Revival of glutathione reductase in human cataractous and clear lens extracts by thioredoxin and thioredoxin reductase, in conjunction with alpha-crystallin or thioltransferase.

Glutathione reductase (GR) plays a key role in maintaining thiol groups in the lens, and its activity decreases with aging and cataract formation. Mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), or the Trx/TrxR system, participates in the repair of oxidatively damaged lens proteins and enzymes. Alpha-crystallin, a molecular chaperone, prevents the aggregation of partially denatured proteins under various stress conditions. Thioltransferase (TTase, or glutaredoxin) can maintain the homeostasis of lens protein thiols thus protecting against oxidative stress. We investigated whether the Trx/TrxR system can revive GR activity in both the cortex and nucleus of human cataract and clear aged lenses and whether alpha-crystallin and TTase can help this effect. The GR activity in the cortex and nucleus of the cataractous lenses was significantly lower than that of the aged clear lenses. The highest activity in the cortex was observed in the clear aged lenses. The combination of Trx and TrxR revived the activity of GR from both the cortex and nucleus of aged clear lenses. However, in cataract lenses (grade II and grade IV), there was a statistically significant recovery of GR activity in the cortex, but not in the nucleus. No recovery was observed when Trx or TrxR were used separately. Alpha-crystallin successfully revived GR activity in the cortex of cataract grade II lenses, but not in the nucleus. The combination of alpha-crystallin and Trx/TrxR gave a further increase of activity. TTase alone revived some of the GR activity but together with the Trx/TrxR system gave no statistically significant enhancement of GR activity. These results indicate that both disulfide bond formation and protein unfolding are responsible for GR inactivation.

Glutathione reductase from human cataract lenses can be revived by reducing agents and by a molecular chaperone, alpha-crystallin.

PURPOSE: The aim of this study was to investigate how glutathione reductase (GR) loses its activity during cataract formation and whether it is possible to revive it back to the normal levels. METHOD: In this study, endogenous as well as synthetic reducing systems (GSH, TTase, DTT, captopril) and alpha-crystallin at different concentrations were incubated with the soluble fraction of human cataract lens protein. The activity of glutathione reductase with or without the reducing agents and alpha-crystallin was tested, and the difference in activity gained was calculated. RESULTS: Five agents (GSH, DTT, TTase, captopril, alpha-low crystallin) were able to revive the activity of GR from human cataract lenses to different extents. CONCLUSION: This study shows that human lens GR activity was revived by different reducing agents as well as by a molecular chaperone (alpha-crystallin).

Revival of inactive glyceraldehyde 3-phosphate dehydrogenase in human cataract lenses by reduction.

In this study, endogenous as well as synthetic reducing systems were shown to reduce the disulphide bonds formed in glyceraldehyde 3-phosphate dehydrogenase, an important glycolytic enzyme previously reported to have lost its activity in human cataract lenses, resulting in reviving the activity of this enzyme. Disulphide bond formation is a non-specific posttranslational modification of proteins, which leads to a loss of function of the affected protein. When an enzyme is targeted, this harmful effect can be easily detected by monitoring the change of activity. Endogenous reducing systems are responsible for breaking these bonds and returning the protein (enzyme) to its natural state, when these mechanisms fail to do so, the loss of enzyme activity will be permanent.