Glycation (non-enzymic glycosylation) inactivates glutathione reductase.
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ABSTRACT:
SUBMITTER: Blakytny R
PROVIDER: S-EPMC1132114 | biostudies-other | 1992 Nov
REPOSITORIES: biostudies-other
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Project description:The secretory enzyme extracellular superoxide dismutase (EC-SOD) is in plasma heterogenous with regard to heparin-affinity and can be divided into three fractions, A that lacks affinity, B with intermediate affinity and C with high affinity. The C fraction forms an equilibrium between the plasma phase and heparan sulphate proteoglycan on the surface of the endothelium. In vitro EC-SOD C could be time-dependently glycated. The enzymic activity was not affected in glycated EC-SOD, but the high heparin-affinity was lost in about half of the studied glycated fraction. Addition of heparin decreased the glycation in vitro, and EC-SOD C modified with the lysine-specific reagent trinitrobenzenesulphonic acid could not be glycated in vitro. The findings suggest that the glycation sites are localized rather far away from the active site and may occur on lysine residues in the heparin-binding domain in the C-terminal end of the enzyme. The proportion of glycated EC-SOD in serum of diabetic patients was considerably higher than in normal subjects. Of the subfractions, EC-SOD B was by far the most highly glycated, followed by EC-SOD A. EC-SOD C was glycated only to be a minor extent. The findings suggest that glycation is one of the factors that contribute to the heterogeneity in heparin-affinity of plasma EC-SOD. Since this phenomenon is increased in diabetes, the cell-surface-associated EC-SOD may be decreased in this disease, increasing the susceptibility of cells to superoxide radicals produced in the extracellular space.
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Project description:A complex of haemoglobin and GSH was prepared by incubating haemoglobin with GSH and acetylphenylhydrazine. GSH could be released from the crude preparation by incubation with NADPH. However, when the haemoglobin preparation was separated from glutathione reductase by DEAE-Sephadex chromatography, NADPH no longer released GSH. Rather, the addition of a combination of either partially purified human erythrocyte or crystalline glutathione reductase and NADPH was required to release GSH from the haemoglobin-GSH complex. This complex is commonly believed to represent a mixed disulphide of GSH and the cysteine-beta-93 thiol group. This interpretation was supported by the finding that prior alkylation of available haemoglobin thiol groups prevented the formation of the complex. By using haemoglobin-[(35)S]GSH complex as a substrate, it was shown that GSH itself released the radioactivity from the complex only very slowly. In contrast, the release of [(35)S]GSH was very rapid in the presence of NADPH and glutathione reductase. This suggests that the cleavage of the haemoglobin-GSH complex is not mediated by GSH with cyclic reduction of GSSG formed, but rather proceeds enzymically through glutathione reductase.
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Project description:The non-enzymic post-translational glycosylation of certain proteins has been implicated in the production of diabetic sequelae. In the present paper the possibility that it is not the glucose aldehyde that binds to proteins but a dicarbonyl autoxidation product is investigated. Earlier experiments may not have distinguished between these two possibilities. The rate of binding of 2-deoxyglucose (a non-autoxidizable sugar) to lens alpha-crystallin is compared with that of glucose (an autoxidizable sugar). The stabilized Schiff-base adducts was investigated by using proton n.m.r. and fast-atom-bombardment mass spectroscopy to distinguish whether they are the product of aldehyde or dicarbonyl addition. We conclude that it is the open-chain aldehyde of glucose that binds initially to amino groups and that there is no participation of dicarbonyl autoxidation products in the initial non-enzymic protein glycosylation reaction.
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Project description:Non-enzymic glycation of collagen involves a series of complex reactions ultimately leading to the formation of intermolecular cross-links resulting in changes in its physical properties. During analysis for the fluorescent cross-link pentosidine we identified the presence of an additional component (Cmpd K) in both glucose and ribose incubations. Cmpd K was formed more quickly than pentosidine in glucose incubations and more slowly than pentosidine in ribose incubations. Cmpd K represented 45% of the total fluorescence compared with 15% for pentosidine in glucose incubations and 25% of the total fluorescence compared with 30% for pentosidine in the ribose incubations. Cmpd K is not an artefact of in vitro incubations, as it was shown to be present in dermal tissue from diabetic patients. Subsequent high-resolution h.p.l.c. analysis of glucose-incubated collagen revealed Cmpd K comprise two components (K1 and K2). Further, a similar analysis of Cmpd K from the ribose incubations revealed two different components (K3 and K4). These differences indicate alternative mechanisms for the reactions of glucose and ribose with collagen. The amounts of these fluorescent components and the pentosidine cross-link determined for both glucose and ribose glycation were found to be far too low (about one pentosidine molecules per 200 collagen molecules after 6 months incubation with glucose) to account for the extensive cross-linking responsible for the changes in physical properties, suggesting that a further additional series of cross-links are formed. We have analysed the non-fluorescent high-molecular-mass components and identified a new component that increases with time of in vitro incubation and is present in the skin of diabetic patients. This component is present in sufficient quantities (estimated at one cross-link per two collagen molecules) to account for the changes in physical properties occurring in vitro.
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Project description:Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.
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Project description:Non-enzymic glycosylation of basic fibroblast growth factor (bFGF, FGF-2) has recently been demonstrated to decrease the mitogenic activity of intracellular bFGF. Loss of this bioactivity has been implicated in impaired wound healing and microangiopathies of diabetes mellitus. In addition to intracellular localization, bFGF is also widely distributed in the extracellular matrix, primarily bound to heparan sulphate proteoglycans (HSPGs). Nonetheless, it is not clear if non-enzymic glycosylation similarly inactivates matrix-bound bFGF. To investigate this, we measured the effect of non-enzymic glycosylation on bFGF bound to heparin, heparan sulphate and related compounds. Incubation of bFGF with the glycosylating agents glyceraldehyde 3-phosphate (G3P; 25 mM) or fructose (250 mM) resulted in loss of 90% and 40% of the mitogenic activity of bFGF respectively. Treatment with G3P and fructose also decreased the binding of bFGF to a heparin column. If heparin was added to bFGF prior to non-enzymic glycosylation, the mitogenic activity and heparin affinity of bFGF were nearly completely preserved. A similar protective effect was demonstrated by heparan sulphate, low-molecular-mass heparin and the polysaccharide dextran sulphate, but not by chondroitin sulphate. Whereas non-enzymic glycosylation of bFGF with G3P impaired its ability to stimulate c-myc mRNA expression in fibroblasts, no such impairment was noticeable when bFGF was glycosylated in the presence of heparin. Taken together, these results suggest that HSPG-bound bFGF is resistant to non-enzymic glycosylation-induced loss of activity. Therefore, alteration of this pool probably does not contribute to impaired wound healing seen in diabetes mellitus.
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