title: Cellular activation of the 72 kDa type IV procollagenase/TIMP-2 complex.
abstract: Members of the collagenase family of enzymes have been implicated as central mediators of a number of both physiologic and pathologic processes. The 72-kDa type IV collagenase is secreted as a latent proenzyme, complexed with tissue inhibitor of metalloproteinase-2 (TIMP-2). Like other members of the collagenase family, this enzyme complex must be converted to a catalytically active form for proteolytic remodeling of extracellular matrix to occur. In the current study we demonstrate an inducible cell-mediated activation of the 72-kDa type IV procollagenase/TIMP-2 complex. Isolation of the 62 kDa activated enzyme/TIMP-2 complex from conditioned media of concanavalin A treated WI-38 fibroblasts demonstrated that the cell activated species was proteolytically active and amino terminal sequencing gave the sequence YNFF. This is identical to that of the 62 kDa species generated following organomercurial activation of purified 72-kDa type IV procollagenase/TIMP-2 complex. We have also isolated biosynthetically 35S-labeled 72-kDa type IV procollagenase/TIMP-2 complex and used this to further study the cellular activation process. In cell lines tested the activator was retained in the residual cell fraction following lysis in the presence of 0.2% (wt/vol) Brij-35. Inhibitor studies demonstrated that processing and activation of 72-kDa type IV procollagenase/TIMP-2 complex by the residual fraction was inhibited by 5 mM ethylenediaminetetraacetic acid and 0.5 mM 1,10-phenanthroline demonstrating a metal atom dependence. The species responsible for activation could be partially recovered in soluble form with 0.5% (vol/vol) Triton X-100 and 0.25% (wt/vol) CHAPS but was not salt extractable.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland.
authors.forename: P D
authors.initials: PD
authors.lastname: Brown
authors.forename: D E
authors.initials: DE
authors.lastname: Kleiner
authors.forename: E J
authors.initials: EJ
authors.lastname: Unsworth
authors.forename: W G
authors.initials: WG
authors.lastname: Stetler-Stevenson
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D017364:Collagenases / Q000737:chemistry / Q000378:metabolism*; D003208:Concanavalin A / Q000494:pharmacology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D020780:Matrix Metalloproteinase 9; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D019716:Tissue Inhibitor of Metalloproteinase-2
chemical_list: D002614:Chelating Agents; D004792:Enzyme Precursors; D009363:Neoplasm Proteins; D015842:Serine Proteinase Inhibitors; D003208:Concanavalin A; D019716:Tissue Inhibitor of Metalloproteinase-2; D017364:Collagenases; C022440:procollagenase; D020780:Matrix Metalloproteinase 9