Affinity preparation of a protein inhibitor recognising a cell surface protease.

Abstract

Epithelial cell surfaces possess a trypsin-like protease, referred to as guanidinobenzoatase (GB). The cytoplasm of these cells contains an extractable protein (I) which recognises the cell surface GB by forming an enzyme-inhibitor complex (GB-I). Rhodamine-agmatine (Rh-Agm) was designed as a red fluorescent probe, directed to the active centre of GB, which can be used to locate cells with GB, employing fluorescence microscopy. Rh-Agm has a high affinity for GB and will displace I from GB-I on the surfaces of cells in frozen sections. Rh-Agm has been used to displace I from immobilised GB-I complexes on the surface of cultured colonic carcinoma cells in an affinity procedure aimed at purifying the inhibitors of GB obtained from cultured carcinoma cells. These inhibitors have been tested on protected frozen sections of normal colon and carcinoma of the colon, the formation of GB-I complexes being followed by a second yellow fluorescent probe which competes for the active centre of GB. The study of the protein-protein interactions to form GB-I has been facilitated by employing two synthetic fluorescent inhibitors of GB with differing affinities for GB and different fluorescent properties. The use of sections of tissue in this study has enabled a sequence of reactions to be carried out on the same cell surface GB, such that reversible inhibition reactions can be quickly demonstrated and recorded by fluorescence microscopy.