Several proteins have been recognized by proteomic analysis as targets of glutathionylation in response to oxidative stress [5-7]. Glutathionylation of -actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. -actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that this mechanism involved formation of a cysteinyl sulfenic acid residue in -actin. Conclusion We conclude that glutathionylation of -actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. We also show that this reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms. Background Protein glutathionylation occurs by the formation of a mixed disulfide Budesonide between a protein cysteinyl residue and glutathione [1,2]. It occurs in response to oxidative stress and has been suggested to be a mechanism to protect against irreversible oxidation of crucial protein cysteinyl residues. Protein glutathionylation is usually reversible and reduction of the mixed glutathione-protein disulfides is usually efficiently catalyzed by glutaredoxins [3,4]. Several proteins have been recognized by proteomic analysis as targets of glutathionylation in response to oxidative stress [5-7]. However, there is also evidence that protein glutathionylation may occur in the absence of exogenous oxidative stress and several studies suggest that it may be an important redox dependent signaling pathway and that glutathionylation directly regulates protein functions em in vivo /em [1,2]. Actin was early identified as one of the most abundant protein that is glutathionylated in cells. Actin glutathionylation was first reported to occur in human neutrophiles stimulated with phorbol diesters to induce production of superoxide [8]. However, subsequent studies have shown that actin is usually constitutively glutathionylated in cells even in the absence of oxidative stress [9,10]. Glutathionylation efficiently inhibits actin polymerization and accordingly affects the cellular cytoskeleton structure [9-11]. Growth factors, such as epidermal growth factor, as well as interactions with the extracelllar matrix via integrin receptors has been shown to regulate actin polymerization by affecting the level of glutathionylation [9,12]. The molecular mechanism that mediates actin glutathionylation em in vivo /em is usually unclear. Proposed mechanisms include oxidation Budesonide of reduced glutathione (GSH) to glutathione disulfide (GSSG), which in turn can undergo thiol-disulfide exchange reactions with protein thiols to form glutathionyated proteins. However, under physiological conditions the concentration of GSH greatly exceeds the concentration of GSSG in cells, and unless the GSSG concentrations reach very high levels, GSSG unlikely glutathionylate proteins based on common redox potentials of cysteinyl residues Budesonide [13]. There is also several lines of experimental evidence against thiol-disulfide exchange with GSSG as the physiological mechanism mediating actin glutathionylation [14,15]. Other proposed mechanism includes formation of reactive glutathione species, such as glutathione-thiyl radicals, that can react with cysteinyl residues to form mixed disulfides. Studies on Budesonide actin glutathionylation have predominantly been performed on cell lines of non-muscle origin. However, several isoforms of actin exists in mammalian cells with differences in tissue distribution: -actin is present in muscle mass cells whereas – and -actin are components of the cytoskeleton in all non-muscle cells [16]. The actin isoforms show structural similarity with Rabbit Polyclonal to A4GNT 90% identical primary structure. We have in the present paper analyzed glutathionylation of skeletal muscle mass -actin and non-muscle -actin em in vitro /em using a highly sensitive enzyme-linked immunosorbant assay for detection of actin glutathionylation. In summary, we provide evidence that glutathionylation of -actin occurs via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. Results A highly sensitive ELISA for detection of actin glutathionylation em in vitro /em We used a monoclonal anti-glutathione antibody to develop an ELISA for detection of actin glutathionylation em in vitro /em . 96-well plates were coated with – or -actin and incubated with DTT to reduce any disulfides present in the samples. Diamide is a strong thiol-specific oxidant and incubation with diamide and GSH has successfully been used to glutathionylate actin em in vitro /em [14,15]. We incubated the actins with combinations of 1 1 mM GSH and/or 1 mM diamide (Physique ?(Figure1A).1A). Actin glutathionylation was detected in the assay with an anti-glutathione antibody. No transmission was detected in the wells.