Metastatic mouse melanoma cells release collagen-gelatin degrading metalloproteinases as components of shed membrane vesicles.
The purpose of this study has been to compare collagen-gelatin degrading enzymes isolated from cancer cell organelles and cytosol to the metalloproteinases released by cancer cells. To this end, metastatic mouse melanoma cell organelles were isolated by sucrose density gradient centrifugation and metalloproteinases were assayed using native and denatured [methyl-3H]collagen substrates. Solubilized proteinases were purified by ammonium sulfate precipitation, anion exchange, concanavalin A affinity and gel-filtration column chromatographic procedures and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conclusions were as follows: malignant melanoma cells have a metalloproteinase (Mr = 59,000) which is shed from cells into conditioned medium as a component of intact membrane vesicles rather than as a soluble enzyme; storage of tumor-conditioned medium leads to the generation of autoactivated soluble metalloproteinases of lower molecular weight; purification of these metalloproteinase species yielded variant collagenases that have considerable gelatinolytic activity and a cleavage preference site for the Gly-Ile bond in a collagen-like synthetic octapeptide substrate which is typical for collagenase-type metalloproteinases. It is proposed that localization of potent proteinases to the surface of cancer cells facilitates the local breakdown of connective tissues during the invasive process.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D003094:Collagen; D003600:Cytosol / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D005780:Gelatin; D008546:Melanoma, Experimental / Q000201:enzymology* / Q000473:pathology; D008666:Metalloendopeptidases; D051379:Mice; D008810:Mice, Inbred C57BL; D003012:Microbial Collagenase / Q000302:isolation & purification / Q000378:metabolism*; D008854:Microscopy, Electron; D008970:Molecular Weight; D009362:Neoplasm Metastasis
D005780:Gelatin; D003094:Collagen; D010450:Endopeptidases; D008666:Metalloendopeptidases; D003012:Microbial Collagenase