Browsing by Subject "Glutathione transferases"

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    Arginine 15 stabilizes an SNAr reaction transition state and the binding of anionic ligands at the active site of human glutathione transferase A1-1
    (Elsevier, 2010) Gildenhuys, Samantha; Dobreva, Marina; Kinsley, Nichole; Sayed, Yasien; Burke, Jonathan; Pelly, Stephen; Gordon, Graeme P; Sayed, Muhammed
    Arg15, conserved in class Alpha GSTs (glutathione transferases), is located at the interface between the G- and H-sites of the active site where its cationic guanidinium group might play a role in catalysis and ligand binding. Arg15 in human GSTA1-1 was replaced with a leucine and crystallographic, spectroscopic, thermodynamic and molecular docking methods were used to investigate the contribution made by Arg15 towards (i) the binding of glutathione (GSH) to the G-site, (ii) the pKa of the thiol group of GSH, (iii) the stabilization of an analog of the anionic transition state of the SNAr reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, and, (iv) the binding of the anionic non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to the H-site. While the R15L mutation substantially diminishes the CDNB–GSH conjugating activity of the enzyme, it has little effect on protein structure and stability. Arg15 does not contribute significantly towards the enzyme's affinity for GSH but does determine the reactivity of GSH by reducing the thiol's pKa from 7.6 to 6.6. The anionic σ-complex formed between GSH and 1,3,5-trinitrobenzene is stabilized by Arg15, suggesting that it also stabilizes the transition state formed in the SNAr reaction between GSH and CDNB. The trinitrocyclohexadienate moiety of the σ-complex binds the H-site where the catalytic residue, Tyr9, was identified to hydrogen bond to an o-nitro group of the σ-complex. The affinity for ANS at the H-site is decreased about 3-fold by the R15L mutation implicating the positive electrostatic potential of Arg15 in securing the organic anion at this site.
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    Clinical and biochemical studies on the gluthathione S-transferases
    (1982) Sherman, Morris; Kirsch, Ralph E
    The glutathione S-transferases (ligandins) are a ubiquitous system of xenobiotic metabolising enzymes. In the rat liver they comprise up to 10% of soluble hepatic protein. Studies in the rat suggested that ligandin was an accurate and sensitive marker of hepatocellular necrosis. and of renal tubular necrosis. The first part of this thesis examines the release of ligandin from liver and kidney in human liver and renal disease in an attempt to determine whether the measurement of ligandin is clinically useful. Ligandin was purified from human liver cytosol using a combination of anion exchange chromatography and gel filtration. The purified protein had similar physicochemical characteristics to ligandin purified by others. The protein was used to raise a monospecific antibody. Ligandin was iodinated by the Chloramine-T method. which yielded a labelled protein of high specific activity. A sensitive and specific radioimmunoassay for human ligandin was developed which had a low intra- and interassay variation. The assay was applied to the study of human liver disease. In acute hepatitis ligandin is released from the liver into serum early in the illness. High serum ligandin levels are seen in the first week of acute hepatitis. The rapid return to normal suggests that ligandin may provide an early indication of recovery. In chronic hepatitis ligandin levels correlated significantly with histological severity of disease. whereas SGOT showed no such correlation. Ligandin may be a better index of severity of disease and for treatment than SGOT. Ligandin was released from the kidney in severe renal ischaemia and in acute tubular necrosis, but was not a reliable predictor or indicator of acute tubular necrosis. Part two examines the distribution of GSH-T activity in organs and in hepatocellular carcinoma. Ligandin was shown to be immunologically similar in all tissues studied. Isoelectric focusing of cytosol separated the three groups of GSH-T activity. Considerable variety in the distribution and activity of GSH-T's was shown in different organs from a single donor, and in the same organs from different donors. Anionic transferase activity was shown to contribute a significant proportion of activity in organs other than the liver. and to be the major source of activity in ovary and lung. In hepatocellular carcinoma cationic GSH-T activity was present in amounts varying from near normal to absent. The anionic and neutral GSH-T's were present in amounts similar to that seen in normal liver. Immunohistochemical studies using a peroxidase-antiperoxidase method showed a rough correlation between tumour differentiation and the amount of ligandin in the tumour.
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    The glutathione S-transferases : kinetics, binding and inhibition
    (1989) Goold, Richard David
    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.
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    Human glutathione S-transferases : characterization, tissue distribution and kinetic studies
    (1988) Corrigall, Anne Vint; Kirsch, Ralph E
    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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    Ligandin in the steroidogenic tissues of the rat : characterisation, distribution and development
    (1982) Eidne, Karin Ann; Gevers, Wieland
    One of the main problems in the field of multifunctional proteins such as ligandin is the possibility that multiple forms and isoproteins may exist. Two forms of liver ligandin [ GSH (reduced glutathione) S-transferase B] have been described, a heterodimeric form consisting of equal amounts of Ya (22000 daltons) and Yc (25000 daltons) subunits, and a homodimeric form containing only Ya. Because rat testis ligandin, prepared by the standard technique of anion-exchange and molecular exclusion chromatography, contains more Yc subunit than Ya, it has been claimed that testis and liver ligandin are different entities (Bhargava, Ohmi, Listowsky and Arias (1980) J. Biol. Chem. 255, 724-727). This thesis investigated the nature and character of ligandin in the steroid-producing tissues of the rat. A comparative study was undertaken to establish whether testis ligandin differed from liver ligandin. Different methods of purification were used to investigate testis ligandin and its relationship to other GSH S-transferases in steroidogenic tissues. Testis ligandin purified by immunoaffinity chromatography using anti-liver YaYa ligandin antiserum yielded a product identical with liver preparations (Yc=Ya). This suggests that the differences previously described may be due to contamination of testis ligandin by a closely related species. Testis ligandin prepared by the standard technique was similar to that previously reported, containing more Yc than Ya. Cross-linking studies of standard testis ligandin preparations with dimethylsuberimidate showed more than one band in the 50000 dalton region, further strengthening the view that these testis ligandin preparations may be contaminated. Since this contaminant was likely to be another GSH S-transferase, sodium dodecyl sulphate/ polyacrylamide-gel-electrophoretic analysis was performed on testis GSH S-transferases separated by CM-cellulose chromatography. GSH S-transferase AA which was present in large amounts, was shown to migrate in the same region as Yc subunit. CM-cellulose chromatography of a 'pure' standard testis ligandin preparation revealed significant amounts of GSH S-transferase AA migrating as Yc subunit, in addition to ligandin consisting of equal amounts of Ya and Yc subunits, indicating that testis ligandin is identical with liver ligandin and that previously described differences are due to a contaminant identified as GSH S-transferase AA. Studies on ligandin in other steroid-synthesising tissues showed that ovary and adrenal ligandin prepared by standard techniques also contained more Yc than Ya. Separation of ovary GSH S-transferases on CM-cellulose showed that GSH S-transferase B, the peak reacting with anti-liver YaYa ligandin antisera contained equal amounts of Ya and Y c subunits, suggesting a situation similar to that in the testis exists. Glutathione peroxidase II activity of testis and ovary GSH S-transferases was investigated. Fractions corresponding to GSH S-transferase AA, A and B exhibited activity with cumene hydroperoxide. The considerable glutathione peroxidase activity of GSH S-transferases in testis and ovary suggest a protective function for the cells of gonadal tissue against oxidative damage to essential intracellular components. Further attempts to clarify the function of ligandin in the steroid-synthesising tissues were made. The pattern of gonadal ligandin development during early life, puberty and pregnancy determined by radioimmunoassay was found to parallel serum steroid hormone concentrations. This correlation was not observed in liver or kidney. Ligandin was localised to specific cells of the steroid synthesising tissues using immunocytochemical techniques. These findings suggest that there may be a functional link between steroidogenic cells, or products of their activity and certain GSH S-transferases. Phenobarbital pre-treatment did not have any effect on developing testis, ovary or adrenal ligand in concentrations. Immunocytochemical localisation of ligandin in rat steroid-producing tissues using a peroxidase anti-peroxidase (PAP) technique with anti-liver YaYa ligandin antiserum as the first antibody, showed staining in the testis to be limited to the interstitial (Leydig) cells. Stromal cells of the ovary and the fascicular, glomerular and reticular zones of the adrenal cortex also contained immunoreactive material. PAP staining with anti-testis ligandin antisera (testis ligandin prepared using the standard technique) showed far greater intensity of staining in these tissues, presumably due to reaction with both ligand in and GSH S-transferase AA. This study has clarified the structural aspects of testis ligandin and demonstrated identity with liver ligandin. Ontogeny of ligandin in the steroidogenic tissues and localisation to specific regions in these tissues suggests a functional link between ligandin, GSH S-transferases, GSH peroxidases and activity of steroidogenic tissue.
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