No description available.

The physiological significance of glutathione (GSH) in the mammalian central nervous system is still uncertain, although some evidence has indicated that GSH may play an important role in the CNS. To address the question of whether GSH may be a candidate for a neuropeptide in the CNS, one step is to establish that GSH receptors are present. In the present study, biotinyl-GSH was synthesized and purified to detect the GSH receptor in the CNS. Histochemical experiments showed that GSH binding sites appeared on the white matter ( such as cingulum, dorsal hippocampal commissure, cerebral peduncle, fasciculus retrbflexus, mammillothalamic tract etc.) of the rat brain. It thus suggested that the GSH receptors might be on astrocytes or oligodendrocytes. Radioactive receptor assays were performed on cultured astrocytocytes using [³⁵S]GSH. Scatchard analysis revealed two binding sites of K₁ = 4.67±0.75 nM, Bmax₂ =70±9.2 fmoles / 6.4 x10⁵ cells (or Bmax₁=6.6 x10⁴molecules / cell), Kd₂=35.14±2.1 nM, Bmax₂=260±12.77 fmole / 6.4 x10⁵ cell (or Bmax₂ = 2.4 x10⁵ molecules / cell). The association and dissociation kinetics studies gave a K₊₁ of 0.003nM⁻¹min¹, and a K₋₁ of 0.0168 min⁻¹for site I. These rate constants gave a K₁ of 5.6 nM, consistent with that from Scatchard analysis. Colloidal gold technique and immunofluorescence double staining also showed the GSH binding sites on cultured astrocytes, and suggested that the binding sites might be GSH receptors. The present study is the first to report the presence of GSH receptors on astrocytes. Based on receptor binding assays and cytochemical experiments, this study not only depicts the biochemical characteristics of GSH receptors in the brain, but also shows the receptor at the cellular level. These results support the view that GSH might be a neuroactively signal substance in the CNS. / Medicine, Faculty of / Graduate

The glutathione S-transferases (ligandins) are a ubiquitous system of xenobiotic metabolising enzymes. In the rat liver they comprise up to 10% of soluble hepatic protein. Studies in the rat suggested that ligandin was an accurate and sensitive marker of hepatocellular necrosis. and of renal tubular necrosis. The first part of this thesis examines the release of ligandin from liver and kidney in human liver and renal disease in an attempt to determine whether the measurement of ligandin is clinically useful. Ligandin was purified from human liver cytosol using a combination of anion exchange chromatography and gel filtration. The purified protein had similar physicochemical characteristics to ligandin purified by others. The protein was used to raise a monospecific antibody. Ligandin was iodinated by the Chloramine-T method. which yielded a labelled protein of high specific activity. A sensitive and specific radioimmunoassay for human ligandin was developed which had a low intra- and interassay variation. The assay was applied to the study of human liver disease. In acute hepatitis ligandin is released from the liver into serum early in the illness. High serum ligandin levels are seen in the first week of acute hepatitis. The rapid return to normal suggests that ligandin may provide an early indication of recovery. In chronic hepatitis ligandin levels correlated significantly with histological severity of disease. whereas SGOT showed no such correlation. Ligandin may be a better index of severity of disease and for treatment than SGOT. Ligandin was released from the kidney in severe renal ischaemia and in acute tubular necrosis, but was not a reliable predictor or indicator of acute tubular necrosis. Part two examines the distribution of GSH-T activity in organs and in hepatocellular carcinoma. Ligandin was shown to be immunologically similar in all tissues studied. Isoelectric focusing of cytosol separated the three groups of GSH-T activity. Considerable variety in the distribution and activity of GSH-T's was shown in different organs from a single donor, and in the same organs from different donors. Anionic transferase activity was shown to contribute a significant proportion of activity in organs other than the liver. and to be the major source of activity in ovary and lung. In hepatocellular carcinoma cationic GSH-T activity was present in amounts varying from near normal to absent. The anionic and neutral GSH-T's were present in amounts similar to that seen in normal liver. Immunohistochemical studies using a peroxidase-antiperoxidase method showed a rough correlation between tumour differentiation and the amount of ligandin in the tumour.

No description available.

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 May 2018 / Proteins exist in equilibrium between the native (N) and the denatured (D) states. In order to form the biologically active native state, the amino acid sequence has to fold to form a stable three-dimensional structure. The large scientific community of biochemists and biophysicists has not yet been able to gain a complete understanding of this process. In this study, the unfolding of the homodimeric detoxification enzyme hGST M1a-1a (WT dimer) was investigated. Additionally, an F56S/R81A double-mutant (mutant monomer) was engineered to create a monomeric form of the protein. The mutant monomer was used to gain a better understanding of the unfolding events occurring at the subunit level, in the absence of quaternary interactions. Data from various techniques indicate the mutant monomer to closely resemble the tertiary structure of the subunits in the WT homodimer, making it a suitable model to study the unfolding mechanism of hGST M1a in the absence of quaternary interactions. A four-state equilibrium unfolding mechanism, involving two stable intermediate species, is proposed. HDX-MS studies indicate that disruption of the conserved lock-and-key motif, as well as the structures surrounding the mu loop, results in a destabilisation of domain 1. However, dimer dissociation cannot occur until the mixed charge cluster at the dimer interface has been destabilised. The destabilisation of domain 1 results in destabilisation of α4 and α5 in domain 2, because the domains unfold in a concerted manner. hGST M1a-1a dissociates to form monomeric intermediate (M), with weak interdomain interactions and compromised short-range contacts. The unstable M intermediate self-associates to form an oligomeric intermediate (I). The destabilisation of α6 and α7 in the hydrophobic core of domain 2 drives the formation of the partially structured denatured state. Further investigation will need to be pursued to determine whether hGST M1a-1a unfolds via transient intermediate states; however, the elucidation of the equilibrium unfolding pathway of a complex homodimeric protein is a valuable addition to the ever-growing knowledge base of protein folding. / MT 2018

No description available.

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, 2019 / This thesis describes a study directed towards the synthesis and full characterization of novel [Pt(phen)(Ln-κS)2] complexes where disubstituted acylthioureato ligands (Ln), (also referred herein as N,N-di(alkyl/aryl)-N’-acylthioureato ligands), coordinate to the platinum(II) metal centre in a monodentate fashion through the sulfur donor atom and establishing the driving factors behind this rare coordination mode. The study included a systematically chosen ligand library where the ligands varied mainly on the electronic influence of acyl substituents balanced with minor steric variations on the amino end of the ligand. [Pt(phen)(Ln-κS)2] complexes were synthesized in excellent yields via a stepwise method where the ligands were first deprotonated using sodium hydride in anhydrous THF and subsequently reacting the resultant solvated salts with the [PtCl2(phen)] precursor under reflux for one hour in a 1:2.2 (precursor/ligand) stoichiometric ratio. The complexes were characterised using FT-IR (ATR) spectroscopy, 1H- and 13C NMR spectroscopy, high-resolution mass spectrometry, UV-vis spectrophotometry, elemental analysis, and single-crystal X-ray diffraction. The complexes were also probed for their solution conformations in correlation with the solid-state structures obtained through single-crystal X-ray diffraction using variable temperature 1H- and 195Pt NMR spectroscopy in order to gain insight into the persistence of intramolecular π-π stacking interactions in solution as a stabilising factor directing the monodentate coordination. This study showed that [Pt(phen)(Ln-κS)2] complexes with acylthioureato ligands bearing aromatic acyl substituents could at least exist in three conformations while complexes with acylthioureato ligands that have non-aromatic acyl substituents could only have one structural conformation at -50 ° C. However, there is no conclusive evidence of the intramolecular π-π stacking interactions in solution at ambient and higher temperatures. Investigations into whether the electronic effects of acyl substituents could influence the nucleophilicity of the other donor sites in the ligands, particularly oxygen, enough to render their reactivity towards platinum(II) ions more preferable was done using conceptual DFT. The study showed that the sulfur donor atom was tenfold more nucleophilic than any other donor atom in the acylthioureato ligands regardless of acyl substituents making it the most probable site to coordinate with platinum(II) ions. Mechanistic insights into how the reaction proceeded were probed through a series of individual experiments that involved the metal precursor, the ligands, the [Pt(phen)(Ln-κS)2] complexes, the byproduct [Pt(phen)(Ln-κO,S)]+ complexes and acids of different degrees of acidity. These experiments showed that the synthetic reaction proceeded most likely via solvolysis of the precursor resulting in a solvento complex and subsequent anation of the solvento complex with disubstituted acylthioureato anions to get [Pt(phen)(Ln-κS)2]. [Pt(phen)(Ln-κS)2] could also be obtained by reacting the cationic byproduct [Pt(phen)(Ln-κO,S)]+ with the ligand after deprotonation with base. It was also shown that the [Pt(phen)(Ln-κS)2] complexes dissociated under different acids resulting in different dissociation products via protonation-anation reactions. The nature of the dissociation products depended on the strength of the acid and the coordinating properties of the acids’ conjugate bases. This coordination mode could be extrapolated to include platinum(II) complexes bearing other co-ligands like bipyridine and triphenylphosphine, however in medium to low yields. A pilot study into the potential antiproliferative activity of [Pt(phen)(L1-κS)2] and [Pt(phen)(L2-κS)2] (where L1 = N,N-di(ethyl)-N’-(1-naphthoylthioureato) and L2 = N,N-di(butyl)-N’-(1naphthoylthioureato) anions) against an A549 lung cancer line showed that these complexes are very active with IC50 values of 6.4 ± 0.9 and 2.4 ± 0.3 μM respectively, killing cancer cells via apoptosis. This study also revealed that these complexes could potentially interact with DNA as a major groove binder. The knowledge obtained through this study should contribute to the fundamental understanding of the coordination chemistry of disubstituted acylthiourea ligands, chemical and physical properties of various platinum(II) complexes bearing disubstituted acylthiourea ligands coordinated in a monodentate fashion and explore possible applications in the fight against cancer. / TL (2020)

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vii, 138 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 123-134).

Thesis (Ph. D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 98 pages. Includes Vita. Includes bibliographical references.

The glutathione S-transferases are a group of enzymes which catalyse the conjugation of reduced glutathione with a variety of electrophilic molecules, and they are therefore thought to play a major role in drug biotransformation and the detoxification of xenobiotics. The cytosolic GSH S-transferase isoenzymes of rat, man and mouse have been assigned to three groups, Alpha, Mu and Pi, based on N-terrninal amino acid sequences, substrate specificities, immunological cross-reactivity and sensitivities to inhibitors. The kinetic mechanism of the GSH S-transferases is controversial, due to the observation of non-Michaelian (non-hyperbolic) substrate-rate saturation curves. The most detailed investigations of the steady-state kinetics of glutathione S-transferase have been performed with isoenzyme 3-3 (class Mu) and the substrate 1,2-dichloro-4-nitrobenzene (DCNB). Explanations for the apparently anomalous non-hyperbolic kinetics have included subunit cooperativity, steady-state mechanisms of differing degrees of complexity and the superimposition of either product inhibition or enzyme memory on these mechanisms. This study has confirmed the biphasic kinetics for isoenzyme 3-3 with DCNB and shown non-hyperbolic kinetics for this isoenzyme with 1-chloro-2,4-dinitrobenzene (CDNB) and for isoenzyme 3-4 with DCNB and CDNB. It is proposed that the basic steady-state random sequential Bi Bi mechanism is the simplest mechanism sufficient to explain the non-hyperbolic kinetics of GSH S-transferases 3-3 and 3-4 under initial rate conditions. Neither more complex steady-state mechanisms nor the superimposition of product inhibition or enzyme memory on the simplest steady-state mechanism are necessary.