The goal of this project is to determine the biochemical and functional characteristics of proteins (CBP) present in bovine corneal endothelial (BCE) cells which bind irreversibly to serine proteinases. First, the CBP will be purified from BCE cells grown on Cytodex beads. Affinity chromatography on native or DIP-thrombin-Biogel-A15 columns will be the primary method of purification. The physicochemical characteristics of the purified CBP will be determined. Using radiolabeled enzymes, the kinetics of CBP binding to a variety of plasma and tissue serine proteinases will be analyzed. It will also be determined whether other endogenous BCE proteinases are inhibited by the CBP. One of these proteinases, plasminogen activator (PA), will be purified using para-aminobenzamidine-Sepharose, and binding of the 125I-PA to CBP will be studied. Based on our previous observation that thrombin increases synthesis and release of CBP, the mechanism for these two phenomena will be studied. The extent to which these processes are modulated by changes in cyclic AMP and/or Ca++ concentration will be determined. It will also be determined whether endocytosis of thrombin is necessary for up-regulation of CBP synthesis. The effect of thrombin on the interaction between BCE cell PA and CBP will be studied as well. The second portion of the project will be to characterize the binding of the CBP-protease complex to BCE cells. As a prototype for these experiments, the thrombin-CBP complex will be used. The complex will be purified by affinity chromatography using anti-thrombin IgG-Sepharose and gel filtration. Binding kinetics of the 125I-thrombin-CBP complex to BCE cells will be studied. The receptor for the complex will be identified using the photoreactive reagent methyl-4-ayedobenzoinidate (MABI) to cross-link the thrombin-CBP complex to the BCE cell receptor. The latter will be localized in autoradiograms of SDS-PA gels by radiolabeling the protease-CBP complex or BCE cell surface proteins. The binding of purified PA-CBP complex to BCE cells will also be characterized. These experiments should provide an improved understanding of the role of CBP in regulating protease activity. The CBP may be important for preventing excessive proteolysis in the ocular cornea as well as in other organs.
| Shuman, M A; Polansky, J R; Merkel, C et al. (1988) Tissue plasminogen activator in cultured human trabecular meshwork cells. Predominance of enzyme over plasminogen activator inhibitor. Invest Ophthalmol Vis Sci 29:401-5 |
| Shuman, M A; Merkel 3rd, C H (1985) Urokinase binding to bovine corneal endothelial cells. Exp Eye Res 41:371-82 |
