The use of substrates with 7-amino-3-trifluoromethylcoumarine (AFC) leaving group in the localization of protease activities in situ.

A method for the localization of activities of proteases using substrates with 7-amino-3-trifluoromethylcoumarine (AFC) leaving group is described. 0.1 ml of 5-20 mMol solution of the respective substrate (Gly-Pro-AFC, Ala-Pro-AFC, Z-Ala-Arg-Arg-AFC, Z-Gly-Arg-Arg-AFC, Z-Gly-Gly-Arg-AFC, D-Val-Leu-Lys-AFC) in dimethylsulfoxide or dimethylformamide was added to 0.9 ml of 0.1 M Tris-HCl buffer, pH 7.4-7.8 or 0.1 M cacodylate buffer, pH 5-5.5. In the case of Z-Ala-Arg-Arg-AFC (cathepsin B substrate) 15 mM EDTA and 12 mM dithiothreitol were added. 7 mM amiloride or 2 mg/1 ml aprotinin were used as inhibitors with Z-Gly-Gly-Arg-AFC (urokinase substrate) and with D-Val-Leu-Lys-AFC (plasmin substrate). Substrate solutions were mixed with an equal amount of 2% agar solution in distilled water or in the respective buffer the pH of which was adjusted according to the pH optimum of the enzyme to be demonstrated. The agar solution was kept in a water bath at a temperature of 50-60 degrees C. After careful mixing, the substrate solution in agar was poured into a cylindrical vessel closed with a semipermeable membrane (Nephrophan) on which unfixed cryostat sections were mounted. 1-5 mM AFC solution in dimethylsulfoxide or dimethylformamide instead of the substrate was used as the control. Quenched samples of rat kidney and jejunum, biopsies of human jejunal mucosa, and of colorectal and uterine tumors were employed for the preparation of sections. After gelification of the medium in a refrigerator the vessels with sections were incubated in the dark at 37 degrees C for 0.5-several h. The reaction was controlled in a fluorescence microscope with an epiillumination adjusted to the FITC fluorescence and documented. A yellowish green fluorescence depicts sites where AFC was set free (sites with enzyme activity). When the reaction reached the required intensity the membranes were cut off, transferred to glass slides, mounted in glycerol, observed and photographed immediately (due to the solubility of AFC in glycerol). An acceptable cellular localization was achieved. The method with AFC substrates can be recommended for comparative biochemical and histochemical studies of proteases using the same substrate and for cases in which no other reliable procedure for the localization of the respective enzyme activity is available (e.g. urokinase, plasmin).