title: Analysis of the role of the COOH-terminal domain in the activation, proteolytic activity, and tissue inhibitor of metalloproteinase interactions of gelatinase B.
abstract: Recombinant human progelatinase B and a COOH terminally truncated version, pro-delta426-688 gelatinase B have been prepared from a myeloma cell expression system. Both proenzymes could be processed to active forms by stromelysin-1 to give an NH2 terminus of Phe88, or by treatment with 4-aminophenylmercuric acetate resulting in an NH2-terminal Met75. The kinetics of activation using either treatment was not affected by removal of the enzyme COOH-terminal domain. The specific activities of both gelatinase B and delta426-688 gelatinase B, activated using either method, were found to be similar using either a quenched fluorescent peptide or gelatin as the substrate. Fibroblast monolayers were shown to mediate processing of both progelatinases at similar rates in the presence of either plasminogen or prostromelysin-1. Active wild-type gelatinase B was inhibited by tissue inhibitor of metalloproteinase (TIMP) -1 at a much faster rate than TIMP-2. COOH-terminal truncation of either enzyme or inhibitor gave a marked reduction in the rate constant for TIMP-1 inhibition but had no effect on the rate of TIMP-2 binding. It can be concluded that the COOH-terminal domain of progelatinase B is not involved in autolytic or cellular activation and does not affect the catalytic activity of the enzyme. However, COOH-terminal domain interactions between active gelatinase B and TIMP-1 significantly enhance the rate of complex formation.
authors.affiliation: Department of Biochemistry, Celltech Ltd., Slough, United Kingdom.
authors.forename: J P
authors.initials: JP
authors.lastname: O'Connell
authors.forename: F
authors.initials: F
authors.lastname: Willenbrock
authors.forename: A J
authors.initials: AJ
authors.lastname: Docherty
authors.forename: D
authors.initials: D
authors.lastname: Eaton
authors.forename: G
authors.initials: G
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017364:Collagenases / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D004789:Enzyme Activation; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D020780:Matrix Metalloproteinase 9; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D011506:Proteins / Q000494:pharmacology; D017384:Sequence Deletion; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases; D014407:Tumor Cells, Cultured
chemical_list: D018076:DNA, Complementary; D006023:Glycoproteins; D061965:Matrix Metalloproteinase Inhibitors; D011506:Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D017364:Collagenases; D020780:Matrix Metalloproteinase 9