AFFINITY PREPARATION OF A PROTEIN INHIBITOR RECOGNIZING A CELL-SURFACE PROTEASE

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 formin g 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 use d to displace I from immobilised GB-I complexes on the surface of cult ured colonic carcinoma cells in an affinity procedure aimed at purifyi ng the inhibitors of GB obtained from cultured carcinoma cells. These inhibitors have been tested on protected frozen sections of normal col on and carcinoma of the colon, the formation of GB-I complexes being f ollowed by a second yellow fluorescent probe which competes for the ac tive centre of GB. The study of the protein-protein interactions to fo rm GB-I has been facilitated by employing two synthetic fluorescent in hibitors of GB with differing affinities for GB and different fluoresc ent properties. The use of sections of tissue in this study has enable d a sequence of reactions to be carried out on the same cell surface G B, such that reversible inhibition reactions can be quickly demonstrat ed and recorded by fluorescence microscopy.