The long-range goal of this research proposal is to determine whether the protein-disulfide interchange enzyme (protein-disulfide isomerase/oxidoreductase) has any function in the normal eye lens and/or in cataract formation, in particular in the generation of disulfide-linked high molecular weight (HMW) aggregates commonly seen in cataracts. The enzyme is referred to here as glutathione-insulin transhydrogenase (GIT), for consistency with previous reports form this laboratory. GIT occurs ubiquitously in tissues, including retina; whether the enzyme is present in the lens or in other ocular components is not known. In the presence of a thiol (e.g., glutathione, cysteamine), GIT catalyzes via disulfide interchange the cleavage or formation of disulfide bonds in certain proteins (e.g., insulin, native or scrambled proinsulin, scrambled ribonuclease, etc.), resulting in inactivation or activation of the protein substrate (including the generation of an aggregated product from one protein, insulin).
Specific aims of this pilot project are to determine whether the lens at any time in life contains (1) GIT or GIT-like enzyme and/or (2) a disulfide-protein(s) which funcitons as a substrate(s) for GIT, resulting in the generation of high molecular weight aggregates (HMW). Experimentally, for aim #1, lenses (human and/or bovine) obtained at different ages will be examined for the presence of GIT both by enzymological and immunological methods (immunodiffusion tests, quantitative antigen-antibody titrations, immunoblots and radioimmunoassay).
For aim #2, the lens (obtained from rats) will be exposed to purified GIT over short to prolonged periods of time in organ culture, and monitored for opacity morphologically and for HMW by SDS-PAGE. These investigations will allow us to test the hypothesis that GIT-mediated (from lens or retina) disulfide interchange of (older) lens proteins contributes to (or triggers) the formation of HMW and thus to the development of certain cataracts.
