title: Modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase.
abstract: We describe a new principle for assessment of the activity of proteolytic enzymes of all classes and show the application of this principle for the quantitative assay of bacterial collagenase and human matrix metalloproteinases (MMPs). Central to this new principle is the presence of a proenzyme that can be activated into an active enzyme by a single proteolytic event. The regular activation sequence in the proenzyme is replaced using protein engineering by an artificial sequence recognized by the proteinase to be determined. The latter can act as an activator for the newly engineered proenzyme. In the present paper a simple colorimetric assay for the determination for MMPs is described based on this principle. With the aid of protein engineering, a modified pro-urokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys upward arrowIle-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly upward arrowIle-Ile-Gly-Gly). The active urokinase resulting from activation of the modified pro-urokinase by a MMP could be measured either directly, using a specific chromogenic peptide substrate for urokinase, or indirectly via urokinase-catalysed plasminogen activation. The response of the assay to equal molar quantities of active MMPs decreases in the order MMP-2>MMP-9>MMP-1>MMP-3>MMP-7. The detection limit for MMP-9 was below 15 pM, corresponding to 3. 75x10(-15) mol per assay. Using the assay, increased MMP activity was detected in synovial tissue extracts from rheumatoid arthritis patients compared with those from osteoarthritis patients, and in stomach tumour extracts as compared with normal stomach tissue extracts.
authors.affiliation: Gaubius Laboratory TNO-PG, P.O. Box 2215, 2301 CE Leiden, The Netherlands.
authors.forename: J H
authors.initials: JH
authors.lastname: Verheijen
authors.forename: N M
authors.initials: NM
authors.lastname: Nieuwenbroek
authors.forename: B
authors.initials: B
authors.lastname: Beekman
authors.forename: R
authors.initials: R
authors.lastname: Hanemaaijer
authors.forename: H W
authors.initials: HW
authors.lastname: Verspaget
authors.forename: H K
authors.initials: HK
authors.lastname: Ronday
authors.forename: A H
authors.initials: AH
authors.lastname: Bakker
mesh_terms: D001172:Arthritis, Rheumatoid / Q000201:enzymology; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D017364:Collagenases / Q000032:analysis / Q000378:metabolism*; D003124:Colorimetry; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013270:Stomach / Q000201:enzymology; D013274:Stomach Neoplasms / Q000201:enzymology; D013379:Substrate Specificity; D013583:Synovial Membrane / Q000201:enzymology; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; D004792:Enzyme Precursors; D016326:Extracellular Matrix Proteins; D061965:Matrix Metalloproteinase Inhibitors; D009363:Neoplasm Proteins; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases; D014568:Urokinase-Type Plasminogen Activator; D017364:Collagenases; D008666:Metalloendopeptidases; C067551:saruplase