose followed by a reverse ammonium sulfate fractionation.

Human red blood cell ghosts were prepared by a modification of the procedure of stepwise hemolysis (57). EDTA (1.0 mM) was included in the washing procedure to remove endogenous ATP and divalent cations. Ghosts resealed with appropriate amounts of ATP, calcium and magnesium were found to have Mg+Ca-ATPase activity and linearity was maintained up to thirty minutes. Active calcium transport could be studied in these ghosts by measuring the change ln the cellular concentration of calcium over time by atomic absorption spectrophotometry. Variation in the concentration of calcium in the loading medium resulted in an activation of Mg+Ca-ATPase and two peaks were evident on the activation curve. The high and low affinity Mg+Ca-ATPase were maximally stimulated at 0.25 and 5.0 mM calcium in the loading medium,respectively. The velocity of calcium transport was also found to be dependent on the concentration of calcium in the loading medium and was activated over the concentration range of 0.1 to 5.0 mM calcium. A change in the concentration of cellular calcium was not evident in the absence of added ATP. In contrast to the activation of Mg+Ca-ATPase two peaks were not obtained, and the activation curve had a sigmoidal appearance. Comparison of the calcium activation curves of Mg+Ca-ATPase and calcium transport revealed, a similarity in the shape and position of the low affinity part of the Kg+Ca-ATPase and calcium transport activation curves. A stoichiometry of two (Ca :ATP) was obtained, in the low affinity activity range. Ruthenium red (0.05 to 0.4 mM) selectively inhibited the low affinity Mg+Ca-ATPase and inhibited calcium transport over the same concentration range to a similar degree. Both low affinity Mg+Ca-ATPase and calcium transport were inhibited by external ruthenium red with an I₅₀ of 0.2 mM. Propranolol, qulnidine and quinine (10⁻⁵ to 10⁻³M) were found to be Ineffective in stimulating or Inhibiting Mg+Ca-ATPase when added to the Internal and external aspects of the ghosts. Manganese, added to the loading medium over a wide concentration range, was unable to substitute for calcium in activating Mg+Ca-ATPase. External divalent cations calcium and magnesium further increased Mg+Ca-ATPase activities when added to the external medium. Maximal stimulation occurred at a concentration of approximately 3.0 mM and calcium was almost twice as effective as magnesium. / Pharmaceutical Sciences, Faculty of / Graduate

Aaueous polymer two phase systems containing dextran T500, PEG 8000, and buffer are widely used to separate and analyse cells and other biological material based on the way they partition between the two phases and their interface. The behaviour of human erythrocytes in such two phase systems was studied in order to characterize some of the physico-chemical interactions important in determining cell partition. Two aspects were studied: the role of electrostatic and affinity ligand effects in determining the relative affinity of the cell for the two phases, and the relationship of this relative affinity to the cell partition. The potential difference produced by the unequal affinity of the buffer cations and anions for each phase was related to the salt partition by a thermodynamic model, which agreed with experimental results obtained in single and mixed salt systems. A thermodynamic theory for the effects of an affinity ligand on the cell surface free energy difference between the phases was derived, and found to agree quantitatively with experimental results using the affinity ligand PEG-palmitate. The change in cell surface free energy difference as a function of potential and ligand concentration was determined by contact angle measurements. This change was very small, based either on previous estimates of the surface charge density, or on the amount of PEG-palmitate bound to the cell surface as determined by adsorption experiments. This was attributed to partial exclusion of the phases from the cell glycocalyx. Cell partition into the upper PEG rich phase increased as this phase was made more positive with respect to the lower phase, or as the amount of an affinity ligand, PEG-palmitate, in the system was increased. Contact angle measurements were used to determine the energy of erythrocyte attachment to the interface between the two phases. The dependence of the cell partition on this parameter showed that thermal energies are far too small to partition cells in these systems. The cell partition was unaffected by the density difference between the phases. This and other results led to the hypothesis that droplet coalescence is the primary process by which large particles (>1 µm dia.) such as cells are distributed between the interface and one of the phases. / Science, Faculty of / Chemistry, Department of / Graduate

Bibliography: pages 110-126. / This thesis describes methodology for the reconstitution of the chicken erythrocyte octamer from acid-denatured histones or the natural H3-H4 tetramer and H2A-H2B dimers. Oligomeric properties of reconstituted octamers were elucidated during column chromatographic and chemical cross-linking studies. The conformational identity of the natural and reconstituted octamers was demonstrated by the ability of all preparations to crystallise as helical octamer tubes. The application of the reconstitution methodology in addressing fundamental problems of chromatin research, was demonstrated during subsequent studies, namely (i) The reconstitution of hybrid histone octamers containing a structural variant of a specific histone. These studies were undertaken to study the effect on histone-histone interactions in hybrid octamers of which erythrocyte H2B was substituted for by sea urchin sperm H2B(l) or erythrocyte H3 and H4 were substituted for by dethiolated H3 and sea urchin sperm H4 respectively. (ii) The reconstitution of an octamer suitable for the sitespecific derivatisation of a specific histone, or covalently labelled with aurothiomalate in a specific histone complex. These studies were concluded to represent general labelling strategies which may be of use in crystallographic or physico-chemical studies of nucleosome structure.

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