GSSG, formation

Figure 15.11 Possible scheme for the formation of free radicals from the metabolism of dopamine. Normally hydrogen peroxide formed from the deamination of DA is detoxified to H2O along with the production of oxidised glutathione (GSSG) from its reduced form (GSH), by glutathione peroxidase. This reaction is restricted in the brain, however, because of low levels of the peroxidase. By contrast the formation of the reactive OH-radical (toxification) is enhanced in the substantia nigra because of its high levels of active iron and the low concentration of transferin to bind it. This potential toxic process could be enhanced by extra DA formed from levodopa in the therapy of PD (see Olanow 1993 and Olanow et al. 1998)...

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

When 4-nitrosophenetol reacted with human red cells formation of acid-stable hemoglobin adducts was observed. The amount of these adducts was markedly increased when the reduction of GSSG was inhibited. These findings suggest GSH-mediated formation of the sulfenamide cation that is not consumed by further GSH-mediated reactions but is available for ring addition reactions56. 

When 1-methyl-2-nitrosoimidazole became available190 it was found that addition of excess GSH to solutions of l-methyl-2-nitrosoimidazole led to a rapid loss of the characteristic absorbance at 360 mn within a few seconds. Preliminary experiments suggested that formation of GSSG and the hydroxylamine was followed by formation of stable thio ethers. It should be noted, however, that detection of free hydroxylamine was unsuccessful57. In cell-free systems l-methyl-2-nitrosoimidazole reacted with excess GSH to form adducts in a 1 3 stoichiometric reaction191. 

Conversely, when 6 was extended N-terminally by a small portion of the prosequence, that is by the Lys-Arg dipeptide to give KR-ET-1 (7), and subjected to oxidation in 0.1 M NH4OAC buffer (pH 9.5) at 25 °C for 24 hours the ratio of native to nonnative-type disulfide isomer increases remarkably (88 12 vs 75 25 for 6), whilst isomer 3 is not detectable. In the presence of GSH/GSSG an additional increase to almost quantitative formation of the native isomer was observed (Table 2). 58 This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of the increase was maintained with similar carboxamide analogues in positions 10 and 18 (Table 2). 

As exemplarily shown in the case of charybdotoxin, a 37-residue peptide with three intramolecular disulfide bonds,[70] operating in redox buffer was crucial for efficient formation of the correct disulfide bonds.[71] When the reduced peptide was oxidized in 0.1 M NHtOAc buffer (pH 8.0) at 0.11 mM concentration in the presence of redox reagents (peptide/GSH/GSSG ratio of 1 60 6), the main product was the native peptide contaminated... 

As already discussed in chapter 4, reactive intermediates can react with reduced GSH either by a direct chemical reaction or by a GSH transferase-mediated reaction. If excessive, these reactions can deplete the cellular GSH. Also, reactive metabolites can oxidize GSH and other thiol groups such as those in proteins and thereby cause a change in thiol status. When the rate of oxidation of GSH exceeds the capacity of GSH reductase, then oxidized glutathione (GSSG) is actively transported out of the cell and thereby lost. Thus, reduced GSH may be removed reversibly by oxidation or formation of mixed disulfides with proteins and irreversibly by conjugation or loss of the oxidized form from the cell. Thus, after exposure of cells to quinones such as menadione, which cause oxidative stress, GSH conjugates, mixed disulfides, and GSSG are formed, all of which will reduce the cellular GSH level. 

Depletion of cellular GSH has been widely studied with hundreds of chemicals including APAP and bromobenzene. These studies demonstrated very clearly that bioactivation followed by GSH adduct formation causes depletion of cytosolic glutathione and oxidative stress as indicated by indicators including enhanced levels of GSSG, lipid peroxidation, and loss of membrane integrity. 

Also, formation of GSNO may lead to either the breakdown of GSNO to form GSSG or reaction with protein thiols to displace NO- and generate a glutathionyl-ated protein. 

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