Print Email Facebook Twitter Modified proenzymes as artificial substrates for proteolytic enzymes Title Modified proenzymes as artificial substrates for proteolytic enzymes: Colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase Author Gaubius Instituut TNO Verheijen, J.H. Nieuwenbroek, N.M.E. Beekman, B. Hanemaaijer, R. Verspaget, H.W. Ronday, K.H. Bakker, A.H.F. Publication year 1997 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 prourokinase has been prepared in which the activation sequence normally recognized by plasmin (Pro-Arg-Phe-Lys ↑ Ile-Ile-Gly-Gly) has been replaced by a sequence expected to be recognized and hydrolysed by many MMPs (Arg-Pro-Leu-Gly ↑ Ile-Ile-Gly-Gly).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 3.75 x 10-15 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. Copyright © 1997 The Biochemical Society Subject Arthritis, RheumatoidBacterial ProteinsCollagenasesColorimetryDNA, ComplementaryEndopeptidasesEnzyme ActivationEnzyme PrecursorsExtracellular Matrix ProteinsHumansMetalloendopeptidasesMutagenesis, Site-DirectedNeoplasm ProteinsProtease InhibitorsRecombinant Fusion ProteinsRecombinant ProteinsStomachStomach NeoplasmsSubstrate SpecificitySynovial MembraneUrinary Plasminogen Activator To reference this document use: http://resolver.tudelft.nl/uuid:5b7769a4-3b33-41cb-ac01-f9fe361dd61f DOI https://doi.org/10.1042/bj3230603 TNO identifier 233895 ISSN 0264-6021 Source Biochemical Journal, 323 (323), 603-609 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.