Abstract
Purpose: :
To investigate the role of αB–crystallin in protecting the enzymes from inactivation and to elucidate the mechanism of αB–crystallin–mediated refolding and reactivation, using glucose–6–phosphate dehydrogenase as a model protein.
Methods: :
Glucose–6–phosphate dehydrogenase (G6PD) was subjected to inactivation either by heat, UV–light or denaturant (GdmCl) in the absence and presence of αB–crystallin. G6PD activity and structural changes, in response to stress, were monitored by spectrophotometric, spectroscopic and size exclusion chromatographic methods. In a set of experiments, αB–crystallin was added to G6PD at different time points of post inactivation and studied refolding and reactivation.
Results: :
G6PD was inactivated either by heat, UV–light or denaturant. Structural changes associated with G6PD inactivation deferred depending upon the inactivating agent. G6PD inactivation by GdmCl resulted from unfolding, via formation of a molten globule–like intermediate, where as heat and UV–light affected pH optima and kinetic properties of G6PD. αB–Crystallin prevented the inactivation of G6PD under the above circumstances when it was present during inactivation but not post–inactivation. Interestingly, the ability of αB–crystallin to refold and reactivate the enzyme was confirmation specific; αB–crystallin was quite effective in refolding and reactivating G6PD from completely unfolded state with GdmCl but not from molten globule–like state. More importantly posttranslational modifications, reported to occur in aged and cataract lenses, decrease the chaperone–like ability of αB–crystallin in protecting the enzymes from inactivation.
Conclusions: :
As a stress protein, protection of enzymes could be one of the important functions of αB–crystallin, apart from suppression of protein aggregation in maintaining the eye lens transparency..
Keywords: chaperones • protein structure/function • protein modifications-post translational