Glutathiolation Enhances the Degradation of γC-crystallin in Lens and Reticulocyte Lysates, Partially via the Ubiquitin-Proteasome Pathway

METHODS. Recombinant human C-crystallin was mixed with various concentrations of glutathione (GSH) and diamide at 25°C for 1 hour. The extent of glutathiolation of the Ccrystallin was determined by mass spectrometry. Native and S-glutathiolated C-crystallins were labeled with I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Far UV circular dichroism, tryptophan fluorescence intensity, and binding to the hydrophobic fluorescence probe 4,4 -dianilino-1,1 -binaphthalene-5,5 -disulfonic acid (Bis-ANS), were used to characterize the native and glutathiolated C-crystallins.