INFLUENCE OF PROTEIN-GLUTATHIONE MIXED DISULFIDE ON THE CHAPERONE-LIKE FUNCTION OF ALPHA-CRYSTALLIN

In an earlier report we showed that incubation of alpha-crystallin wit h oxidized glutathione results in significant loss of its chaperone-li ke activity. In the present study, we determined the effect of protein -glutathione mixed disulfides (PSSG), formed at Cys-131 in bovine alph a A-crystallin, and Cys-131 and Cys-142 in human alpha A-crystallin, o n the function of alpha-crystallin as a molecular chaperone. After inc ubation of calf and young human alpha(L)-crystallin fractions with oxi dized glutathione, levels of PSSG were determined by performic acid ox idation of the mixed disulfides followed by reversed-phase high pressu re liquid chromatography separation of phenylisothiocyanate-derivatize d glutathione sulfonic acid, Levels of PSSG increased from 0.01 to 0.1 4 nmol/nmol (20 kDa) in bovine alpha(L)-crystallin and from 0.022 to 0 .25 nmol/nmol in human alpha(L)-crystallin. The presence of glutathion e adducts at Cys-131 and Cys-142 mere confirmed by mass spectral analy sis. The chaperone-like activity was determined by the heat denaturati on assay using beta(L)-crystallin as the target protein, To examine th e reversibility of the effect of mixed disulfides on chaperone activit y, studies were done before and after reduction with the glutathione r eductase system. Increased levels of PSSG resulted in lower chaperone activities, Treatment with the glutathione reductase system led to 80% reduction in PSSG levels with a concomitant recovery of the chaperone activity, These results suggest that cysteine(s) in the alpha A-cryst allin subunit play an important role in the function of alpha-crystall in as a molecular chaperone.