Investigations of Glutathione Conjugation in Vitro by 1H NMR Spectroscopy. Uncatalyzed and Glutathione Transferase-Catalyzed Reactions

Abstract

Conjugation reactions of glutathione (GSH) and related thiols with diethyl maleate (DEM) and other alpha, beta-unsaturated carbonyl compounds have been investigated by 1H NMR spectroscopy. The products from the reaction with DEM and diethyl fumarate (DEF) are shown to be the diastereomers of S-(alpha,beta-diethoxycarbonylethyl)glutathione. During the course of the reaction, DEM isomerized to DEF, and the rate of isomerization was dependent upon whether the solvent was 1H2O or 2H2O. The observed rate data exhibit apparent second order kinetic behavior. The reaction of maleate with GSH was considerably slower, and solvent-dependent isomerization was observed, while little reaction of fumarate with GSH was observed at pH 6.5. Reaction of DEM with N-acetyl-L-cysteine followed a similar course to that of GSH, and although L-cysteine reacted rapidly with DEM, it did not promote the isomerization of DEM. Reactions involving penicillamine and N-acetylpenicillamine were considerably slower. Conjugation reactions catalyzed by commercial GSH transferases and selected rat and human purified isoenzymes were also investigated. Of those isoenzymes studied, rat GSH transferase 4-4 was found to exert the greatest degree of stereo control in conjugation reactions with DEF.

Redox Regulation of GA-binding Protein-α DNA Binding Activity

“…Previous reports have demonstrated that DEM treatment increases the relative GSSG levels of cells (55,60,61), and our results show that DEM treatment of 3T3 cells leads to inactivation of GABP DNA binding activity, possibly by direct oxidation by GSSG. To test the ability of GSSG to directly oxidize GABP and thereby inhibit its DNA binding activity, 3T3 cell nuclear extracts were treated with DTT, GSSG, or GSH, and the effect on GABP dimer and tetramer DNA binding activities was determined by EMSA analysis using the dPEA3-0 probe (Fig.…”

“…NAC may protect cells from the action of DEM by direct chemical conjugation with DEM, by antioxidant action, or by replenishing GSH levels through biosynthesis of GSH from NAC, its immediate biosynthetic precursor (55). Chemical (nonenzymatic) reaction of NAC with DEM is relatively slow (Ͻ20% after 1 h), whereas enzymatically catalyzed DEM depletion of GSH in cultured cells is nearly 80% complete in Ͻ1 h (60,61). Under the conditions of our experiments, only a small fraction of the DEM would likely be lost to direct chemical conjugation with NAC, suggesting that the protective effect of NAC on DEM inhibition of GABP DNA binding is the restoration of GSH levels.…”

Glutathione Depletion Down-regulates Tumor Necrosis Factor α-induced NF-κB Activity via IκB Kinase-dependent and -independent Mechanisms

“…DEM (32,33) is a commonly used agent for acute GSH depletion because it diffuses freely into cells due to its hydrophobicity and forms glutathione conjugates in reactions catalyzed by glutathione S-transferase (Fig. 3A).…”

Antigenicity and immunogenicity of sulphamethoxazole: demonstration of metabolism‐dependent haptenation and T‐cell proliferation in vivo

“…Diethyl maleate binds irreversibly to glutathione and is frequently used to deplete intracellular glutathione (Boyland & Chasseaud, 1967;Kubal et al, 1995). In these studies, diethyl maleate caused a signi®cant depletion of hepatic (14.6+9.6% of control levels; P50.05) and extrahepatic [PBMC (55.0+22.2% of control levels; P50.05); splenocytes (51.4+17.6% of control levels; P50.05)] glutathione.…”