Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver.
ABSTRACT: A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.
Project description:Cytoplasmic thioltransferase purified from rat liver [Axelsson, Eriksson & Mannervik (1978) Biochemistry 17, 2978--2984] catalyses the formation and decomposition of mixed disulphides of proteins and glutathione. The enzyme was found to catalyse the reversible thiol-disulphide interchange between glutathione disulphide and a crude thiol-containing protein fraction from rat liver. This finding indicates a role of the thioltransferase in the regulation of the 'glutathione status' of the cell. Specifically, it was found that thioltransferase catalyses the reactivation of pyruvate kinase from rat liver that had previously been inactivated by glutathione disulphide. It is suggested that thioltransferase may have a general role in regulatory processes involving thiol-disulphide interchange.
Project description:Two enzyme systems capable of reducing disulphide bonds both in low-Mr compounds and in polypeptides and proteins exist. One consists of thioltransferase in combination with reduced glutathione and glutathione reductase, and the second consists of thioredoxin in combination with thioredoxin reductase. Their relative effectiveness in catalysing disulphide reduction of various substrates in rat liver cytosol was evaluated in the present study. The thioltransferase-dependent system was found to be more efficient in reducing small molecules. Insulin was most effectively reduced by the thioredoxin system. Bovine trypsin was a better substrate for thioltransferase, and partially proteolysed bovine serum albumin was equally good for the two systems. Thus, in the case of protein disulphide bonds, the nature of the particular substrate used determines which of the two reducing systems is the more important.
Project description:A Mu-class glutathione S-transferase purified to electrophoretic homogeneity from bovine lens displayed thioltransferase activity, catalysing the transthiolation reaction between GSH and hydroxyethyldisulphide. The thiol-transfer reaction is composed of two steps, the formation of GSSG occurring through the generation of an intermediate mixed disulphide between GSH and the target disulphide. Unlike glutaredoxin, which is only able to catalyse the second step of the transthiolation process, glutathioneS-transferase catalyses both steps of the reaction. Data are presented showing that bovine lens glutathione S-transferase and rat liver glutaredoxin, which was used as a thioltransferase enzyme model, can operate in synergy to catalyse the GSH-dependent reduction of hydroxyethyldisulphide.
Project description:The reduction of mixed disulphides of some proteins and GSH [Protein(-SSG)n] is accomplished with GSH as a reductant and a thioltransferase from rat liver as a catalyst, thus: See article. The spontaneous reaction is negligible in comparison with the enzymic reaction in vivo, and any direct reduction with glutathione reductase is not detectable with the substrates used. The reduction is only indirectly dependent on NADPH, which is required for the regeneration of GSH from GSSG. Other protein disulphides apparently are reduced via analogous GSH-dependent reactions
Project description:Trypanothione: glutathione disulphide thioltransferase of Try-panosoma cruzi (p52) is a key enzyme in the regulation of the intracellular thiol-disulphide redox balance by reducing glutathione disulphide. Here we show that p52, like other disulphide oxidoreductases possessing the CXXC active site motif, catalyses the reduction of low-molecular-mass disulphides (hydroxyethyl-disulphide) as well as protein disulphides (insulin). However, p52 seems to be a poor oxidase under physiological conditions as evidenced by its very low rate for oxidative renaturation of reduced ribonuclease A Like thioltransferase and protein disulphide isomerase, p52 was found to possess a glutathione-dependent dehydroascorbate reductase activity. The kinetic parameters were in the same range as those determined for mammalian dehydroascorbate reductases. A catalytic mechanism taking into account both trypanothione- and glutathione-dependent reduction reactions was proposed. This newly characterized enzyme is specific for the parasite and provides a new target for specific chemotherapy.
Project description:1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 22.214.171.124) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 126.96.36.199). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 188.8.131.52) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.
Project description:Two types of 25 000-Mr subunits are present in rat lung glutathione S-transferase I (pI 8.8). These subunits, designated Yc and Yc', are immunologically and functionally distinct from each other. The homodimers YcYc (pI 10.4) and Yc'Yc' (pI 7.6) obtained by hybridization in vitro of the two subunits of glutathione S-transferase I (pI 8.8) were isolated and characterized. Results of these studies indicate that only the Yc subunits express glutathione peroxidase activity and cross-react with the antibodies raised against glutathione S-transferase B (YaYc) or rat liver. The Yc' subunits do not express glutathione peroxidase activity and do not cross-react with the antibodies raised against glutathione S-transferase B of rat liver. The amino acid compositions of these two subunits are also different. These two subunits can also be separated by the two-dimensional gel electrophoresis of glutathione S-transferase I (pI 8.8) of rat lung.
Project description:The purification of human placenta and rat liver protein disulphide-isomerase (PDI, EC 184.108.40.206) and the production of a panel of monoclonal antibodies against these proteins are described. The physical and enzymic properties of human PDI and rat PDI were similar; immunological characterization revealed the presence of unique, as well as shared, antigenic determinants. Although purified rat liver PDI was present as three forms differing slightly in Mr value, evidence was presented that the multiple forms represent proteolytic degradation products of a single 59,000-Mr species. Purified human PDI had an apparent Mr of 61,200. Two of the monoclonal antibodies against human PDI partially inactivated the enzyme, and one of these in indirect immunoprecipitation led to the precipitation of all glutathione:insulin transhydrogenase activity from a crude extract of human placenta. Results of immunofluorescence experiments with HT-29 human colon carcinoma cells were consistent with localization of PDI in the nuclear membrane and cell cytoplasm.
Project description:Although trypanothione [T(S)2] is the major thiol component in trypanosomatidae, significant amounts of glutathione are present in Trypanosoma cruzi. This could be explained by the existence of enzymes using glutathione or both glutathione and T(S)2 as cofactors. To assess these hypotheses, a cytosolic fraction of T. cruzi epimastigotes was subjected to affinity chromatography columns using as ligands either S-hexylglutathione or a non-reducible analogue of trypanothione disulphide. A similar protein of 52 kDa was eluted in both cases. Its partial amino acid sequence indicated that it was identical with the protein encoded by the TcAc2 cDNA previously described [Schoneck, Plumas-Marty, Taibi et al. (1994) Biol. Cell 80, 1-10]. This protein showed no significant glutathione transferase activity but surprisingly catalysed the thiol-disulphide exchange between dihydrotrypanothione and glutathione disulphide. The kinetic parameters were in the same range as those determined for trypanothione reductase toward its natural substrate. This trypanothione-glutathione thioltransferase provides a new target for a specific chemotherapy against Chagas' disease and may constitute a link between the glutathione-based metabolism of the host and the trypanothione-based metabolism of the parasite.
Project description:Mixed disulphide formation in the presence of oxidized glutathione reversibly inactivates rabbit skeletal muscle aldolase. Inactivation is allosteric, preferentially modifying Cys-72 on the surface of the aldolase homotetramer distant from active-site locations and subunit interfaces. Ion-exchange chromatography fractionates partly inactivated aldolase into three distinct enzymic species: unmodified enzyme, inactive fully modified enzyme corresponding to one thiol reacted per subunit, and inactive singly modified enzyme in which only one thiol has reacted. Acid-precipitable enzymic intermediates formed in the presence of substrate, D-fructose 1,6-bisphosphate, and product, dihydroxyacetone phosphate, indicates that active site binding is unaffected upon modification. The absence of enamine carbanion formation in the presence of substrate but not product is consistent with mixed disulphide formation's blocking -C-C- cleavage and/or subsequent D-glyceraldehyde 3-phosphate release. Inactivation upon single subunit modification and substrate protection against modification denotes that the blocked step is associated with a long-range conformational transition involving highly co-operative subunit behaviour.