Protein S-thiolation and redox regulation of membrane-bound glutathione transferase

Membrane-bound GST transferase (GSTm) occurs in hepatic microsomal and plasma membranes as well as in the outer mitochondrial membrane, and it is known to be activated by N-ethylmaleimide. We recently analysed the activation by GSSG in some detail. The approximately 5-fold stimulation is reversed upon reduction of GSSG by GSSG reductase. In steady-state experiments, the Kox value was determined to be 0.05, i.e. 20 times more GSSG than GSH produces half-maximal activation. Kox is independent of the total glutathione concentration, indicating that S-thiolation by mixed disulfide formation, rather than interchain or intrachain disulfide bridge formation, is responsible for activation. In Western blots, a 17.7 kDa band, in addition to the 17.3 kDa band, was detected upon treatment with GSSG or with GSH plus t-butyl hydroperoxide. We suggest that under oxidative stress, GSTm is activated through direct S-thiolation of the enzyme. Dethiolation occurs via thiol disulfide exchange governed by the cellular glutathione redox state.