title: Purification of a gelatin-degrading type IV collagenase secreted by ras oncogene-transformed fibroblasts.
abstract: Connective tissue matrix-degrading metalloproteinases play an important role in cancer invasion. In this report we describe the isolation of a metalloproteinase exhibiting both type IV collagenolytic and gelatinolytic activities from the conditioned medium of NIH-3T3 fibroblasts transformed with DNA containing an activated c-Harvey-ras oncogene from T24 bladder cancer cells. This tumor proteinase was purified by anion exchange chromatography, zinc-chelate Sepharose chromatography, and gel permeation chromatography. The final product was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (relative molecular mass = 67,000). Gelatin zymography revealed two bands of gelatinolytic activity, corresponding to molecular weights of 67,000 and 62,000. Upon immunoblotting with the use of an affinity-purified polyclonal rabbit antibody to a peptide region of type IV collagenase that lacks homology with interstitial collagenase or stromelysin, the purified tumor enzyme was identified as type IV collagenase.
authors.affiliation: Department of Research, Veterans Administration Medical Center, Northport, NY 11768.
authors.forename: C
authors.initials: C
authors.lastname: Spinucci
authors.forename: S
authors.initials: S
authors.lastname: Zucker
authors.forename: J M
authors.initials: JM
authors.lastname: Wieman
authors.forename: R M
authors.initials: RM
authors.lastname: Lysik
authors.forename: B
authors.initials: B
authors.lastname: Imhof
authors.forename: N
authors.initials: N
authors.lastname: Ramamurthy
authors.forename: L A
authors.initials: LA
authors.lastname: Liotta
authors.forename: H
authors.initials: H
authors.lastname: Nagase
mesh_terms: D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000032:analysis*; D005347:Fibroblasts / Q000032:analysis; D011905:Genes, ras; D003012:Microbial Collagenase / Q000302:isolation & purification*; D008970:Molecular Weight
chemical_list: D003012:Microbial Collagenase