FLUORESCENT ASSAY OF PROTEINASES IN CULTURED MAMMALIAN-CELLS

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphtylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of 3T3 arylamidase activities in BALB/c 3LT3 and C3H 10T 1/2 cells. With low magnification these enzymes activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.