The two goals of this project are: 1) to determine age and cataract related changes in the levels of human lens proteinases and trypsin inhibitor activities, a 5500 dalton trypsin inhibitor protein (5.5 K TIP) and degraded crystallins; and 2) to determine whether the 5.5 K TIP and degraded crystallins participate in the formation of heavy molecular weight (HMW) proteins in aging human lenses. During the course of this project, the following questions will be addressed: 1) What are the specific and sensitive substrates to assay human lens proteinases? 2) What are levels of proteinases with trypsin specificity and trypsin inhibitors, 5.5 K TIP and degraded crystallins in aging or cataractous lenses? 3) Do degraded crystallins and 5.5 K TIP play a role in the formation of HMW protein aggregates? Specific assay methods with highly sensitive fluorogenic substrates will be used to assay trypsin-like proteinases and trypsin inhibitors. Methods will be sought to fully activate these proteinases in the shortest time possible. Age and cataract specific changes in the levels of the proteinase and trypsin inhibitor activities will be determined by direct assays. The radio-immunoassay method will be developed to determine similar changes in the level of 5.5 K TIP. Similarly, the origin of degraded crystallins and changes in their levels will be determined by immunological methods in aging and cataractous lenses. The proteinase-inhibitor complexes in the crude lens homogenate will be identified by their reactivity with anti-5.5 K TIP serum or with serum prepared against 25 K and 43 K proteinases of bovine lenses. The role of degraded crystallins and the 5.5 K TIP in the formation of HMW protein aggregate will be determined. First the presence of degraded crystallins and 5.5 K TIP will be established among the aggregates of the HMW proteins. Attempts will then be made to generate such HMW protein aggregates in vitro by an interaction among degraded crystallins, 5.5 K TIP and native crystallins.
