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B.R. Geereddy, M.S. Kumar, P.Y. Reddy; Relevance of In Vitro Aggregation Assays to Study the Influence of Modifications on Chaperone Function of -Crystallin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2374.
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Purpose: To understand the mechanism of increased chaperone activity due to modification by methylglyoxal and nutritional factors. Methods: Bovine α-crystallin was incubated with different concentrations of methylglyoxal (MG) and glyoxal (G) for three days and dialysed. Chaperone activity was measured by various in vitro aggregation and functional assays. Secondary/ tertiary structure and hydrophobicity were monitored by CD and fluorescence, stability by DSC and fluorescence unfolding and oligomeric size by HPLC. Results: Various modifications are known to occur to α-crystallin during aging and most of them are accelerated in diabetic conditions. It is of significant importance to investigate the effect of these modifications on chaperone function of α-crystallin and modulation by various dietary/ nutritional factors. Modification of α-crystallin with MG and G, whose levels are shown to increase several fold in cataract and diabetes, has lead to increased chaperone activity as measured by various in vitro aggregation methods. Data indicate that there is a loss of secondary/ tertiary structure, decreased stability and hydrophobicity upon incubation with MG and G. In addition oligomeric size of the crystallin is increased due to modification. Increased chaperone activity in the absence of considerable structure along with decreased stability and hydrophobicity indicates that increased oligomeric size of the protein upon modification may be providing a surface non-specifically to the unfolding substrates. Therefore, we have assessed the chaperone activity of modified protein against UV-induced inactivation of glucose-6-phosphate dehydrogenase (non-aggregation assay). Contrary to aggregation assays, modified crystallin has not displayed higher chaperone activity in non-aggregation system rather showed a marginally reduced activity. Furthermore, α-crystallin isolated from the eye lens of rats fed with 50% vitamin restricted diet also showed greater chaperone activity, while aggregation tendency of ß- and γ-crystallins isolated from these animals is higher. Conclusion: These preliminary results raise concern about the relevance of in vitro aggregation assays to assess in vivo chaperone potential of α-crystallin.
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