3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Ferro- kinetic studies in a variety of haematological disorders, acute porphyria and scurvy(1960) Kramer, SydneySince changes occur in size, shape and haemoglobin content of red cells in disease a classification of the anaemias based on the morphology of the red cell has been widely used ( Wintrobe, 1956 ) . While such classification has a limited usefulness from the diagnostic and therapeutic approach it has two serious defects..Item Human red cell NADP-dependent xylitol dehydrogenase: kinetic and genetic studies(1984) Lane, Anthony BruceA deficiency of the enzyme NADP dependent xylitol dehydrogenase (L-xylulose reductase) has previously been found to be the cause of chronic essential pentosuria. Essential pentosuria is a recessively inherited condition which is marked by the continual excretion of relatively large amounts of the enzymes substrate, L-xylulose. The major objective of the study described was to find a simple method for the identification of individuals who are heterozygous for the "pentosuria" and normal alleles. The pentosuria allele could then be used as a gene marker in linkage studies aimed at mapping the L-xylulose reductase locus. A L-xylulose reductase assay suitable for the identification of carriers of essential pentosuria was developed and tested on members of a South African Lebanese family in which the inheritance of pentosuria had previously been suggested to be dominant. It was found that family members could, on the basis of their L-xylulose reductase activities, be classified as either normal, heterozygous or homozygous for the pentosuria allele. Measurements of serum L-xylulose concentrations revealed that pentosuria is, contrary to the previous report, . recessively inherited in this family. A sample of the local Ashkenazi Jewish population was screened for pentosuria carriers. Six out of the 237 individuals screened were found (on the basis of their L-xylulose reductase activities and from the results of a loading test), to carry the pentosuria allele. The frequency of the pentosuria allele in this population was estimated from the apparent heterozygote frequency to be 0.0127. Linkage analyses were carried out on the families of the identified heterozygotes and on members of the Lebanese family mentioned above. No evidence of tight linkage was found between the pentosuria allele's locus and those coding for various red cell antigens, red cell enzymes and serum proteins. Kinetic, chromatographic and electrophoretic studies revealed that the red cells of normal individuals contain two distinct L-xylulose reductases, a minor and a major isozyme. Pentosurics lack the major isozyme but appear to have approximately normal amounts of the minor isozyme. The minor isozyme is e1ectrophoretica 1 1 y distinct from the major isozyme, has markedly higher Michael is constants for the substrates L-xylulose and xylitol and shows a lower pH optimum when catalysing the oxidation of xylitol. Electrophoresis also revealed that liver tissue contains two L-xylulose reductases which occur in similar proportions to those of red cells but which migrate at slightly different rates.Item Kinetic and thermodynamic characterization of the South African subtype C HIV-1 protease : implications for drug resistance(2008-03-27T07:11:42Z) Mosebi, SalerweABSTRACT The magnitude of the AIDS epidemic is well documented. It has been shown that Africa constitutes about 70 % of people infected with HIV worldwide. Efforts to control the AIDS epidemic have focused heavily on studies pertaining to the biology, biochemistry and structural biology of HIV and on the interactions between HIV proteins and new drugs. One of the most challenging problems in AIDS therapy is that HIV develops drug-resistant variants rapidly. Extensive research has been dedicated to designing resistance-evading drugs for HIV-1 protease (predominantly subtype B), which is crucial for the maturation of viral, structural and enzymatic proteins. There are 10 subtypes of HIV-1 within the major group of the virus, with subtype C accounting for about 95 % of infections in South Africa. Since HIV-1 antiretroviral treatment has been developed and tested against the B subtype, which is prevalent in North America, Western Europe and Australia, an important question relates to the effectiveness of these drugs against the C subtype. At this point, however, little is known about inhibitor-resistant mutations in the subtype C. The study, therefore, looked at the two active site mutations (V82A and V82F/I84V) in the South African HIV-1 subtype C protease (C-SA) emerging from the viral population circulating in patients. These mutations are well-characterized within the framework of the subtype B and are known to cause cross-resistance to most of inhibitors currently in clinical use. Protein engineering techniques were used to generate the V82A and the V82F/I84V variants. Comparative studies with the wild-type HIV-1 C-SA protease were performed. The spectral properties of the V82A and the V82F/I84V variants indicated no changes in the secondary structure in the respective variant proteins. Tryptophan and tyrosine fluorescence indicated a major difference in the intensities at the emission maxima for all three proteins. The fluorescence intensity of the V82F/I84V variant, in particular, was significantly enhanced indicating the occurrence of tertiary structural changes at/near the flap region. Both mutations did not impact significantly upon catalytic function. Both variants also had the same Km values comparable to that of the wild-type enzyme. The catalytic efficiencies and the kinetic constants were lowered 3.6-fold for the V82A mutation and 6-fold for the V82F/I84V mutation relative to the wild-type C-SA protease. Inhibition studies were performed using four inhibitors in clinical use (saquinavir, ritonavir, indinavir and nelfinavir). For the V82A variant, IC50 and Ki values for saquinavir and nelfinavir iv were not affected, whilst those for ritonavir and indinavir were 5- and 9-fold higher than the wild-type C-SA protease, respectively. Against the V82F/I84V variant, however, the inhibition constants were drastically weaker and characterized by IC50 and Ki ratios ranging from 50 to 450. Isothermal titration calorimetry (ITC) was also used to determine the binding energetics of saquinavir, ritonavir, indinavir and nelfinavir to the wild-type C-SA, V82A and V82F/I84V HIV-1 protease. The V82A mutation lowered the Gibbs energy of binding for the respective four clinical inhibitors by 0.4 kcal/mol, 1.3 kcal/mol, 1.5 kcal/mol and 0.6 kcal/mol, respectively, relative to the wild-type C-SA HIV-1 protease. The affinity of V82A HIV-1 protease for saquinavir, ritonavir, indinavir and nelfinavir (Kd = 1.85 nM, 2.00 nM, 12.70 nM and 0.66 nM, respectively, at 25 °C) was in the range of 2- to 13-fold of magnitude weaker than that of the wild-type C-SA protein. The clinical inhibitors exhibited the highest binding affinity to both the wild-type and the V82A enzymes, but were extremely sensitive to the V82F/I84V mutation. The V82F/I84V mutant reduced the binding of saquinavir, ritonavir, indinavir and nelfinavir 117-, 1095-, 474- and 367- fold, respectively. A drop in Kd values obtained for the V82F/I84V in association with saquinavir, ritonavir, indinavir and nelfinavir was consistent with a decrease of between 2.8 - 4.2 kcal/mol in ΔG, which is equivalent to at least 2 to 3 orders of magnitude in binding affinity. Taken together, thermodynamic data indicated that the V82A and V82F/I84V active site mutations in the C-SA subtype lower the affinity of the first-generation inhibitors by making the binding entropy less positive (unfavorable) and making the enthalpy change slightly less favorable.