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1
Corrigall, Anne Vint. "Human glutathione S-transferases : characterization, tissue distribution and kinetic studies." Doctoral thesis, University of Cape Town, 1988. http://hdl.handle.net/11427/27205.
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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.
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Goold, Richard David. "The glutathione S-transferases : kinetics, binding and inhibition." Doctoral thesis, University of Cape Town, 1989. http://hdl.handle.net/11427/27175.
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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.
3
Mosi, Renée M. "Mechanistic studies on Ã-glycosyl transferases." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ34594.pdf.
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Dixon, David Peter. "Glutathione transferases in maize (Zea mays)." Thesis, Durham University, 1998. http://etheses.dur.ac.uk/4788/.
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The glutathione transferases (GSTs) of maize have been the most studied GSTs in plants, however much is still not known about these enzymes. In the course of the current study six GST subunits (Zm GSTs I, II and III, which have been reported previously, and Zm GSTs V, VI and VII, which have not been previously reported) have been identified in the dimers Zm GST I-I, I-II, I-III, V-V, V-VI and V-VII. Maize GSTs are known to be important in herbicide detoxification and the purified maize enzymes were each found to have differing activities toward a number of herbicides, and also a range of other potential GST substrates. Additionally, Zm GST I II and Zm GST V-V possessed glutathione peroxidase activity. The developmental regulation and chemical inducibility of maize GSTs were studied in maize seedlings using western blotting, with different subunits showing markedly different responses. Zm GST I was constitutively present in all plant parts and unaffected by chemical treatment, Zm GST II was only detected in young roots but was induced in roots and shoots by many different chemical treatments, and Zm GST V was present at low levels throughout maize plants, with levels enhanced greatly by treatment with the safener dichlormid but not by other chemicals tested. cDNA clones corresponding to Zm GST subunits I, III, V, VI and VII were isolated by library screening using antibody or DNA probes. The cDNA sequences for Zm GST subunits V, VI and VH were different from those of previously cloned type I (theta class) maize GSTs and were most similar to the auxin-regulated GST family (type III or tau class GSTs) previously only identified in dicotyledonous species. The cloned GSTs were expressed as recombinant proteins in E. coli, allowing further characterisation, including detailed kinetic analysis for recombinant Zm GST I-I and Zm GST V-V.
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Barrozo, Alexandre. "Promiscuity and Selectivity in Phosphoryl Transferases." Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-279693.
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Phosphoryl transfers are essential chemical reactions in key life processes, including energy production, signal transduction and protein synthesis. They are known for having extremely low reaction rates in aqueous solution, reaching the scale of millions of years. In order to make life possible, enzymes that catalyse phosphoryl transfer, phosphoryl transferases, have evolved to be tremendously proficient catalysts, increasing reaction rates to the millisecond timescale. Due to the nature of the electronic structure of phosphorus atoms, understanding how hydrolysis of phosphate esters occurs is a complex task. Experimental studies on the hydrolysis of phosphate monoesters with acidic leaving groups suggest a concerted mechanism with a loose, metaphosphate-like transition state. Theoretical studies have suggested two possible concerted pathways, either with loose or tight transition state geometries, plus the possibility of a stepwise mechanism with the formation of a phosphorane intermediate. Different pathways were shown to be energetically preferable depending on the acidity of the leaving group. Here we performed computational studies to revisit how this mechanistic shift occurs along a series of aryl phosphate monoesters, suggesting possible factors leading to such change. The fact that distinct pathways can occur in solution could mean that the same is possible for an enzyme active site. We performed simulations on the catalytic activity of β-phosphoglucomutase, suggesting that it is possible for two mechanisms to occur at the same time for the phosphoryl transfer. Curiously, several phosphoryl transferases were shown to be able to catalyse not only phosphate ester hydrolysis, but also the cleavage of other compounds. We modeled the catalytic mechanism of two highly promiscuous members of the alkaline phosphatase superfamily. Our model reproduces key experimental observables and shows that these enzymes are electrostatically flexible, employing the same set of residues to enhance the rates of different reactions, with different electrostatic contributions per residue.
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Hayes, Peter C. "Glutathione S-transferases in the pancreas." Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/19832.
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Glutathione S-transferase (GST) is an important xenobiotic metabolising enzyme which has been extensively studied in the liver. In the first part of this study immunohistochemistry was used to identify the presence and histological localisation of different GST isoenzymes in various gastrointestinal tract tissues in the human in health and disease. GSTP was found throughout the gastrointestinal and biliary tract whilst the position and quantity of other isoenzymes varied locally. Increased expression of GSTP was observed in cholangiocarcinoma and colonic adenocarcinoma, but not hepatocellular carcinoma. In the pancreas GSTP was present in ductal and centroacinar cells, whilst GSTA was present in acinar cells. GSTM was universally present in the cells of islets of Langerhan, not demonstrating genetic polymorphism. In both chronic pancreatitis and pancreatic carcinoma increased expression of GSTP was demonstrated. Using affinity chromatography and high performance liquid chromatography GSTA, P and M were purified from human pancreatic tissue. A novel GST isoenzyme, which ran on SDS/PAGE, similar to GSTP, was identified, purified and confirmed by Western blot analysis to be a GSTA. Feeding rats exclusively on raw soya flour resulted in pancreatic hypertrophy and eventually carcinoma. Serial measurements of GST activity showed only a minor reduction with short term feeding which returned to normal with chronic administration contrary to what has been proposed (Ross & Barrowman, 1987). No selective change in GST isoenzymes was identified. A dominant cytoplasmic protein, shown both enzymatically and by Western blot analysis to be α-amylase fell dramatically with short term administration recovering only marginally with chronic administration.
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DAWOOD, KUTAYBA F. "New physiological roles of glautathione transferases." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/923.
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Glutathione transferases (GSTs) are enzymes able to conjugate GSH to a lot of toxic compounds thereby favoring their excretion. Recently, other protective roles of these enzymes have been discovered. In particular, it has been observed that a peculiar and strong interaction exists between some mammalian GSTs and an endogenous carrier of nitric oxide, the dinitrosyl-diglutathionyl iron complex (DNDGIC). This iron complex is a paramagnetic molecule with a characteristic EPR spectrum centered at g = 2.03, that is spontaneously formed when NO enters the cell. This complex is a strong irreversible inhibitor of glutathione reductase. The present work explores the possible role of GSTs like a protection system against DNDGIC. Actually, mammalian GSTs bind DNDGIC with extraordinary affinity (KD = 10-9-10-10 M). When rat hepatocytes are incubated in the presence of GSNO, a natural source of NO, a rapid formation of 0.1 - 0.2 mM intracellular DNDGIC has been observed. This concentration would be lethal for glutathione reductase. However the complex does not appear like a free species but completely bound to GSTs, that are present at the cytosolic level of 0.8 mM. In this form the complex is completely harmless for glutathione reductase.
Surprisingly, electron paramagnetic data, reveal that DNGIC-GST is partially associated to subcellular fractions and in particular to nuclei. Our data indicate that about 10% of the cytosolic pool GST is electrostatically associated with the outer nuclear membrane, and a similar quantity is compartmentalized inside the nucleus. Mainly Alpha class GSTs, in particular GSTA1-1, GSTA2-2 and GSTA3-3, are involved in this double modality of interaction. Confocal microscopy and immunofluorescence experiments have been used to detail the electrostatic association in hepatocytes. A quantitative analysis of the membrane-bound Alpha GSTs suggests the existence of a multilayer assembly of these enzymes at the outer nuclear envelope that could represent a potent protection shell for the nucleus and an amazing novelty in cell physiology.
A second target of this study is represented by the particular GST isoenzyme expressed by the Plasmodium falciparum (PfGST), the parasite causative of malaria. This enzyme is characterized by a peculiar dimer/tetramer transition that occurs in the absence of GSH and that causes a total loss of its enzymatic activity. Moreover PfGST binds hemin with high affinity and this interaction is finalized to the protection of the parasite against this toxic compound. Binding of hemin is regulated by a cooperative mechanism and does not occur in the tetrameric enzyme. Side directed mutagenesis, steady-state kinetic experiments, fluorescence anisotropy and X-ray crystallography were used to verify the involvement of some protein segment in the tetramerization process and in the cooperative phenomenon. Actually the loop 113-118 represents one the most prominent structural difference between PfGST and other GSTs. Our results demonstrate that truncation, increased rigidity or even a simple point mutation of this loop cause a dramatic change of the tetramerization kinetics that becomes hundred times slower than that observed in the native enzyme. Furthermore all mutants loose the positive cooperativity for hemin binding found in the native structure suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process, that is very fast in the absence of GSH in the native enzyme, is prevented not only by GSH but even by GSSG. This result indicate that the protection of the parasite against free hemin is independent of the redox status of the cell.
8
Dolan, Catherine. "Regulation of mouse hepatic glutathione S-transferases." Thesis, University of Edinburgh, 1991. http://hdl.handle.net/1842/23855.
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The glutathione S-transferases (GST) are a multi-gene family of dimeric proteins which catalyse the conjugation of glutathione to a wide range of electrophilic compounds. Three classes of mouse cytosolic GST have been isolated, alpha, mu and pi, comprising Ya-, Yb- and Yf-type subunits respectively. A marked sexual dimorphism in mouse hepatic GST has been observed. The YfYf GST is the most abundant form in the male, constituting approximately 70% of total hepatic GST content. By contrast, the Yf subunit represents only a minor form in the livers of female mice. The hormonal controls which regulate the expression of the YfYf GST in mouse liver have been investigated. Testosterone, the major male sex hormone, is found to regulate the levels of Yf in mouse liver. Castration of the male leads to a decline in the levels of Yf to that observed in females. Replacement therapy with testosterone partially restores the levels of Yf. Testosterone treatment induces expression of this subunit in the female. Growth hormone secretion from the pituitary gland differs markedly between the sexes. Androgens act to produce the male pattern of growth hormone secretion which regulates the sex-specific expression of numerous hepatic proteins. Male 'little mice', specifically defective in the production of growth hormone, exhibit a feminine pattern of GST expression, despite having normal levels of testosterone. Testosterone treatment has no effect on the expression of YfYf in little mice. In contrast, growth hormone replacement therapy, administered to simulate the male-specific pattern causes an increase in the expression of the Yf subunit. These findings strongly suggest that testosterone regulates the hepatic expression of the Yf subunit indirectly through the male-specific pattern of growth hormone secretion. The effects of the xenobiotics, phenobarbital, dexamethasone and 1,4-Bis[2-(3,5-dichloropyridyloxy)]-benzene (TCBOP) on mouse hepatic GST content have been investigated in two strains of mice, C57BL/6 and DBA/2. All three compounds were found to induce hepatic GST in both strains and sexes, predominately affecting expression of members of the mu class. TCBOP was the most potent inducer. Hypophysectomy did not significantly affect induction of GST by these compounds.
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Meikle, Ian. "Glutathione S-transferases in the adrenal cortex." Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/19139.
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Data available prior to this thesis had shown that, of all bovine organs examined, the adrenal cortex exhibited the second highest level of glutathione S-transferase (GST) expression behind the liver. This finding, along with increasing evidence implicating the importance of GST in endogenous detoxification processes, formed the basis for a further extensive investigation of the GST isoenzymes expressed by the adrenal cortex. Investigation of the GST isoenzymes expressed by a number of different bovine organs using affinity chromatography on S-hexylglutathione-Sepharose 6B (S-hexG-Ag) revealed a marked organ-specific distribution of these enzymes. Bovine adrenal cortex, in particular, expressed isoenzymes from each GST class, as determined by immunoblotting experiments. GST activity determinations of these enzyme pools using a number of model substrates revealed the bovine enzymes to possess a specificity distinct to that of rat and human GST. Isoelectric focusing of the bovine adrenal cortex isoenzymes showed them to possess pl values similar to those found in other species. The affinity-purified mu- and pi-glass isoenzymes were resolved using anion-exchange chromatography, followed by reverse-phase hplc. Using this approach, at least 3 mu-class GST subunits and 1 pi-class GST subunit were identified. Ion-exchange chromatography failed to resolve the affinity-purified alpha-class GSTs, and reverse-phase hplc analysis resolved 2 polypeptides, designated Ya1 and Ya3 respectively. Analysis of the protein that failed to bind to the S-hexG-Ag column revealed that about 35% of GST activity remained in this fraction. Application of this material to glutathione-Sepharose 6B (GSH-Ag) resulted in the purification of an abundant alpha-class GST (1.3% total cytosolic protein). This GST was found to exhibit marked peroxidase and Δ5-ketosteroid isomerase activities, in addition to high activity with 4-hydroxynonenal. SDS/PAGE analysis revealed 2 distinct polypeptides of Mr 25900 and 26500, the former being equivalent to the Ya3 subunit purified on S-hexG-Ag, and the latter named Ya2. Ion-exchange chromatography of the GSH-Ag purified alpha-class GST isoenzyme pool resulted in a complex picture, suggesting there to be at least 3 distinct subunits in this pool.
10
Hill, Alison Elspeth. "Regulation of glutathione S-transferases during stress." Thesis, University of Edinburgh, 1994. http://hdl.handle.net/1842/19846.
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Elevation of glutathione S-transferases (GST) in tumour cells can be responsible for resistance to a variety of chemotherapeutic agents. It has been hypothesised that GSTs may be induced as part of a stress response, similar to that of the prokaryotic adaptive response. To further this investigation I studied the induction of GSTs in a variety of permanent and transient stress models. A chlorambucil resistant CHO cell line which was known to express increased levels of Alpha-class GST was studied to determine the nature of the increase in protein. Northern and Southern blot analysis revealed a 4-8 fold amplification in the DNA encoding the Alpha-class GSTs with an accompanying increase in mRNA levels. Elevated levels of an Alpha-class GST were noted in oxygen resistant CHO cells. Transient exposure to 98% oxygen also induced the same Alpha-class GST. A heat shocked lung tumour cell line as well as heat selected sublines showed some changes in the levels of Pi- and Mu-class GSTs. A novel putative Mu-class GST subunit has been identified in the nucleus of heat shocked cells. The nature of the GST level variations at the RNA and DNA levels were studied. These studies do not suggest co-ordinate regulation of the GSTs as part of a general stress response. It does not exclude the possibility of GST π and perhaps the nuclear Mu-class GST are induced as part of a more limited response either to heat or in certain tissues. Inconsistencies in the data from the preliminary induction experiments led to the investigation of the effect of growth conditions on GST levels. Unexpectedly isoenzymes from three classes of GST were found to be elevated by increased confluence and a low frequency of feeding. This response was found to be mediated through the culture media.
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Entova, Sonya. "Structure-function relationships in monotopic phosphoglycosyl transferases." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/122206.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019Cataloged from PDF version of thesis.
Includes bibliographical references.
Complex glycans play essential roles in prokaryotic and eukaryotic biology. While this ubiquitous post-translational modification takes a diversity of forms, many glycoconjugate biosynthesis pathway across domains of life follows a common logic. Glycan assembly is initiated by a phosphoglycosyl transferase (PGT) that transfers a phosphosugar from a nucleotide donor to a polyprenol phosphate (PrenP) chain embedded in the membrane. The PrenPP-sugar product is elaborated by downstream glycosyltransferases, transferred across the membrane and ultimately appended to various acceptor molecules. The PGTs initiating glycan assembly adopt diverse membrane architectures. An extensive superfamily of PGTs, elucidated in part by this thesis, is exemplified by PglC from the Gramnegative pathogen, Campylobacterjejuni. PglC comprises a globular cytosolic domain and an N-terminal membrane-resident domain.
Recent structural and biochemical analyses determined that this domain forms a helix-break-helix motif, termed the reentrant membrane helix (RMH), that enters and exits on the same face of the membrane, resulting in a monotopic topology. The RMH anchors the PglC fold in the membrane in a manner not previously observed among other monotopic membrane proteins. This thesis focuses on structure-function relationships in the RMH and associated domains. Two conserved motifs are shown to drive formation of a reentrant topology for PglC, and to exemplify common principles of topology determination among diverse monotopic proteins. These principles are further applied to the identification of reentrant domains in an extensive superfamily of monotopic lipid A acyltransferases previously thought to be membrane-spanning. The next section of the thesis explores the highly conserved role of PrenP in complex glycan biosynthesis.
The significance of PrenP geometry in mediating substrate binding and modulating the local membrane environment is presented. Additionally, a conserved proline residue in the PglC RMH is determined to drive PrenP binding and specificity. Molecular insights from this study shed new light on the roles of PrenP in facilitating diverse glycoconjugate biosynthesis pathways. Finally, a cell-free methodology for expression of PglC directly into model membrane lipid Nanodiscs is described. This system has valuable applications for the study of interactions between PglC and downstream glycosyltransferase enzymes, and for further structural characterization of PglC in a membrane environment.
by Sonya Entova.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
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Thumser, Alfred Ernst Adolf. "The glutathione S-transferases : inhibition, activation, binding and kinetics." Thesis, University of Cape Town, 1990. http://hdl.handle.net/11427/28958.
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Eklund, Birgitta I. "Role of Multiple Glutathione Transferases in Bioactivation of Thiopurine Prodrugs : Studies of Human Soluble Glutathione Transferases from Alpha, Kappa, Mu, Omega, Pi, Theta, and Zeta Classes." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7102.
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Smith, Lisa Stievater. "Factors influencing glutathione S-transferases in (Daphnia magna)." College Park, Md. : University of Maryland, 2006. http://hdl.handle.net/1903/3801.
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Thesis (M.S.) -- University of Maryland, College Park, 2006.Thesis research directed by: Marine, Estuarine, Environmental Sciences Graduate Program. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Howie, Alexander Forbes. "Measurement of human glutathione S-transferases by radioimmunoassay." Thesis, University of Edinburgh, 1990. http://hdl.handle.net/1842/24005.
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A purification scheme was devised for human glutathione S-transferase (GST) pi that resulted in a high yield of homogeneous protein from human placenta, lung and erythrocytes. Polyclonal antisera against GST pi from lift lung and placenta was raised in eight rabbits. The GST pi proteins purified from placenta, lung and erythrocytes were found to have identical isoelec-tric points (pl= 4.8), subunit molecular weights (Mr= 24 800) and were found to be immunologically identical. The antibody demonstrating the best specificity and sensitivity was employed to develop a radioimmunoas-say (RIA) for the measurement of GST pi. Using the RIA,GST pi concentrations were measured in the plasma of patients diagnosed as having cancer of the bronchus. Concentration of the enzyme were significantly higher (P< 0.01) than those measured in a control group of patients with respiratory disorders other than malignancy. To prevent spuriously high results for GST pi concentrations caused by platelet release, the blood for analysis was collected into 'Thrombotect' tubes, which contain inhibitors of platelet activation. The RIA developed for GST pi and also existing RIA for GST BI, GST 82 and GST p were used to determine GST levels in a variety of biological fluids and tissues. Measurement of the individual GST isocnzymes expression in normal and tumour cytosol prepared from human lung, colon and stomach showed that the concentration of GST pi was significantly increased in tumour tissue relative to paired normal tissue. The expression of GST BI and GST Bj, the predominant isoenzymes in normal liver, kidney and stomach, decreased dramatically in tumour tissue from these organs. In breast cancer cytosols significant differences in GST expression were also observed between estrogen receptor-rich and estrogen receptor-poor tumours. The proportion of individuals with cancer found to express the polymorphic GST isoenzyme GST \i was not significantly different from the expression rate of the isoenzyme in control populations. The expression of the various GST isoenzymes in development showed that there was a decrease in GST pi expression in liver and lung as the fetus matured, while the alpha class GST showed an increase in expression throughout development. Purification of GST isoenzymes from bronchoalveolar lavage and gall bladder bile by affinity chromatography, showed that GST pi constituted the major isoenzyme in bile, and it is postulated that GST pi acts as a carrier protein of toxic, non-substrate, ligands to remove as yet unidentified substances from biliary epithelial cells and prevent their reabsorption. Concentrations of GST BI and GST Bj were found to be significantly raised (/>< 0.02) in bronchoalveolar lavage fluid obtained from the suspected abnormal area of lung compared with the presumed normal area of lung, in patients later diagnosed as having cancer of the bronchus, whilst in patients with non-malignant respiratory disorders no significant difference in GST concentration was found. Plasma GST BI measurements were used to investigate hepatocellular integrity in adults following hypoglycacmia and in children exposed to isoflurane or halothane anaesthesia. Hypoglycaemia produced a significant increase in plasma GST B i. Abnormalities in plasma GST B i were greater in children receiving halothane compared to the children who received isoflurane. These studies indicate that GST pi measurements by RIA may provide a useful tumour marker for cancer of the bronchus, whilst GST BI measurements in bronchoalveolar lavage may be useful in the diagnosis of suspected lung malignancy. The studies on plasma GST BI measurements confirm previous studies that suggested such measurements provide an extremely sensitive index of hepatocellular integrity.
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Matas, Nada. "Molecular genetics of human arylamine N-acetyl transferases." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338241.
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Thom, Russell. "Structural studies of glutathione S-transferases from plants." Thesis, University of Glasgow, 2000. http://theses.gla.ac.uk/6648/.
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The aim of this thesis was to structurally characterise a range of plant GSTs to increase the understanding as to which structural features determine substrate specificity. During the course of this work, a total of ten plant GSTs from species including Maize, Wheat, Carnation, Petunia and Arabidopsis thaliana were overexpressed in E. coli. Nine of these overexpressed GSTs were purified the produce protein for crystallisation using a range of matrices such as Orange A agarose, S-hexyl glutathione and metal chelate followed by ion-exchange and/or gel filtration chromatography depending on the particular GST. Initial crystallisation conditions for seven of these purified GSTs were found by sparse matrix screening. Crystallisation conditions providing crystals suitable for X-ray diffraction experiments were determined for four of the GSTs under study. The data gained from these diffraction experiments enabled the solution of four different plant GST three-dimensional structures. The first structure to be solved was ZmGSTF1, a GST isoenzyme constitutively expressed in Maize. The second structure to be solved was AtGSTZ1 from Arabidopsis thaliana. The third GST structure determined (AtGSTT1), again from Arabidopsis thaliana, was found to share significant homology with the mammalian Theta class GSTs. The fourth structure determined was a GST isoenzyme from wheat, able to metabolise the commercially important herbicide fenoxaprop. These structural models provide a detailed understanding of the structure determinants of a variety of GSTs which dictate the different substrate specificities of GSTs and provide suggestions for the rational design of GSTs to improve herbicide selectivity in crops. In addition, this study presents material relating to the in-vivo role of plant GSTs and their binding to endogenous substrates.
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Andrews, Christopher John. "Glutathione transferases in soybean Glycine max (L.) Merr." Thesis, Durham University, 1999. http://etheses.dur.ac.uk/4857/.
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Glutathione transferases, also known as Glutathione S-transferases (GSTs), are a diverse group of enzymes that catalyse the conjugation of the tri-peptide glutathione to a wide range of electrophilic substrates. Their biological function in endogenous metabolism in plants is not well characterised, although their role in herbicide metabolism and herbicide selectivity is well documented. Many herbicides used in soybean. Glycine max (L.) Merr., are selective against weeds due to their rapid detoxification in the crop through conjugation with homoglutathione (γ-glu-cys-β-ala), the predominant free thiol in many legumes. However, an in depth characterisation of the GSTs which can potentially catalyse these reactions in soybean has never been performed. This work describes the biochemical and molecular characterisation of GSTs in soybean with emphasis on the identification of specific isoenzymes involved in herbicide metabolism. GST activity toward the chloroacetanilide herbicides acetochlor and metolachlor, the diphenyl ethers acifluorfen and fomesafen and the sulphonyl urea chlorimuron-ethyl were all detected in crude protein extracts from five-day-old suspension cultured soybean cells. GST activity was also determined in five-day-old soybean seedlings, though this activity was significantly lower than that observed with the cell suspension cultures. Treatment of soybean plants with herbicides and herbicide safeners resulted in increased GST activity toward the model substrate l-chloro-2,4-dinitrobenzene (CDNB), but no change in activity toward herbicide substrates. In both plant and cell cultures GST-catalysed conjugation of the diphenyl ethers acifluorfen and fomesafen was over five-fold greater in the presence of homoglutathione as compared with glutathione. The preferential detoxification of these herbicides in the presence of homoglutathione appeared to be an important determinant of their rapid detoxification in soybean and an important factor in herbicide selectivity. GSTs were purified from five-day-old soybean cell cultures using S-hexylglutathione affinity chromatography and anion-exchange chromatography. A combination of reversed-phase HPLC, SDS-PAGE and MALDI-TOF mass spectrometry of the purified fractions indicated the presence of nine putative GST subunits, each with a molecular mass between 25 and 29 kDa. Soybean GST cDNA clones were obtained using a combination of RT-PCR, utilising degenerate oligonucleotides designed to conserved regions within plant GSTs, and screening of cDNA libraries prepared from soybean plants and cell cultures. This process failed to identify any theta-type GSTs, the class associated with herbicide detoxification in maize. In contrast, seven distinct tau-type GSTs were isolated together with a number of clones showing minor variations in individual sequences. Expression of these cDNAs in Escherichia coli showed the purified recombinant GSTs were active toward a diverse range of substrates, and possessed additional glutathione peroxidase activity. GST activities for each recombinant enzyme varied with substrate and thiol type, with a marked preference for homoglutathione with selected substrates. From the work reported in this study it would appear that the tau-type GSTs of soybean are at least as complex as those previously reported in cereals and have an important role in determining herbicide metabolism and selectivity in this major crop.
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Banger, Kulwinder Kaur. "Glutathione S-transferases of the rat nasal cavity." Thesis, Liverpool John Moores University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261475.
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Harris, Jonathan Malcolm. "The glutathione S-transferases of rat liver mitochondria." Thesis, University College London (University of London), 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325084.
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Taylor, James Philip. "Synthesis of novel acceptor substrates for mannosyl transferases." Thesis, University of Exeter, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317390.
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Guelfi, Andréa. "Bioinformática estrutural aplicada à evolução das glutationas transferases." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-04052006-161903/.
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As glutationas transferases compõem uma superfamíla de proteínas que atuam na fase II do sistema de desintoxicação das células. Participam principalmente através do processo de conjugação da glutationa com moléculas hidrofóbicas e eletrofílicas, como por exemplo os herbicidas. No entanto, outras funções foram descritas como a tolerância ao estresse oxidativo, inseticidas, antibióticos microbianos, transporte de produtos secundários tóxicos, sinalização da célula durante as respostas ao estresse e fenômenos de resistência envolvendo agentes de quimioterapia contra o câncer. Nesta tese procurou-se estabelecer uma relação entre a seqüência, estrutura, função e afinidade das GSTs. A estrutura, de modo geral, determina a função da enzima, mas por si só, não dita sua especificidade. Esta última informação é fundamental para o desenvolvimento de novos agroquímicos ou para o desenho racional de novas proteínas. A relação entre a seqüência, estrutura, função e afinidade mostra que o paradigma estrutura-função deveria ser ampliado para incluir a seqüência de aminoácidos e a afinidade da enzima. Apesar da grande diversidade de substratos e seqüências encontradas nas GSTs há pelo menos um caso de convergência funcional em duas classes distintas desta superfamília. Uma encontrada apenas no reino Animalia (classe Pi) e outra exclusiva do reino Plantae (classe Phi). Ferramentas da bioinformática estrutural, como docking molecular e minimização de energia foram utilizadas para analisar as interações entre a enzima e o substrato. Estas ajudam a explicar como duas proteínas com aproximadamente 22% de identidade de seqüência apresentam afinidades semelhantes. Finalmente, foram propostos mutantes da GST de Saccharum officinarum utilizando a informação estrutural da enzima, visando uma alteração na afinidade da mesma.Glutathione Transferases comprehend a superfamily of proteins that plays the phase II of the detoxification system of the cells. Their major catalysis is the conjugation of glutathione with hydrophobic and eletrophilic molecules, for example herbicides. However, other functions were described like oxidative stress, insecticides, microbial antibiotics, transport of secondary products, cells signalization during response to stress and resistance of chemotherapy drugs against cancer. This work aimed to establish a relation between sequence, structure, function and affinity of GSTs. The structure, in general, determines the function, but alone, can not determine the enzyme specificity. This last information is essential to the development of new agrochemicals or for the rational design of proteins. The relation between sequence, structure, function and affinity shows that the paradigm of structure-function should be enlarged in order to include the information of amino acid sequences and the enzyme affinities. Despite the wide variety of substrates and sequences found in the GSTs, there is at least one case of functional convergence between two distinct classes in this superfamily. One is found in the Animalia kingdom (class Pi) and the other is exclusively found in Plantae (class Phi). The structural bioinformatic tools, such as molecular docking and energy minimization were used to analyze the interactions between the enzyme and the substrate. These help to understand how two enzymes with approximately 22% of sequence identity can show the same affinities. Finally, GST mutants of Saccharum officinarum were proposed, using the enzyme structural information in order to modify the enzyme affinities.
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Stockman, Paul Kennedy. "The glutathione S-transferases in human liver cytosol." Thesis, University of Edinburgh, 1988. http://hdl.handle.net/1842/26977.
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A purification scheme is described for the basic and neutral glutathione S-transferases which occur in human liver. Three forms with basic isoelectric points, B-|Bi (pl'8.9), B-|B2 (pi 8.75) and B2B2 (pi 8.4) and two forms with neutral isoelectric points, N-j (pi 6.1) and N2 (pi 4.6), were obtained. Not every liver examined expressed transferase N-] or transferase N2^ An acidic enzyme from human lung, GST \ (pi 4.8), was included in the study for the purpose of comparison. Sodium dodecylsulphate/polyacrylamide-gel electro? phoresis demonstrated that each enzyme comprises two subunits of identical size. As a group the human glutathione S-transferases are composed of three distinct types of subunit with different molecular weights. The basic enzymes (B-]B-j, B1B2 and B2B2) were shown to have a subunit molecular weight of Mr 25 900, whereas, the neutral enzy? mes (N-] and N2) were found to comprise subunits of molecu? lar weight Mr 26 500 and the acidic transferase from lung ( X) was shown to be composed of subunits of molecular weight Mr 24 800. Antisera were raised against each of these enzymes and they were shown to cross-react only with enzymes from the same charge/molecular weight group. Contrary to previous work (Kamisaka e^t_ al_., ( 1975) Eur. J. Biochem. 60_: 153-161) data are presented to demonstrate that two distinct basic subunits are expressed in human liver (B^ and B2). Reversible dissociation and reassociation experiments demonstrated that transferases B-|Bi and B2B2 could be formed from trans? ferase B-1B2. Tryptic-di gest peptide 'maps' showed that the B-| and B2 subunits possess extensive sequence homo? logy, however, seven peptides were recovered from trans? ferase B-|Bi that were not recovered from transferase B2B2. Conversely, four peptides were recovered from transferase B2B2 that were not recovered from transferase B1B1. However, all these peptides were recovered from trans? ferase B*i B2; this is consistent with the hypothesis that B1B2 is a hybrid enzyme. A comparison of the substrate specificities of the enzymes demonstrated that transferases B -| B1, B-] B2 and B2B2 had a high peroxidase activity with cumene hydro? peroxide, transferases N-| and N2 had a high activity with trans-4 -phenyl-3 -but en-2- one and transferaseX had a high activity with ethacrynic acid. The IC50 values obtained for the basic enzymes demonstrated that the B1 subunit was much more potently inhibited by tributyltin acetate than the B2 subunit. The isoelectric point, molecular weight and high spe? cific activity with trans-4-phenyl-3-buten-2-one of trans? ferase N-| suggest that it is identical to transferase ]x, a form that has been previously characterised by Warholm et al., ( 1983) Biochemistry 22_: 361 0?3617- However, transferanse N2 is a novel enzyme that has not been described previously. The properties of this new enzyme indicate that it also belongs to the neutral group of enzymes.
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Copp, Janine Naomi Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Catalysed activation of cyanobacterial biosynthetic pathways by phosphopantetheinyl transferases." Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2005. http://handle.unsw.edu.au/1959.4/33254.
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Cyanobacteria produce a diverse array of natural products with significant potential in many biotechnological, clinical and commercial applications. These include pharmaceuticals, such as antitumour products, antibiotics, immunosuppressants, anticholesterolemics and anti-parasitic agents, as well as veterinary therapies and agrochemicals. These compounds are synthesised by complex secondary metabolism pathways involving polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS), both of which require an essential phosphopantetheinyl transferase (PPT) for their activity. PPTs activate the acyl, aryl and peptidyl carrier proteins within various biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. Phylogenetic analysis of the large superfamily of PPTs has revealed two separate families based on substrate specificity, which have been designated AcpS and Sfp-like. The AcpS PPT family activate acyl carrier proteins of fatty acid synthesis, while the Sfp-like PPT family, typified by the Bacillus subtilis PPT Sfp, has diverse roles in primary and secondary metabolism. The majority of cyanobacterial PPTs are of the Sfp-like PPT family and occur in genomes lacking an AcpS PPT. Phylogenetic analysis uncovered a distinct clade of cyanobacterial PPTs involved in heterocyst differentiation. Heterologous expression and functional analysis of NsPPT, the heterocyst-associated PPT in Nodularia spumigena NSOR10, represented the first characterisation of a cyanobacterial PPT. PCR-based screening was utilised to identify NsPPT and Southern hybridisation suggested this was the only PPT encoded by the N.spumigena NSOR10 genome. Enzymatic analyses demonstrated the ability of NsPPT to phosphopantetheinylate PKS and NRPS carrier proteins from a range of metabolism pathways and cyanobacterial species. Nostoc punctiforme ATCC 29133 encodes three PPTs. One of these PPTs, NgcS, is also a heterocyst-associated PPT and is homologous to NsPPT of N.spumigena NSOR10. Expression and enzymatic analysis of NgcS from N. punctiforme ATCC 29133, revealed contrasting phosphopantetheinylation activity to that seen for NsPPT, and indicated that NgcS may have evolved to have a strict specificity for the glycolipid biosynthesis pathway. Although the Sfp-like family of PPTs are normally associated with secondary metabolite biosynthesis, Synechocystis sp. PCC 6803 harbours a unique Sfp-like PPT (Sppt) but does not produce NRPS or PKS compounds. Genetic disruption of Sppt was attempted and expression of Sppt allowed the characterisation of its enzyme kinetics. Sppt displayed the ability to activate non-cognate cyanobacterial carrier proteins from NRPS and PKS biosynthetic pathways, although only at a low level of activity. This suggested that wild-type Synechocystis sp. PCC6803 would not be suitable for heterologous expression of cyanobacterial secondary metabolites. These results have important implications regarding the expression and manipulation of cyanobacterial bioactive compounds in heterologous hosts. Applications of this research may provide a biotechnological platform for the sustainable production of cyanobacterial natural products.
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Ati, Jihen. "Leishmanian Galactofuranosyltransferases as promising versatile tools for therapeutic and chemoenzymatic approaches." Thesis, Orléans, 2018. http://www.theses.fr/2018ORLE2043.
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Les cellules exposent à leurs surface glycoconjugués qui jouent important dans des événements biologiques importants tels que la communication entre cellules, la croissance de cellules saines ou cancéreuses et les processus d'infection d'agents pathogènes. Certaines structures polysaccharidiques qui contiennent le résidu Galf ont attiré beaucoup d’intérêt au cours des dernières décennies. En effet, le galactofuranose peut être exprimé chez de nombreuses espèces pathogènes, telles que Mycobacterium tuberculosis, Aspergillus et Leishmania, mais il est absent chez les mammifères. Par conséquent, ces glycoconjugués sont considérés comme des cibles intéressantes pour des approches thérapeutiques.Les galactofuranosyltransférases (GalfT) catalysent le transfert des résidus de galactofuranose dans les structures des glycoconjugués. Cependant, ces GalfT sont des enzymes faiblement décrites, malgré leur rôle crucial dans la virulence ainsi que dans la pathogénicité de nombreux micro-organismes. Jusqu'à présent, seule la GalfT2 de Mycobacterium tuberculosis a été entièrement caractérisée.Dans cette thèse, quatre GalfTs de Leishmania major, l'agent responsable de la leishmaniose, ont été caractérisées. Elles ont été d'abord clonées, surexprimées dans E. coli et purifiées. Ensuite, leurs paramètres cinétiques respectifs ont été déterminés. De plus, puisque ces GalfT sont situées dans l'appareil de Golgi de Leishmania, nous avons supposé que leur glycosylation pourrait être un élément important pour leur stabilité etleur activité. Ainsi, des GalfT glycosylés ont été produites à l'aide de Leishmania tarentolae et les résultats préliminaires de leur activité enzymatiques ont été obtenus.Les GalfT leishmaniennes démontrent des résultats prometteurs pour le développement de nouvelles stratégies chimio-enzymatiques pour la synthèse de glycoconjugués contenant du Galf, ainsi que pour la conception de nouveaux médicaments contre la leishmanioseCells are heavily decorated by diverse glycoconjugates that are involved in important biological events such ascell-cell communication, growth of healthy or cancerous cells and pathogens infection process. Among these polysaccharidic structures, Galf-containing glycans have been the subject of increasing interest in the last decades. Indeed, the galactofuranose can be found in many pathogenic species, such as Mycobacteriumtuberculosis, Aspergillus and Leishmania, but is absent in mammals. Therefore, these glycoconjugates are considered as interesting targets for therapeutic approaches.Galactofuranosyltransferases (GalfTs) catalyse the transfer of galactofuranose residues into glycoconjugatesstructures. However, GalfTs are poorly described enzymes despite their crucial role in the virulence and the pathogenicity of numerous microorganisms. Up to date, only one mycobacterial GalfT has been fully characterized.In this thesis, four putative GalfTs of Leishmania major, the causing agent of leishmaniosis diseases, were characterized. They were first cloned, overexpressed in E. coli and purified. Then, their respective kineticparameters were determined. In addition, since these GalfT are located in the Golgi apparatus of Leishmania, we assumed that their glycosylation could be an important element for their stability and activity. So, glycosylatedGalfTs were produced using, Leishmania tarentolae, and preliminary results of their enzymatic activity were obtained.Still, leishmanian GalfTs demonstrate promising results for the development of new chemoenzymatic strategies for Galf-containing glycoconjugates synthesis, as well as the design of new drugs against leishmaniasis
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Molin, Eva U. "In vitro characterization of glutathione transferases from Sarcoptes scabiei /." Uppsala : Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, 2009. http://epsilon.slu.se/200980.pdf.
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Fedulova, Natalia. "Alpha-class Glutathione Transferases from Pig: a Comparative Study." Doctoral thesis, Uppsala universitet, Institutionen för biokemi och organisk kemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-144119.
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Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. This thesis contributes to knowledge about glutathione transferases from Sus scrofa (pig). The study is needed for better understanding of biochemical processes in this species and is desirable for drug development, for food industry research and in medicine. A primary role of GSTs is detoxication of electrophilic compounds. Our study presents porcine GST A1-1 as a detoxication enzyme expressed in many tissues, in particular adipose tissue, liver and pituitary gland. Based on comparison of activity and expression profiles, this enzyme can be expected to function in vivo similarly to human GST A2-2 (Paper II). In addition to its protective function, human GST A3-3 is an efficient steroid isomerase and contributes to the biosynthesis of steroid hormones in vivo. We characterized a porcine enzyme, pGST A2-2, displaying high steroid-isomerase activity and resembling hGST A3-3 in other properties as well. High levels of pGST A2-2 expression were found in ovary, testis and liver. The properties of porcine enzyme strengthen the notion that particular GSTs play an important role in steroidogenesis (Paper I). Combination of time-dependent and enzyme concentration-dependent losses of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. Enzyme adsorption to surfaces was found to be the main explanation of high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Several approaches to improved functional comparison of highly active GSTs were proposed (Paper III).Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 733
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Hatton, Pamela J. "Role of glutathione transferases in herbicide detoxification in weeds." Thesis, Durham University, 1996. http://etheses.dur.ac.uk/5443/.
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Glutathione transferases (GSTs) catalyse the conjugation of the electrophilic herbicides atrazine, metolachlor, alachlor and fluorodifen with the tripeptide glutathione (GSH). Maize (Zea mays L), contains multiple GSTs with differing substrate specificities which confer tolerance to a variety of herbicides. In contrast far less is known regarding the GSTs in competing weed species. In vivo metabolism studies using seedlings of maize and the weeds Panicum miliaceum. Digitaria sanguinalis, Sorghum bicolor. Setaria faberi. Abutilon theophrasti and Echinochloa crus-galli demonstrated that all species were capable of metabolising radiolabelled atrazine to GSH conjugates and the relative rates of metabolism related well to GST activities. Similarly, GST activities toward atrazine, metolachlor and alachlor correlated well with herbicide tolerance, with GSH availability being less important. GST activities towards metolachlor, alachlor and atrazine were highest in young maize plants and decreased with age, whilst GST activities in S.faberi remained unchanged. At 35 days GST activities were similar in the two species and the atrazine selectivity was lost. GSH content decreased with age in both species. Protein purification studies showed that S.faberi contains 4 GST isoenzymes with differing substrate specificities. The major GST was estimated to account for 0.1 % of die total soluble protein in S.faberi. PCR-amplification of a cDNA prepared from mRNA showed that S.faberi contains a GST with 88% identity to GST I from maize at the nucleotide level and 82% identity at the amino acid level. Similarly antibodies raised to maize and wheat GSTs recognised GSTs in S.faberi. It is concluded that GSTs determine the relative tolerance to chloroacetanilides and atrazine in weed seedlings but may be less important in older plants. The GSTs in S.faberi are similar in complexity to those determined in maize but are expressed at lower levels.
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Flaherty, Christopher. "Glycosyl derivatives as acceptors and inhibitors of mycobacterial transferases." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341460.
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Todorova, Tatina. "Glutathione S-transferases and oxidative stress in Saccharomyces cerevisiae." Université Louis Pasteur (Strasbourg) (1971-2008), 2007. https://publication-theses.unistra.fr/public/theses_doctorat/2007/TODOROVA_Tatina_2007.pdf.
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Les glutathion S-transférases répresentent une famille d'enzymes impliquée dans la détoxification de composés d’origine éxogène et endogène. Les GSTs peuvent avoir des activités péroxidase ou isomérase et également d’exprimer fonctions non-catalytiques, comme l’interaction avec differents ligands ou la modulation de la signalisation cellulaire. Un éventail des mutants de S. Cerevisiae a été choisi pour étudier le rôle des GSTs dans la réponse au peroxide et à l’arsenic. Notre criblage a montré l’existance de deux GSTs, qui peuvent avoir un rôle dans la détoxification de ces composés. Tef4p est un facteur de transduction dont le mutant correspondant est sensibile à l'As(V) et au H2O2. Au contraire, Ure2p joue un rôle dans la détoxification de l’As(III). Ce rôle, déterminé par la partie GST de la molécule est résultat de la fonction de la protéine dans la régulation du type GATA. La fonction régulatrice est dans l’origine également de la sensibilité aux oxydants du mutant ure2ΔGlutathione S-transferases are an enzyme family playing an important role in cellular detoxification of exogenous and endogenous toxic compounds. In addition, GSTs can serve as peroxidases, and isomerases or have non-catalytic functions, among which binding of non-substrate ligands and the modulation of signaling processes. A systematic approach, using defined yeast mutants, has been taken to demonstrate the connection between GSTs and the oxidative stress caused by peroxide and arsenic. This screening revealed that two yeast GSTs, Ure2p and Tef4p, may play a role in arsenic and oxidant detoxification. Tef4p is a translatation factor and the corresponding disruption mutant is sensitive to H2O2 and As(V). In contrast, Ure2p is required for the detoxification of As(III) in S. Cerevisiae. This protection role is determined by the GST domain of the molecule and is a result of the GATA repression role of Ure2p. GATA regulation is also in the origin of oxidant sensitivity of the mutant ure2Δ
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Todorova, Tatina Vuilleumier Stéphane Kujumdzieva Anna. "Glutathione S-transferases and oxidative stress in Saccharomyces cerevisiae." Strasbourg : Université Louis Pasteur, 2007. http://eprints-scd-ulp.u-strasbg.fr:8080/814/01/Todorova_Tatina_2007.pdf.
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Thèse de doctorat : Aspects moléculaires et cellulaires de la biologie : Strasbourg 1 : 2007. Thèse de doctorat : Aspects moléculaires et cellulaires de la biologie : Sofia - Bulgarie : 2007.Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Bibliogr. f. 143-155.
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Lyon, Robert Patrick. "Enzymology at the dimer interface of cytosolic glutathione S-transferases /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/8165.
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Bamber, Dianne Elizabeth. "Polymorphism in loci encoding detoxyfying enzymes : its role in cancer susceptibility and outcome." Thesis, Keele University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246867.
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Scott, Trevor Robert. "The identification, purification and characterization of the fetal rat liver glutathione S-transferase isoenzyme YcYfetus." Doctoral thesis, University of Cape Town, 1988. http://hdl.handle.net/11427/27171.
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This study has examined the expression of the glutathione S-transferases (GSH S-T) in fetal rat livers in order to provide more information about the role played by this important group of enzymes in the fetus. The study commenced with an examination of the subunit composition of adult and fetal rat liver GSH S-T using affinity chromatography followed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Adult livers contained four major GSH S-T subunits. An additional and previously unidentified subunit was detected in fetal livers. This subunit, which differed from that found in rat placenta, had a Mᵣ of approximately 25 500. Densitometric measurements suggest that the newly detected subunit accounts for as much as 26% of the GSH S-T in fetal livers. The novel fetal isoenzyme comprising this subunit was purified using a combination of affinity chromatography, carboxymethyl-cellulose column chromatography and chromatofocusing. The six major basic rat liver GSH S-T were purified for reference and comparative purposes. The fetal isoenzyme is composed of two non-identical subunits, namely, subunit Yc (Mᵣ 28 000) and the fetal subunit referred to as 'Yfetus'· The enzyme which I have termed GSH S-transferase Yc Y fetus has an isoelectric point of approximately 8.65 and has GSH S-T activity towards a number of substrates. Significantly, the fetal isoenzyme has one of the highest glutathione peroxidase activities yet described for the purified rat liver GSH S-T towards the model substrate, cumene hydroperoxide. Kinetic studies reveal that the fetal isoenzyme has a catalytic efficiency for the peroxide substrate which is four fold higher than that of the adult rat liver isoenzyme, GSH S-T YcYc. The in vitro effect of the GSH S-T substrate and teratogen, acrolein, on this fetal isoenzyme was investigated and compared with acrolein's effect on some of the adult rat liver GSH S-T isoenzymes in the standard 1-chloro-2,4-dinitrobenzene assay. Surprisingly, acrolein was identified as a non-competitive inhibitor of the GSH S-T. Exposure to acrolein in various guises could therefore result in inhibition of the fetal isoenzyme and its subsequent failure in inhibiting lipid peroxidation. Inhibitor studies were performed to look at the effect of acrolein, as well as other substrate and non-substrate ligands, on the glutathione peroxidase activity of GSH S-T YcY fetus and YcYc. The glutathione peroxidase activity of the fetal isoenzyme was far less susceptible to acrolein inhibition than the YcYc isoenzyme and the fetal isoenzyme was found to retain significant glutathione peroxidase activity despite saturating concentrations of non-substrate ligand. This study suggests that the fetal isoenzyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.
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Mutanda, Taurai. "Purification, characterisation and application of inulinase and transferase enzymes in the production of fructose and oligosaccharides." Thesis, Rhodes University, 2008. http://hdl.handle.net/10962/d1007734.
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Inulin hydrolysis can occur as a result of the action of exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing β-(2, I) end of inulin releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F₃), inulotetraoses (F₄) and inulopentaoses (F₅) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short chain fructooligosaccharide. The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a challenge. Oligosaccharides of chain length up to degree of polymerisation (DP) 5 and fructose were produced using preparations of three commercial microbial enzymes. Production of these novel oligosaccharides was achieved by employing response surface methodology (RSM) with central composite experimental design (CCD) for optimising product yield. Using a crude Novozyme 960 endoinulinase preparation isolated from Aspergillus niger, the following conditions gave a high inulooligosaccharide (lOS) yield, temperature (60 ºC), 150 g/L inulin concentration, 48 h incubation; pH 6.0 and enzyme dosage of 60 U/ml. Under these conditions, inulotrioses (70.3 mM), inulotetraoses (38.8 mM), and inulopentaoses, (3.5 mM) were produced. Response surface regression predicted similar product levels under similar conditions. The crude endoinulinase was purified through a three step purification procedure with a yield of 1.11 % and 3.5 fold purification. The molecular weight of this endoinulinase was estimated to be 68 .1 kDa by SDS-PAGE and its endoinulinase nature was confirmed by native PAGE. The purified endoinulinase was more efficient in production of lOS than the crude endoinulinase preparation. The purified endoinulinase demonstrated a high affinity for the inulin substrate (Km[subscript] 3.53 mM, Vmax[subscript] 666.67 μmol/min/ml). Pectinex Ultra SP-L, a commercial crude enzyme preparation isolated from Aspergillus aculeatus is a cocktail of several enzymes including a fructosyltransferase. The crude enzyme showed both transfructosylation and hydrolytic activity in 200 to 600 g/L sucrose. The main fructooligosaccharides produced from sucrose were l-kestose (GF₂), nystose (GF₃) and fructofuranosyl nystose (GF₄). After the first RSM, with the coded independent variables of temperature, incubation time, pH and sucrose concentration, the highest levels of GF₂, was 68.61 mM, under sucrose concentration 600 g/L, temperature 60°C, enzyme dosage 20 U/ml , pH 5.6, after 4 h incubation. A sucrose concentration of 400 g/L favoured the synthesis of high levels of GF₃ and GF₄. In the second RSM the maximal yields of GF₂, GF₃ and GF₄ were 152.07 mM, 131.38 mM and 43.99 mM respectively. A purified fructosyltransferase did not synthesise GF₄. Ammonium ions were demonstrated to enhance the yield of FOS. A mixture of glucose and fructose was used as substrate for FOS synthesis and no FOS were formed. Glucose was shown to be an end product inhibitor of the fructosyltransferase and therefore hinders the formation of high FOS yield. Fructozyme, isolated from Aspergillus ficuum is a mixture of exo and endoinulinases with the former being predominant was used for fructose production from inulin hydrolysis. The exoinulinase was purified to electrophoretic homogeneity by a three step purification procedure. The molecular weight of the enzyme was estimated to be 53 kDa with a 2 I % yield and 4.2-fold. Response surface regression was used to predict the maximum fructose levels achievable under the combinations of temperature, enzyme dosage and incubation time. A reaction time (48 h), enzyme dosage (100 U/ml) and inulin concentration (150 g/l) at pH 5.0 at 50°C gave higher fructose levels (106.6 mg/ml) using crude exoinulinase as compared to 98.43 mg/ml using the purified exoinulinase. These findings indicate that higher levels of fructose require longer incubation periods and higher inulin substrate concentrations with higher enzyme dosage. The crude exoinulinase preparation gave fairly higher levels of fructose than the purified exoinulinase and this is due to the presence of other hydrolytic enzymes in the crude preparation. The conditions established by RSM and CCO were adequate in producing high yield of oligosaccharides and fructose and can therefore be applied for their industrial production since they are in high demand due to their health benefits as prebiotics.
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Kurtovic, Sanela. "Directed Evolution of Glutathione Transferases Guided by Multivariate Data Analysis." Doctoral thesis, Uppsala University, Department of Biochemistry and Organic Chemistry, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8718.
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Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates.
Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates.
Several multivariate analysis tools, e.g. principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.
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Caniard, Anne M. "Glutathione transferases : probing for isoform specificity using dynamic combinatorial chemistry." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/4731.
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Cytosolic glutathione transferases (GSTs) are a large family of enzymes that play an important role in detoxification of xenobiotics. They catalyse the conjugation of the glutathione tripeptide (GSH) to a wide range of toxic electrophilic acceptors. The overall 3D folds and architectures of the catalytic sites of many GSTs are conserved. They are composed of a well conserved glutathione binding site (G-site) and a promiscuous hydrophobic binding site (H-site). The 3D structure and ligand specificity has allowed the sub-classification of the multiple isoforms within the soluble GST superfamily. GSTs are involved in the drug detoxification and so are the target of medicinal chemistry programmes but it has proven difficult to generate isoform-specific inhibitors due to their inherent promiscuity. In this project, Venughopal Bhat (University of Edinburgh, laboratory of Dr. Mike Greaney) and I have explored a new platform to probe enzyme specificity. Protein-directed dynamic combinatorial chemistry (DCC) allows the assembly and amplification of a ligand within the confines of a binding site. DCC was used as a tool to explore the promiscuous H-site of four eukaryotic GSTs. I purified recombinant forms of SjGST, hGST P1-1, mGST M1-1 and mGST A4-4 from E. coli and assayed them with the universal, synthetic GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Venughopal Bhat prepared a ten-member, thermodynamically-controlled, dynamic combinatorial library (DCL) of acyl hydrazones from a 1-chloro-2-nitrobenzene aldehyde and ten acylhydrazides. This DCL was incubated with each of the four GST isozymes (spanning diverse classes) and distinct amplification effects were observed for SjGST and hGST P1-1. I subsequently carried out several biophysical experiments in an attempt to rank each of the ligands. These experiements, coupled with molecular modelling, provided insight into the basis of the observed selectivity. Bacterial GSTs are thought to play a role in primary metabolism and display a different GSH-conjugation mechanism compared to the eukaryotic GSTs. A recombinant form of the beta-class GST from the pathogenic bacterium Burkholderia cenocepacia was isolated, purified and biochemically characterised. The same ten-member acylhydrazone DCL was interfaced with the bacterial GST which was shown to amplify a hydrophobic library member that shared structural features with the known substrate 2-hydroxy-6-oxo-6-phenyl-2,4-dienoate (HOPDA). With the collaboration of Venughopal Bhat, I attempted to explore the putative active site of a GST-like protein with an unknown function using the same DCL. Although no amplification was observed, a new aldehyde template was suggested for future DCC experiments on this protein. GSTs are widely employed in biotechnology as protein fusion tags to enhance target protein solubility coupled with a facile enzyme assay. Manish Gupta and Juan Mareque-Rivas (University of Edinburgh) used the N-terminal, hexahistidine-tagged SjGST to demonstrate that quantum dots (QDs) coated with nitrilotriacetic acid (NTA) bound to Ni2+ ions can be used to reversibly and selectively bind, purify, and fluorescently label a His6-tagged GST in one step with retention of enzymatic activity. For this prupose, I purified and characterized both the untagged and hexahistidinetagged – SjGST prior to their experiments.
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Li, Yaxiao. "Diverse roles of protein S-acyl transferases in Arabidopsis thaliana." Thesis, University of Bath, 2017. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.715280.
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S-acylation, commonly known as S-palmitoylation, is a reversible posttranslational lipid modification in which fatty acid, usually palmitic acid, covalently attaches to specific cysteine residues of proteins via thioester bonds. Palmitoylation enhances the hydrophobicity of proteins and contributes to their membrane association. It plays roles in protein trafficking, signalling, protein-protein interaction, protein stability and other important cellular functions. A family of Protein S-acyl Transferases (PATs) is responsible for this reaction. PATs are multi-pass transmembrane proteins that possess a catalytic Asp- His-His-Cys cysteine rich domain (DHHC-CRD) of ~50 amino acids. In Arabidopsis there are at least 24 such DHHC-CRD containing PAT proteins and they are named as AtPAT01 to AtPAT24. The function of only 2 AtPATs, AtPAT10 and AtPAT24 were studied in some detail, and a recent survey showed the ubiquitous expression pattern and different membrane localization habit of all 24 AtPATs. However, the biological function of the remaining 22 AtPATs in Arabidopsis was not reported when I started my project. Therefore, we carried out an initial screen of all the available T-DNA insertion lines of the 22 Arabidopsis PATs and identified transcriptional null mutants of 18 of the AtPATs. Among them, the k/o mutant plants of only 3 genes showed significantly altered phenotypes compared to wild-type Arabidopsis, and the mutants are named as atpat14, atpat21 and plp1(PAT-like Protein 1). This project aims to characterize these three putative PATs in details in terms of their PAT activities, catalytic domains, expression patterns, subcellular localizations and biological functions. AtPAT14 was proved as a PAT by yeast complementary and in vitro auto-acylation assays. Mutagenesis studies clearly demonstrated that the cysteine residue in the DHHCmotif is essential for the enzyme activity of AtPAT14. Transgenic Arabidopsis plants expressing AtPAT14-GFP were observed and it was shown that AtPAT14 is predominantly localized at the Trans-Golgi. The phenotype was observed in both atpat14-1 and atpat14-2 mutant lines and this showed that the leaves of both lines were aging much faster than the WT. Analysis of the levels of different phytohormones revealed that the mutant leaves contained much higher salicylic acid (SA) than the WT. This coincided with the increased transcript levels of genes involved in SA biosynthesis and signalling. Therefore, AtPAT14 mediated protein S-acylation plays important roles in leaf senescence via the regulation of SA biosynthesis and signalling pathways. AtPAT21 was also confirmed as a PAT and the DHHC its functional domain by similar approaches as for AtPAT14. The plasma membrane (PM) localized AtPAT21 plays essential roles in both male and female gametogenesis. As such, loss-of-function by TDNA insertion in AtPAT21 leads to the plant being completely sterile. Therefore, AtPAT21-mediated S-acylation of proteins(s) plays important roles in the reproduction of Arabidopsis. AtPLP1 (PAT-like Protein 1) contains the signature DHHC-CRD. However, it does not rescue the growth defects of akr1, pfa3 and swf1, the 3 yeast PAT mutants used in enzyme activity assays of other known PATs from plant and animals. Further, the cysteine residue in the DHHC motif was not essential for the function of AtPLP1 as mutated variant containing serine in place of cysteine of the DHHC motif can still rescue the growth defects of atplp1-1. Seedling establishment of atplp1-1 was impaired without external carbon source. This is because the efficiency in converting the seed storage lipid to sugar in the mutant is much lower than WT due to the defective β-oxidation process involved in the degradation of free fatty acids released from lipid during post-germinative growth. In addition, atplp1-1 seedlings are also de-etiolated in the dark, and this was coincided with more cytokinin (CK) and less active gibberellin (GA) related pathway in the mutant. Other defects were also found in atplp1-1, such as hypersensitive to abscisic acid (ABA) and sugar during seed germination and abnormal shoot apical meristem (SAM) in older plants. Therefore, protein S-acyltransferases play distinct and diverse roles throughout the life cycle, from seed germination, seedling growth to seed production in Arabidopsis. This is most likely through the palmitoylation of an array of proteins they modify. Hence, our results provide vital clues for future studies on the molecular mechanism as to how AtPATs operate in plant.
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Schmidt, Marie-Joëlle. "Characterisation of cytoplasmic uridyl transferases in yeast and human cells." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:e3831ff6-2b5f-4456-a91c-4ac0cca52594.
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Regulation of gene expression by terminal addition of uridyl residues to RNA substrates has recently emerged as a widespread phenomenon in eukaryotes. Studies in organisms ranging from fission yeast to human cells have shown that uridylation of RNA 3' ends stimulates rapid RNA degradation. However, many questions regarding the specificity of the uridyl transferases, the broad range of their substrates and the consequences of their loss are still unanswered. In light of this, the uridyl transferases Cid1 in Schizosaccharomyces pombe and ZCCHC11 in human cells and their roles in the regulation of gene expression were further characterised in this study. To begin with, the biochemistry of the Cid1 protein complex responsible for uridylation in Schizosaccharomyces pombe was analysed in more detail by mass spectrometry and in vitro assays. These experiments provided insights into the modulation of Cid1 activity by accessory factors. Next, the role of the human uridyl transferase ZCCHC11 in the regulation of replication- dependent histone mRNAs was examined. Results showed that ZCCHC11 is required for efficient destabilisation of histone mRNAs following inhibition or completion of DNA replication. In agreement with this finding, cDNA sequencing experiments showed that ZCCHC11-mediated uridylation is particularly prevalent at the end of S phase. Finally, this thesis also explored the phenotype resulting from ZCCHC11 knock-down with respect to the human cell cycle. Depletion of ZCCHC11 led to the occurrence of DNA damage and activation of the DNA integrity checkpoint, which in turn resulted in cell cycle delay. Taken together, the data presented in this thesis extend current knowledge of the uridyl transferases and their actions in fission yeast and human cells and provide a link between RNA regulation and cell cycle control.
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Favrot, Lorenza. "Structure and Enzymatic Characterization of Mycobacterium tuberculosis Transferases." University of Toledo / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1404588587.
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Sparrow, Helen. "The role of glutathione transferases in the detoxification of TNT." Thesis, University of York, 2010. http://etheses.whiterose.ac.uk/1470/.
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Manufacture, use, storage and improper disposal of the explosive 2,4,6-trinitrotoluene (TNT) have led to widespread, global contamination of soil and groundwater. TNT is highly toxic and recalcitrant to degradation resulting in environmental build-up with far reaching ecological and health implications. It is therefore a priority to remove contaminating TNT from the environment. Phytoremediation is a promising solution; suitable plants possess some natural ability to transform TNT, high biomass, deep root systems, requirement for minimal nutrient input and ability to reduce contamination spread by wind or water erosion; making them an attractive remediation system. Key genes involved in the detoxification of TNT by plants have been recently identified by expression studies and the encoded enzymes characterised. This has lead to the thorough investigation of the enzymes in the pathway of TNT detoxification; Phase I transformation includes oxophytodienoate reductases, with uridine diphosphate (UDP) glycosyltransferases (UGTs) playing a role in the Phase II conjugation step. The expression studies identified additional enzymes also likely to be involved in these phases including glutathione transferases (GSTs). GSTs are known to detoxify compounds by conjugation to glutathione (GSH), and like UGTs are Phase II detoxification enzymes. This thesis presents an investigation into whether plant GSTs play a role in the detoxification of TNT. In vitro analysis of recombinant GSTs was performed to elucidate the activity of GSTs towards TNT. Seven GSTs were cloned, expressed and purified from Escherichia coli. TNT assays performed with pure enzyme indicated that at least two of the GSTs were able to transform TNT. Analysis of the reaction product by mass spectrometry showed that TNT was conjugated to glutathione through substitution of a nitro-group, a highly desirable reaction as the removal of a nitro group from TNT is likely to increase the likelihood of subsequent mineralisation of the pollutant. This is the first identification of enzymes capable of this transformation. The two GSTs which exhibited activity towards TNT were overexpressed in Arabidopsis to clarify if the conjugating activity observed in vitro was able to confer increased tolerance to TNT to the transformed plants. Transgenic lines showed no enhanced growth compared to wild type plants on TNT amended media, root lengths appeared slightly shorter while TNT uptake and biomass were reduced. The role of GSTs in the detoxification of TNT remains unresolved however it is likely that GSTs do not play an integral role in TNT detoxification in plants. Nonetheless, the two GSTs characterised in the project are the first examples of plant enzymes which are able to catalyse the removal of nitro groups from TNT. Engineering these GSTs to improve their ability to transform TNT could offer an opportunity for effective environmental remediation.
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Sharath, B. "Structure and kinetics of gamma glutamyl transferases from bacillus species." Thesis(Ph.D.), CSIR-National Chemical Laboratory, Pune, 2008. http://dspace.ncl.res.in:8080/xmlui/handle/20.500.12252/2665.
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Bastos, Frederico Freire. "Melatonina, isoenzimas de glutationa S-transferases e estresse oxidante em pacu Piaractus mesopotamicus (Holmberg, 1887)." Universidade do Estado do Rio de Janeiro, 2010. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=2991.
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Conselho Nacional de Desenvolvimento Científico e TecnológicoO oxigênio é fundamental para os vertebrados. No entanto, variações dos níveis de oxigênio na água podem provocar estresse oxidante em peixes porque privação de oxigênio seguida de reoxigenação forma espécies reativas de oxigênio (ERO) em células. Níveis intracelulares de ERO aumentados favorecem que moléculas de proteínas, fosfolipídios e ácidos nucleicos sofram alterações, vindo a prejudicar muitas funções celulares. No Pantanal, habitat do pacu, o nível de oxigênio varia circadianamente na água das lagoas rasas que acabam isoladas dos rios na seca. O pacu evoluiu sob a pressão contínua da exposição aos efeitos prejudiciais das ERO causados pelos pulsos de inundação. A melatonina, uma indolamina produzida na glândula pineal, influencia os níveis de atividade de enzimas antioxidantes que reduzem ERO, além de ser capaz de doar elétrons ou captar radicais livres de forma não enzimática. Os níveis de melatonina no pacu são mais altos no verão e menores no inverno. Isoenzimas de glutationa S-transferases que conjugam o tripetídeo glutationa com o 4-hidroxinonenal, aldeído derivado da peroxidação de ácidos graxos por ERO, são importantes para evitar alteração funcional de proteínas por ligação do 4-hidroxinonenal à sua estrutura. Neste trabalho procuramos relação entre estresse oxidante, níveis de atividades de glutationa S-transferase e melatonina, para estabelecer se a melatonina ajudaria pacus a superar os efeitos deletérios das espécies reativas de oxigênio. Ensaiamos atividades de isoenzimas de glutationa S-transferases no citosol de fígado de pacus mantidos em normoxia, hipoxia, reoxigenação e hiperoxia no inverno e no verão. Medimos o efeito da melatonina in vitro e in vivo sobre as atividades de isoenzimas de glutationa S-transferase. Medimos os efeitos do estresse oxidante sobre a ligação do 4-hidroxinonenal com proteínas nos fígados de pacus tratados com melatonina. Somente as isoenzimas que conjugam 4-hidroxinonenal com glutationa mostraram menor atividade no inverno em relação ao verão; outras isoenzimas de glutationa S-transferases não alteram suas atividades sazonalmente. In vitro a melatonina não alterou a atividade de isoenzimas de glutationa S-transferase que conjugam o 4-hidroxinonenal, mas inibiu outras isoenzimas de glutationa S-transferase. In vivo a melatonina aumentou a atividade encontrada no inverno das isoenzimas que conjugam o 4-hidroxinonenal para os níveis do verão. A ligação de 4-hidroxinonenal com proteínas foi menor em pacus inoculados com melatonina. Nossos resultados mostram que a melatonina pode influenciar os efeitos de ERO em fígado de pacus. Ficou claro que a melatonina do plasma mantém os níveis de atividade conjugadora de 4-hidroxinonenal do fígado em pacus e que a baixa produção de melatonina no inverno não é adequada para a conjugação do 4-hidroxinonenal em fígado de pacus.
Oxygen is vital for vertebrates. However, changes in the levels of dissolved oxygen in water might cause oxidative stress in fishes because the shortage of oxygen followed by reoxygenation originates reactive oxygen species (ROS) inside cells. Higher intracellular levels of ROS favor alterations of proteins, phospholipids and nucleic acid molecules, which result in impairment of many cell functions. In Pantanal, the pacus habitat, circadian variation of the oxygen levels occurs in water of the shallow lagoons that ended up isolated from the rivers along the dry season. Pacu has evolved under the pressure of continuous exposition to harmful effects of ROS caused by the annual inundation pulses. Melatonin, an indolamine produced by the pineal gland, influences the levels of activity of antioxidant enzymes that reduce ROS, and is capable of donating electrons or scavenge free radicals nonenzymatically. Pacus melatonin levels are higher during summer than in winter. Glutathione S-transferases isoenzymes that catalyze the conjugation of the tripeptide glutathione with 4-hydroxynonenal, an aldehyde derived from peroxidation of fat acids by ROS, are important to avoid functional alterations of proteins consequential to the binding of 4-hydroxynonenal to their structures. In this work, we searched for facts that linked oxidative stress, levels of activity of glutathione S-transferase and melatonin, in order to establish whether melatonin could help pacus to overcome the pernicious effects of reactive oxygen species. We carried out assays of glutathione S-transferases in liver cytosol of pacus kept under normoxia, hypoxia, reoxygenation and hyperoxia, in the summer and in the winter. We measured the effect of melatonin in vitro and in vivo on isoenzymes of glutathione S-transferases. We measured the effects of oxidative stress on the binding of 4-hydroxynonenal to proteins in liver of pacu treated with melatonin. Only isoenzymes that conjugate 4-hydroxynonenal with glutathione showed less activity during the winter in comparison to the summer; other isoenzymes did not have their activities changed seasonally. In vitro, melatonin did not change the activity of glutathione S-transferases isoenzymes that conjugate 4-hydroxynonenal, but inhibited other isoenzymes of glutathione S-transferase. In vivo, melatonin enhanced the liver activity of the glutathione S-transferase that conjugate 4-hydroxynonenal found in winter up to the levels found in summer. The binding of 4-hydroxynonenal to proteins was lower in liver cytosol from pacus injected with melatonin. Our findings show that melatonin can influence the effects of ROS in liver of pacu. It became evident that plasma melatonin maintains the liver levels of the conjugating activity of 4-hydroxynonenal and that the lower production of melatonin during winter is not adequate to the conjugation of 4-hydroxynonenal.
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Jonsson, Stefan. "The mechanism of Formyl-Coenzyme A transferase, a Family III CoA transferase, from Oxalobacter formigenes." [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0007068.
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Ricagno, Stefano. "Formyl-coenzyme A transferase, structure and enzymatic mechanism /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-028-1/.
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Hilton, John Frederick. "The molecular basis of glutamate formiminotransferase deficiency /." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33776.
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Glutamate formiminotransferase deficiency (OMIM 229100) is an autosomal recessive disorder marked by clinical heterogeneity. The severe phenotype, first identified in patients of Japanese descent, includes high levels of formiminoglutamate (FIGLU) in the urine in response to histidine loading, megaloblastic anemia, and mental retardation. The mild phenotype is marked by high levels of FIGLU in the urine in the absence of histidine loading, mild developmental delay and no hematological abnormalities. The gene for human glutamate formiminotransferase-cyclodeaminase consists of 15 exons and is located at 21q22.3. The protein consists of a tetramer of dimers, with dimerization essential for both formiminotransferase and cyclodeaminase activity.Genomic DNA extracted from cell lines from three patients with suspected glutamate formiminotransferase deficiency was analyzed by PCR and sequencing of individual exons. Cell lines WG 1758 and WG 1759 are from two siblings of Germanic descent. Both siblings are heterozygous for the mutations c457 C → T and c940 G → C. The c457 C → T changes a conserved arginine to a cysteine in a loop involved in the binding of formiminotetrahydrofolate to the enzyme. The c940 G → C mutation converts an arginine to a proline in an alpha-helix essential for the dimerization of the formiminotransferase domain. Cell line WG 1795 is from a patient of Danish descent. The patient appears to be hemizygous for a c1033 insG mutation. Quantitative PCR suggests the presence of a deletion on the other chromosome, which minimally encompasses exon 9. All of the FTCD gene changes were absent in 100 control individuals (200 alleles).
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Jiang, Hong, and 姜紅. "Effects of estrogen on human catechol-O-methyl transferase." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31241864.
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Jiang, Hong. "Effects of estrogen on human catechol-O-methyl transferase." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23273744.
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Lu, Weiya Douglas. "Photophysical consequences from interactions of glutathione S-transferases with the photodynamic sensitizer hypericin /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/8638.
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Street, Ian Philip. "Fluorinated carbohydrates as probes of mechanism and specificity in glycosyl transferases." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/29434.
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The Compounds 2-deoxy-2-fluoro-β-D-glucosyl fluoride (1), 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2), 2-deoxy-2-fluoro-β-D-galactosyl fluoride (3) and 2-deoxy-2-fluoro-β-D-mannosyl fluoride (4) were all found to be potent covalent inhibitors of β-glucosidase from Alcaligenes faecalis (pABG5 β-glucosidase), which function through the accumulation of a stable glycosyl-enzyme intermediate. The mechanism of action for these inhibitors was investigated and found to be both specific and active site directed, involving a 1:1 stoichiometric formation of an enzyme inhibitor complex.
Investigation of the pre-steady state kinetics for the inhibition reaction provided values for the rates of formation (kon) and hydrolysis (koff) of the glycosylated-enzyme intermediate. The values of kon determined in this manner are 5.9, 25, 3.6 and 5.6 min.⁻¹ for compounds 1,2,3 and 4 respectively, while the values of koff were found to be much smaller; 1.5x10⁻⁵, 5.4 x 10⁻³ and 1.0 x 10⁻³ min.⁻¹, respectively for the intermediates formed from compounds 1, 3 and 4.
The isolated intermediates were also found to be competent in the catalysis of transglycosylation reactions and evidence for the participation of a specific binding site for the acceptor compound in this process was gained. The results of these kinetic experiments were corroborated by data obtained from ¹⁹F-NMR spectra of the glycosyl-enzyme intermediate and by isolation and subsequent characterization of the transglycosylation products. The stereochemistry of the inhibition reaction was investigated by a simple ¹⁹F-NMR experiment, and was found to be consistent with a double displacement reaction mechanism, as would be expected from the generally accepted reaction mechanism for this type of enzyme.
The compound D-glucal was found to be a substrate for pABG5 β-glucosidase (kcat = 2.28 min.⁻¹ Km = 0.85 mM). Hydration of this compound by pABG5 β-glucosidase in deuterated buffers demonstrated that the double bond of D-glucal was deuterated stereo-specifically from below the α-face. The compound 2-fluoro-D-glucal was found to be a weak competitive inhibitor (Ki = 30 mM) of pABG5 β-glucosidase. Glycogen phosphorylase catalyses the reversible phosphorolysis of glycogen. A series of deoxy analogues of the enzymes natural substrate, α-D-glucose 1-phosphate have been prepared and along with a number of deoxyfluoro analogues tested as substrates. All were found to act as substrates but at exceedingly slow rates. The large rate reductions when compared with the normal substrate can be attributed to a deleterious combination of electronic and binding effects in the modified substrates reducing the stability of the enzymic transition states. A linear free energy relationship between kcat and the first order rate constant for the acid catalysed hydrolysis of the same series of deoxy and deoxyfluoro glucopyranosyl phosphates was demonstrated, suggesting similar transition states for the two reactions and implicating an oxocarbonium ion-like transition state in the enzymic reaction. The binding data obtained from the steady state kinetics of these analogues suggests that hydrogen bonding interactions are qualitatively conserved in the glucopyranose binding site during the T- to R-state conformational transition of the enzyme and that interactions between the enzyme and the hydroxyl groups at the 3- and 6-positions of the glucopyranose ring of the substrate are potentially important for stabilization of the enzymic transition state.
The specificity of the substrate phosphate binding site has been probed using the compounds 2-deoxy-2-fluoro-α-D-glucopyranosyl phosphate (5), (1-deoxy-α-D-glucopyranosyl) methylphosphonate (6) and 2-deoxy-2-fluoro-α-D-glucopyranosyl phosphofluoridate (7). The results suggested that phosphorylase b can bind both the monanionic and dianionic forms of its substrate with approximately equal affinity. NMR studies of the ternary enzyme-ligand complexes formed with glycogen phosphorylase b and 5 or 6 indicated that no proton donation occurred in the ground-state active site complex.
A preliminary investigation into the ability of the cellulase complex from a number of different wood-degrading fungi to hydrolyse p-nitrophenyl β-glucoside has been carried out. This work is aimed at producing environmentally safe fungicides, which are activated by the β-glucosidase component of the cellulase complex in these organisms and this study was carried out in conjunction with Forintek Canada.Science, Faculty of
Chemistry, Department of
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