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BR Geereddy, S Narayanan, PY Reddy, A Surolia; Abrin, A New Model Substrate for Alpha-crystallin In Vitro Chaperone Activity . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3565.
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Purpose: To demonstrate that DTT-induced aggregation of abrin serves as a model aggregation system for studying α-crystallin chaperone function in vitro. Methods: Suppression of DTT-induced aggregation of abrin B-chain by recombinant αA- and αB-crystallin was monitered by light scattering method and complex formation between α-crystallin and abrin B-chain was assessed by SDS-PAGE and gel filtration. Results:Eye lens αA- and αB-crystallins are subject to changes with regard to conformational stability, hydrophobicity and chaperone activity under non-native conditions. Therefore, it is important to assess the protective effect of αA- and αB-crystallins under native conditions. With the exception of insulin, α-lactalbumin, and few enzyme assays, not many model substrates are available to investigate the chaperone mechanism of α-crystallin under native conditions. In this study, we demonstrate that abrin, a ribosome inactivating lectin like protein isolated from Abrus precatorius, aggregates upon reduction of the disulfide bridge with DTT in a similar fashion to insulin as monitored by light scattering. This aggregation process could be suppressed by, recombinant αA- and αB-crystallin in a concentration dependent manner, mostly by binding to aggregation prone abrin B-chain. SDS-PAGE and gel filtration results indicate that there is a soluble complex formation between α-crystallin and abrin B-chain. Interestingly, incontrast to insulin, there is no significant difference between αA- and αB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures. Conclusions: These results suggest that due to relatively larger size of its B-chain (33 kDa), compared to insulin B-chain (about 3 kDa), abrin may serve as a better model substrate for in vitro chaperone studies of α-crystallin. In addition, the data also aid in addressing the substrate specificity and hydrophobicity with respect to differential chaperone activity of αA- and αB-crystallins.
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