For many years, a major goal of lens protein chemists has been the identification and quantitation of posttranslational modifications occurring in all major proteins of the aging and cataractous human lens. Accomplishment of this goal will provide the basis for understanding, on a molecular level, the nature of biochemical processes that may contribute towards the opacification process. As a first step towards the accomplishment of this objective total alpha-A and alpha-B crystallins will be purified from total cataractous and normal human lens proteins. both these proteins have been implicated in possible etiologies of human cataractogenesis, through decreases in their molecular chaperon properties and through their involvement in protein aggregation. Following endoprotease digestion of the purified crystallins, the mixture of peptides will be screened for the presence of posttranslationally modified sequences, using various procedures that take advantage of recent advances in mass spectrometry and reverse phase HPLC. After purification of the modified peptide, the exact chemical nature of the modification will be identified by mass spectrometry, and the quantitative extent o the modification will be determined by reverse phase HPLC. The above mentioned experimental approach will then be used to identify and quantitate the extent of the same modifications in lens of animal cataract model systems, to determine whether the time course of these modifications precedes the development of lens opacification. Together, the studies outlined in this proposal will identify and quantitate the major posttranslational modification occurring in alpha-A and alpha-B crystallins during human senile cataractogenesis, and will provide the basis for understanding the temporal sequence of these modifications during development of lens opacifications in animal model systems.
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