Extraction of type-IV collagenase/gelatinase from plasma membranes of human cancer cells.
Tumor proteinases are considered to be important in the process of cancer invasion and metastasis. We have proposed that the surface membrane localization of these proteinases places them in an optimal site to facilitate the invasion of surrounding extracellular matrix. In this study, we have used the organic solvent, n-butanol, and the detergent, n-octyl-glucoside, to sequentially extract metalloproteinases from crude plasma membranes of human RWP-I pancreatic cancer cells. Anion exchange chromatography and gel permeation chromatography were employed to further purify enzymes with the capacity to degrade gelatin, type-IV collagen, and carboxymethylated transferrin. Gelatin zymography was used to demonstrate proteinase bands of 92, 70 and 62-kDa. Immunoblotting of solubilized, partially purified pancreatic cancer plasma membrane proteins using polyclonal rabbit antibodies, which have specificity for type-IV collagenase/gelatinase, resulted in the recognition of a 70-kDa protein, but not the 92-kDa gelatinase. A type-IV collagenase/gelatinase of 68-kDa was similarly identified in A2058 human melanoma cancer cell plasma membranes.
Zucker
S
S
Department of Research, Veterans Administration Medical Center, Northport, NY 11768.
Moll
U M
UM
Lysik
R M
RM
DiMassimo
E I
EI
Stetler-Stevenson
W G
WG
Liotta
L A
LA
Schwedes
J W
JW
D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D020780:Matrix Metalloproteinase 9; D008545:Melanoma / Q000201:enzymology; D003012:Microbial Collagenase / Q000302:isolation & purification*; D008970:Molecular Weight; D009369:Neoplasms / Q000201:enzymology*; D010190:Pancreatic Neoplasms / Q000201:enzymology; D014407:Tumor Cells, Cultured / Q000201:enzymology
D003012:Microbial Collagenase; D020780:Matrix Metalloproteinase 9