FIG. 5.

Mutation of Cys346 abolishes glutathionylation of CBS. (a) Wild-type and Cys346Ser CBS were treated with 1 mM DA and 1.5 mM GSH for 30 min at room temperature, subjected to nonreducing SDS-PAGE followed by immunoblotting with anti-SSG antibody (top) or anti-CBS antibody (bottom). The CBS monomer (63 kDa) and a dimeric species were detected with anti-SSG antibody in the DA+GSH-treated wild type but not in Cys346Ser CBS. In contrast, anti-CBS antibody was observed in both the monomeric and cross-linked dimeric species in samples that were oxidized with DA. Wild-type and Cys346Ser CBS were treated with±1 mM DA and 1.5 mM GSH for 30 min at room temperature (b) or with±25 μM H₂O₂ for 30 min followed by 125 μM GSH (c). The samples were then dialyzed, and CBS activity was measured using the methylcysteine assay as described in Figure 1 legend. *p<0.05 or **p<0.01 versus controls. N.S., not significant.