title: Assessment of the role of the fibronectin-like domain of gelatinase A by analysis of a deletion mutant.
abstract: The properties of a deletion mutant delta V191-Q364 of gelatinase A, which represents the removal of the fibronectin-like type II repeats defined by exons 5-7, were compared with those of full-length gelatinase A. Both enzymes underwent self-activation over a similar time course in the presence of 4-aminophenylmercuric acetate. The fully active enzymes had similar kcat/Km values for the cleavage of an octapeptide substrate, but the deletion mutant had 50% of the activity of wild type gelatinase A against beta-casein and 10% of the activity against gelatin. The cleavage pattern for gelatin was similar for both enzymes but differed for type IV collagen. Comparison of the rates of association of the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 and their N-terminal domains to both forms of gelatinase indicated that the fibronectin-like domain plays little role in TIMP binding. The deletion mutant failed to bind to collagen, while the wild type gelatinase bound tightly, indicating that the fibronectin-like domain is the sole site of collagen binding. Both gelatinases could be activated by concanavalin A-activated fibroblasts, suggesting that the fibronectin-like domain is not required for the membrane-mediated activation process.
authors.affiliation: Strangeways Research Laboratory, Cambridge, United Kingdom.
authors.forename: G
authors.initials: G
authors.lastname: Murphy
authors.forename: Q
authors.initials: Q
authors.lastname: Nguyen
authors.forename: M I
authors.initials: MI
authors.lastname: Cockett
authors.forename: S J
authors.initials: SJ
authors.lastname: Atkinson
authors.forename: J A
authors.initials: JA
authors.lastname: Allan
authors.forename: C G
authors.initials: CG
authors.lastname: Knight
authors.forename: F
authors.initials: F
authors.lastname: Willenbrock
authors.forename: A J
authors.initials: AJ
authors.lastname: Docherty
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D017931:DNA Primers; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005091:Exons; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D005353:Fibronectins / Q000378:metabolism*; D018093:Gelatinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000494:pharmacology; D007700:Kinetics; D020778:Matrix Metalloproteinase 2; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010662:Phenylmercuric Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D019714:Tissue Inhibitor of Metalloproteinases; D014162:Transfection
chemical_list: D017931:DNA Primers; D004792:Enzyme Precursors; D005353:Fibronectins; D006023:Glycoproteins; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D003208:Concanavalin A; C010024:4-aminophenylmercuriacetate; D003094:Collagen; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D010662:Phenylmercuric Acetate