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Biochemical Society Transactions ( 1 99 1 ) 19 427s Exchange of inhibitors and proteases at tumour cell surfaces. FRANK S STEVEN, MARGARET M GRIFFIN, NEIL J BULLEID and BERNARD S BROWN Department of Biochemistry and Molecular Biology, School of Biological Sciences, Stopford Building, Oxford Road, Manchester MI3 9PT, U.K. Tumour cells possess a cell-surface protease, referred to as guanidinobenzoatase (GB), which binds the yellow-fluorescent probe 9-aminoacridine (9-AA) at the active centre as a competitive inhibitor [I]. It has now been shown that tissue plasminogen activator (t-PA) cleaves guanidinobenzoates [2] in a similar manner to GB and that GB and t-PA are closely similar, if not identical proteases [3]. The GB of intact rat leukaemia cells has been shown to be located at the external surface of the cell, and this was recognised by a cytoplasmic protein inhibitor of this cell-surface GB [4], the latter forming an enzyme-inhibitor complex which failed to bind 9-AA. For this study we required frozen sections of easily recognisable tumour cells: we chose the squamous cell carcinoma obtained from the oral cavity to illustrate our findings. To avoid problems associated with the cytoplasmic inhibitor of GB [4] we prepared protected sections [5] in which the GB remained in the native state on the tumour cell surface whilst the cytoplasmic inhibitors were extracted from thin sections (Figure 1). The GB on these protecteij sections was then used as a target for potential inhibitors of GB, this interaction being followed by use of 9-AA and fluorescent microscopy of the section. Protected sections were used to demonstrate the transfer of cytoplasmic inhibitors from fresh frozen sections to the protected sections resulting in the formation of a reversible enzyme-inhibitor complex. The GB on sections was inhibited by dansylglutamyl-glycylarginylchloromethyl ketone, an inhibitor of t-PA [6] which was not displaced by 9-AA. Y u w Fig. 2. Fibrin removes proteases but not receptor The GB on protected sections of tumour cells was removed by contact with fibrin fibrils, as demonstrated by the cells' subsequent failure to bind 9-AA (Figure 2). We assumed that the GB on the cell surface was originally bound by a receptor protein [7] which would remain attached to the cell surface after fibrin treatment. If this concept was valid then these cells might bind pure t-PA in place of GB. We treated such sections with t-PA that had been labelled with Texas red, and demonstrated the binding of this red-fluorescent probe to the surface of the tumour cells. The binding of the fluorescent t-PA was shown to be competitive with the binding of unlabelled tPA. We suggest that p r o t e c a sections containing tumour cells may provide a good test system for: (a) the interaction of proteases with secreted inhibitors, (b) the exchange of proteases which bind to the putative receptor protein on the cell surface, and (c) studies on the receptor protein. 1. 2. 3. 4. 5. 6. 7. GI3 Fig. 1. Preparation of protected cells Steven, F.S., Griffin, M.M. & Al-Ahmad, R.K. (1985) Eur. J. Biochem. 149, 35-40. Geiger, M. & Binder, B.R. (1987) Biochim. Biophys. Acta, 912, 34-40. Steven, F.S., Griffin, M.M., Cederholm-Williams, S.A., Mangel, W.F. & Maier, H. (1991) Anticancer Res. in press. Steven, F.S., Maier, H. & Amdt, J. (1989) J. Enz. Inhibition, 3, 145-157. Steven, F.S. & Griffin, M.M. (1991) J. Enz. Inhibition in press. Kettner, C. & Shaw, E.N. (1981) Methods in Enzym. 80, 826-842. Stopelli, M.P., Tacchetti, C., Cubellis, M.V., Corti, A., Heating, V.J., Cassani, G . , Appella, E. & Blasi, F. (1984) Cell, 45, 675-684.