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 with oxidized glutathione results in significant loss of its chaperone-like activity. In the present study, we determined the effect of protein-glutathione mixed disulfides (PSSG), formed at Cys-131 in bovine alphaA-crystallin, and Cys-131 and Cys-142 in human alphaA-crystallin, on the function of alpha-crystallin as a molecular chaperone. After incubation of calf and young human alphaL-crystallin fractions with oxidized glutathione, levels of PSSG were determined by performic acid oxidation of the mixed disulfides followed by reversed-phase high pressure liquid chromatography separation of phenylisothiocyanate-derivatized glutathione sulfonic acid. Levels of PSSG increased from 0.01 to 0.14 nmol/nmol (20 kDa) in bovine alphaL-crystallin and from 0.022 to 0.25 nmol/nmol in human alphaL-crystallin. The presence of glutathione adducts at Cys-131 and Cys-142 were confirmed by mass spectral analysis. The chaperone-like activity was determined by the heat denaturation assay using betaL-crystallin as the target protein. To examine the reversibility of the effect of mixed disulfides on chaperone activity, studies were done before and after reduction with the glutathione reductase 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 alphaA-crystallin subunit play an important role in the function of alpha-crystallin as a molecular chaperone.
Cherian
M
M
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912-2100, USA.
Smith
J B
JB
Jiang
X Y
XY
Abraham
E C
EC
D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000378:metabolism*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D018832:Molecular Chaperones / Q000378:metabolism*
D003459:Crystallins; D018832:Molecular Chaperones; C031586:cysteine-glutathione disulfide; D005980:Glutathione Reductase; D005978:Glutathione; D003545:Cysteine