The objective of this research is to investigate the complex interplay between intrinsic and extrinsic factors that determine the stability of the crystallins, the major vertebrate eye lens proteins. It therefore relates to the problem of cataract development, and also has implications for the study of aging and turnover of lens proteins in general. It has recently been found that several crystallins are very similar or event identical to regular enzymes. However, they must have specific properties to function as extremely stable and long-lived """"""""structural"""""""" proteins in the lens. To identify these specific properties we intend to analyse intrinsic and extrinsic factors that contribute to the stability and activity of crystallins and homologous enzymes. To achieve this objective we proposed two parallel lines of investigation. 1. To compare the in vitro stability properties of crystalline with their enzyme homologues. Kinetic and stability properties as well as distribution and quantities of different eye lens crystallins will be compared with the same properties of the identical or related enzymes, isolated from other tissues. Furthermore, the effects of posttranslational modifications on stability and activity will be taken into account. This should provide novel insights into the intricate problem of lens protein aging and turnover. 2. To determine factors in the intracellular environment, which influence the stability and turnover of eye lens proteins. The effects of extrinsic factors on structure and stability of the crystallins/enzymes will be determined, by in vitro incubations of lenses and purified proteins in media which contain factors that are known to affect lens transparency and lead to cataract development.
