Specific catalytic activities of
cysteine proteinases including
cathepsins B (EC 3.4.22.1) and L (EC 3.4.22.15) in human
melanoma cell lines SK-MEL-28, SK-MEL-30, MEL-HO and in fibroblasts of different origin are reported. Cell line-specific pH profiles of these
cysteine proteinases were determined fluorometrically with benzyloxycarbonyl-phenylalanyl-
arginine-amidomethylcoumarine (
Z-Phe-Arg-AMC) under saturated conditions. Single activities of
cathepsins B and L were inactivated by
urea and by benzyloxycarbonyl-phenylalanyl-
phenylalanine-diazomethylketone (Z-Phe-Phe-CHN2) in order to describe the activities of these
enzymes separately. The
melanoma cell line MEL-HO, which originated from a primary lesion, showed highest activity of an unknown
cysteine proteinase. This
enzyme is not inactivated by
urea and
Z-Phe-Phe-CHN2 and has a Michaelis constant (K(M) value) of approximately 1 mM. The specific characteristics suggest that it is a
tumor-associated
cathepsin B. In addition, high invasive subpopulations of SK-MEL-28 and SK-MEL-30 cell lines isolated by an invasion assay showed higher
proteinase activities than the low invasive subpopulations. Furthermore, in fibroblasts originating from
melanoma tissue
cysteine proteinase activities were increased compared to normal skin fibroblasts. In conclusion, these results indicate that these
cysteine proteinases shown here are
tumor-associated
proteinases, possibly facilitating invasion and dissemination of
melanoma cells.