THE INTERACTIONS OF PROTEIN INHIBITORS WITH TUMOR PROTEASES STUDIED IN SOLUTION AND IMMOBILIZED ON CELL-SURFACES IN FROZEN-SECTIONS

The cell surface protease guanidinobenzoatase (GB) has been purified f r om human colonic and lung carcinoma tissue by an affinity step invol ving the binding of the enzyme either onto fibrin fibrils or onto agma tine-sepharose. The inhibitor protein (I) was extracted from the cytop lasm of tumour cells and isolated by an affinity step involving the bi nding of I to GB on the surface of cultured carcinoma cells. The inter action of GB and I in solution was followed by kinetic studies employi ng the release of the fluorescent 4-methylumbelliferone (MU) from the synthetic substrate 4-methylumbelliferyl-p-guanidinobenzoate (MUGB). T he interaction of soluble I with membrane bound GB was followed by usi ng the yellow fluorescent probe 9-aminoacridine (9AA) which binds to a ctive GB but not to GB-I. The results of these studies demonstrated th e presence of isoenzymic froms of GB which were recognised specificall y by their appropriate isoinhibitor, isolated from the appropriate cel l type. This high degree of selectivity suggests a cell specific regul atory role for the inhibitors and the possibility that they might be u sed for the delivery of cytotoxic molecules to the surface of specific types of tumour cells.