Background: Cysteinyl residues in actin are glutathionylated, ie. form a mixed disulfide with glutathione, even in the absence of exogenous oxidative stress. Glutathionylation inhibits actin polymerization and reversible actin glutathionylation is a redox dependent mechanism for regulation of the cytoskeleton structure. The molecular mechanism that mediates actin glutathionylation in vivo is unclear.
Results: We have studied glutathionylation of alpha- and beta-actin in vitro using an enzyme-linked immunosorbant assay with a monoclonal anti-glutathione antibody. alpha- and beta-actin were both glutathionylated when incubated with reduced glutathione (GSH) combined with diamide as a thiol oxidant. However, beta-actin was also glutathionylated by both glutathione disulfide (GSSG) and GSH in the absence of diamide whereas alpha-actin was poorly glutathionylated by GSH or GSSG. Glutathionylation of beta-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. beta-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in beta-actin.
Conclusion: We conclude that glutathionylation of beta-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 the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.