Properties of a plasma membrane-associated cathepsin B-like cysteine proteinase in metastatic B16 melanoma variants.
Activities of a cathepsin B-like cysteine proteinase have previously been observed to correlate with the malignancy of several animal and human tumors. Plasma membrane fractions of some of these tumors have been found to be enriched in cathepsin B-like activity. We have determined the subcellular distribution of this enzyme and three additional lysosomal hydrolases ( cathepsin H, beta-hexosaminidase, and beta-glucuronidase) in normal murine liver and six metastatic variants of the B16 melanoma. The tissues were fractionated initially by differential centrifugation followed by Percoll density gradient centrifugation of the light mitochondrial fraction. Two fractions were obtained: an L-2 fraction enriched in all four lysosomal hydrolases; and an L-1 fraction enriched in a marker enzyme for the plasma membrane. Cathepsin B-like and beta-hexosaminidase activities, but not the other hydrolase activities, were also found to be enriched in the L-1 fractions of the metastatic B16 tumors. We explored the nature of the association of the cathepsin B-like activity with the plasma membrane using fractions from the spontaneously metastatic B16 amelanotic melanoma. Activity could not be dissociated from the plasma membrane fraction by washing with a physiological salt solution suggesting that it was not adsorbed to this fraction nonspecifically, nor could it be displaced by mannose 6-phosphate or other sugars which compete for binding to the known lysosomal receptors. High salt concentrations, low concentrations of the mild detergent saponin, mild acidification, or phosphatidylinositol-specific phospholipase C did not elute the cathepsin B-like activity. However, activity was eluted by exposure to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a detergent used in the purification of integral membrane proteins. The B16 amelanotic melanoma plasma membrane-associated cathepsin B-like activity had a slightly higher pH optimum and was resistant to inactivation by neutral pH and to inhibition by three low molecular weight inhibitors of cysteine proteinases. The Ki values for inhibition by leupeptin and stefin A were 20-fold higher. The presence of a cathepsin B-like cysteine proteinase at the surface of metastatic tumor cells, particularly in a form which can retain activity at physiological pH and retain activity in the presence of extracellular proteinase inhibitors, may contribute to the focal dissolution of the extracellular matrix observed at sites of contact with invading tumor cells.[1]References
- Properties of a plasma membrane-associated cathepsin B-like cysteine proteinase in metastatic B16 melanoma variants. Rozhin, J., Robinson, D., Stevens, M.A., Lah, T.T., Honn, K.V., Ryan, R.E., Sloane, B.F. Cancer Res. (1987) [Pubmed]
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