Binding and processing of trypsin by cultured bovine corneal endothelial cells

Bovine corneal endothelial cells in culture bind internalize and degrade [¹²⁵I]-trypsin. Binding involves the active site of trypsin and increases as a function of [¹²⁵I]-trypsin concentration. Saturation is observed at a concentration of 0·5-1·0 μg ml^-1. The cell surface binding of [¹²⁵I]-trypsin is specific: a seven-fold excess of unlabeled trypsin abolishes about 60% of the total cell surface-associated radioactivity. In addition, thrombin competes poorly with [¹²⁵I]-trypsin cell surface binding and only 20% of the specific cell surface binding of [¹²⁵I]-trypsin is subjected to competition with thrombin. This fraction of the cell surface-bound [¹²⁵I]-trypsin which is accessible to competition with thrombin appears in a covalent complex of [¹²⁵I]-trypsin · protease-nexin with a molecular weight of 64 000 daltons. The cells, when incubated at 37°C, appear to internalize the cell surface-bound [¹²⁵I]-trypsin at a rate of 0·15-0·25 ng (10⁶ cells)^-1 min^-1. Both the non-covalently cell surface-bound and the proteasenexin (PN) mediated-bond [¹²⁵I]-trypsin are internalized by the cells, but the [¹²⁵I]-trypsin · PN complexes contribute about 75% of the total amount of [¹²⁵I]-trypsin internalized by the cells. The internalized [¹²⁵I]-trypsin is degraded by the cultures at a rate of about 0·05 ng (10⁶ cells)^-1 min^-1 and the degradation products are released by the cells into the incubation medium as a trichloroacetic acid non-precipitable material. Chloroquine inhibits about 60% of the internalization of [¹²⁵I]-trypsin by the cells, and inhibits more than 80% of the degradation process of [¹²⁵I]-trypsin, which indicates that the degradation of the ligand is taking place in lysosomes. Bovine corneal endothelial cells in culture have demonstrated the binding and metabolism of the serine protease trypsin. This described process may indicate the ability of corneal endothelial cells to control the activity of serine proteases in their microenvironment.