Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiv, 133 p.; also includes graphics (some col.). Includes bibliographical references (p. 124-133).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-145).

In this study the purification of human basic and near-neutral liver, and human basic and acidic lung glutathione S-transferases (GSH S-T) was undertaken. Purification of the basic and near-neutral GSH S-T was achieved using a combination of affinity chromatography, chromatofocusing and immunoaffinity chromatography. Affinity and ion exchange chromatography were employed in the purification of the basic and acidic lung forms. The purified proteins had similar physicochemical characteristics to the GSH S-T purified by others. The binding of 1-chloro-2,4-dinitrobenzene (CDNB) to the 3 classes of human GSH S-T, viz. basic, near-neutral and acidic and the effects of such binding, if any, were examined. Human acidic lung GSH S-T is irreversibly inactivated by CDNB in the absence of the co-substrate glutathione (GSH). The time-dependent inactivation is pseudo-first order and demonstrates saturation kinetics, suggesting that inactivation occurs from an EI complex. GSH protects the enzyme against CDNB inactivation. In contrast, the basic and near-neutral GSH S-T are not significantly inactivated by CDNB. Incubation with [¹⁴C]-CDNB indicated covalent binding to all 3 classes of GSH S-T. When the basic and acidic GSH S-T were incubated with [¹⁴C]-CDNB and GSH, cleaved with cyanogen bromide, and chromatographed by HPLC, a single peptide fraction was found to be labelled in both classes. Incubation in the absence of GSH yielded 1 and 2 additional labelled peptide fractions for the basic and acidic transferases, respectively. These results suggest that while CDNB arylates all 3 classes of human GSH S-T, only the acidic GSH S-T possesses a specific GSH-sensitive CDNB binding site, which when occupied leads to time-dependent inactivation of the enzyme. The tissue distribution and localization of the 3 classes of human GSH S-T in normal and tumour tissue was examined. Antibodies to representatives of the 3 classes were raised in rabbits, and radial immunodiffusion employed to quantitate their concentrations in the cytosol of 18 organs from 9 individuals. The data provide the first direct, quantitative evidence for the inter-individual and inter-organ variation suggested by earlier workers. The absence of the near-neutral GSH S-T in 5 of the 9 individuals studied confirms an earlier suggestion of a "null" allele for this transferase. Basic and acidic GSH S-T (apart from in a single liver), were always present. Near-neutral GSH S-T, when present, were found in all tissues examined. The marked inter-organ and inter-individual variation observed in this study may explain individual and organ susceptibility to drugs, toxins and carcinogens. The immunohistochemical localization of the 3 classes of GSH S-T reveals important differences in their localization, and may provide insight into their functions in various organs and tissues.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2017. / Glutathione S-transferase A3-3 is the most catalytically efficient steroid isomerase enzyme known in humans, transforming Δ5-androstene-3-17-dione into Δ4-androstene-3-17-dione. Though its mechanism of action remains unsolved. GSTA3-3 catalyses this reaction with at least ten-fold greater efficiency than GSTA1-1, its closest competitor in the Alpha class of GSTs. In order to examine the differences between Alpha class GSTs and to better elucidate the mechanism of GSTA3-3 the roles of Tyr9 and Arg15 were examined. Tyr9 is the major catalytic residue of Alpha class GSTs and Arg15 is proposed to be catalytically important to GSTA3-3 but never before experimentally examined. While the structure and stability of the Alpha class enzymes are highly comparable, subtle differences at the G-site of the enzymes account for GSTA3-3 having a ten-fold greater affinity for the substrate GSH. Y9F and R15L mutations, singly or together, have no effect on the structure and stability of GSTA3-3 (the same effect they have on GSTA1-1) despite the R15L mutation removing an interdomain salt-bridge at the active site. Hydrogen-deuterium exchange mass spectrometry also revealed that neither mutation had a significant effect on the conformational dynamics of GSTA3-3. The R15L and Y9F mutations are equally important to the specific activity of the steroid isomerase reaction; however, Arg15 is more important for lowering the pKa of GSH. Lowering the pKa of GSH being how GSTs catalyse their reactions. This suggests an additional role for Tyr9, with an important mechanistic implication. Factoring in the inability to detect an intermediate during the reaction, all data are in agreement with the mechanism being concerted and that Tyr9 acts as a proton shuttle. Additionally, there is evidence to suggest that Arg15 is integral to allowing GSTA3-3 to differentiate between Δ5-androstene-3-17-dione and Δ4-androstene-3-17-dione, indicating that Arg15 is a more important active-site residue than previously recognized. / LG2018

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 143-154).

In rodent models as well as in fish models there are significant species differences in the susceptibility toward aflatoxin B₁ (AFB₁) carcinogenesis. Mouse is less susceptible toward AFB₁ carcinogenesis than rat. Researchers have come to the conclusion that the lower susceptibility of mice is not a result of less effective activation of AFB₁, but rather of more effective inactivation of the toxic intermediate AFB₁-2,3-epoxide, and especially inactivation through the glutathione (GSH) conjugation of the epoxide. Rainbow trout fed Oregon Test Diet are more sensitive toward AFB₁ hepatocarcinogenesis than coho salmon, or, than trout fed the inhibitors β-naphthoflavone (BNF), 1ndole-3-carbinol (I3C), or Aroclor 1254 (PCB). This study examined the role of AFB1-glutathione (AFB₁-SG) conjugation In these differences. A tritiated AFB₁-glutathione conjugate standard (³H-AFB₁-S6) was produced in vitro using mouse liver S-9 fraction as a source of GSH transferase. It was purified by reverse phase HPLC, and its structure verified by amino acid analysis and mass spectrometry. Coho salmon and rainbow trout fed the various diets were injected i.p. with ³H-AFB₁ (49μCi, 50 μg/ kg fish); bile, liver and kidney were collected at 24 h. Recovery of total aflatoxin radioactivity was determined for all three tissues, and the hepatic AFB₁-DNA binding was also determined. Bile metabolites were quantitated by reverse phase HPLC using the mouse ³H-AFB₁-SG as a standard. The resistance of coho salmon toward AFB₁ carcinogenicity was supported by a 20-fold lower hepatic AFB₁-DNA binding compared to control trout. AFB₁-SG was detected in bile only in control, BNF, and I3C fed trout, at < 1% of total recovered metabolites, and at < 0.2% of the original dose, being highest in control trout. The major conjugates were glucuronides of aflatoxicol (AFL) and aflatoxicol-M₁ (AFL-M₁) (80-902 of the total recovered metabolites). In vitro metabolism studies using isolated liver cell fractions supported the in vivo metabolism results. Less than 0.531 of the original AFB₁ dose was converted to AFB₁-SG conjugate in salmon and trout samples. In contrast, with isolated mouse liver cell fractions approximately 25% of the original AFB₁ dose was conjugated with GSH. The GSH concentration of control trout liver was 2.9 mmol/ kg. Coho salmon had GSH concentration 70% of that found in control trout. In BNF pre-fed trout liver GSH concentration was enhanced by 25% compared to controls. Liver GSH transferase activity using l-chloro-2,4- dlnitrobenzene as substrate was 1.15 μmol/min/mg protein in control trout. This enzyme activity In salmon was only 26% of that found in control trout. A 62% elevation In GSH transferase activity compared to controls was detected in trout fed BNF diet. There is no apparent correlation between liver GSH or GSH transferase activities among the various groups, and their relative sensitivities to AFB₁ carcinogenesis. This study indicates that AFB₁-SG conjugation is not a significant pathway in salmon and trout fed control diets, or trout fed various inhibitors, and cannot account for the variation In AFB₁ sensitivity. / Graduation date: 1986

A dissertation submitted in fulfilment of the requirements for the degree of Master of Science at the University of the Witwatersrand. Johannesburg, October 1996. / Glutathione S-transferase (GST) class Pi has two tryptophan residues which are conserved within domain one. Trp38 plays a functional role in sequestering glutathione at the active site, whereas Trp28 plays a structural role. The effects of the sterically-conservative substitution of Trp28 to Phe were investigated. When the W28F mutant was compared with the wild-type enzyme, mutation of Ttp28 to Phe was not well tolerated and resulted in a dimeric protein with impaired catalytic function and conformational stability. [Abbreviated Abstract. Open document to view full version] / AC2017

Domain interfaces are important to the folding, stability, structure and function of multidomain proteins. In the case of human glutathione S-transferase A1-1 (hGSTA1-1) site-directed mutagenesis studies have previously implicated the interdomain Arg13 residue of the protein in maintaining the proper catalytic function of the GST though its exact role was never determined (Stenberg et al., 1991). In this study it was shown by structural and sequence alignment of many representatives of the GST family and other thioredoxin-fold containing proteins that Arg13 is also highly conserved throughout the Alpha, Mu, Pi, Plasmodium falciparum and Sigma classes, all of which are Y-GSTs, and that it forms an interdomain salt bridge. This study therefore chose to evaluate the contribution of Arg13 towards the structure, stability and function of hGSTA1-1 by mutating the Arg residue to an Ala and performing comparative studies between wild-type and R13A hGSTA1-1. The spectral properties of R13A hGSTA1-1 monitored using far-ultraviolet circular dichroism and fluorescence indicated no significant changes in the secondary structure as compared to the native protein though fluorescence did indicate local tertiary structural changes around Trp21. Additionally, the catalytic activity of the R13A variant was reduced by 70% as compared to that of the wild-type enzyme further indicating local tertiary structural changes at and possibly near the active site which is located near the Trp21 residue. Conformational stability studies were performed by monitoring both thermal- and chemical-induced protein unfolding. The stability of the R13A variant was lower than that of the wild-type protein as revealed by a thermal-induced unfolding study which indicated that the melting point (Tm) of the R13A variant was 6 °C lower than that of the wild-type. Thermal-induced unfolding was shown not to be reversible however and the thermodynamic parameters of unfolding could not be determined. Urea-induced equilibrium unfolding studies on the other hand were reversible and displayed a variant-induced destabilisation of the conformation of the protein with a ΔΔG(H2O) of 16.7 kJ.mol−1 between the mutant and native protein. Additionally urea-induced equilibrium unfolding studies in the presence of ANS indicated that the equilibrium unfolding of both wild-type and R13A hGSTA1-1 was three-state. In summary the Arg13 residue is more important to the function of the protein than it is for its global stability or structure. Also since the Arg13 residue was found to be highly conserved in all the Y-GSTs and that it forms an interdomain interaction, the residue most likely performs a similar role in each of the Y-GSTs as well.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy. / The thermodynamic stability and the properties of the unfolding/refolding pathways of homodimeric human glutathione transferase A1-1 (hGST A1-1) were investigated. The conformational stability, assessed by urea- and temperature-induced denaturation studies, was consistent with a folded dimer/unfolded monomer transition with no stable intermediates. The high energy of stabilisation and the highly co-operative transition implies that the subunit-subunit interactions are necessary to maintain the three-dimensional state of the individual subunits. The stopped-flow-unfolding pathway, monitored using Trp fluorescence, was biphasic with a fast and slow unfolding event. Urea-dependence and thermodynamic activation parameters suggest that the transition state for each phase is well structured and is closely related to the native protein in term., of solvent exposure. The unfolding pathways monitored by energy transfer or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding phase (described above). A two-step sequential unfolding mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed. The crystal structures of all cytosolic glutathione transferases show that the alpha helices 5, 6 and 7 pack tightly against each other to form the hydrophobic core of' domain II. Leu164 in class alpha glutathione transferase is a topologically conserved residue in the alpha helix 6. The replacement ofLeu164 with alanine did not impact on the functional or gross structural properties of hGST A1-1. The urea-induced equilibrium and kinetic unfolding pathways were similar to those observed for the wild-type protein. The free energy change of unfolding was equivalent to the energetic cost of deleting three methylene groups. Furthermore, the decreased co-operativity of the unfolding transition is consistent with a decrease in co-operativity of the forces that maintain the native state of hGST A1-1. The biphasic kinetic unfolding pathway indicated that the fast phase was destabilised to a greater extent than the slow unfolding phase. ( Abbreviations abstract) / Andrew Chakane 2019

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy