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Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay

Author: Beekman, B. · El, B. van · Drijfhout, J.W. · Ronday, H.K. · TeKoppele, J.M.
Type:article
Date:1997
Institution: Gaubius Instituut TNO
Source:FEBS Letters, 3, 418, 305-309
Identifier: 234219
doi: doi:10.1016/S0014-5793(97)01371-9
Keywords: Biomedical Research · Fluorogenic substrate · MMP-3 · Rheumatoid arthritis · Stromelysin · Synovial fluid · Stromelysin · Controlled study · Enzyme activity · Enzyme assay · Human tissue · Priority journal · Rheumatoid arthritis · Synovial fluid · Technique · Arthritis, Rheumatoid · Biological Assay · Biological Markers · Fluorescence · Humans · Matrix Metalloproteinase 3

Abstract

Stromelysin-1 (MMP-3) is an important member of the matrix metalloproteinase family. In joint-degrading diseases like arthritis, elevated levels of MMP-3 protein are detected in synovial fluid using immunological methods. However, these methods do not discriminate between active and inactive enzyme. In the present study, a specific stromelysin activity assay was developed using the selective fluorogenic substrate TNO003 (Dabcyl-Gaba-Arg-Pro-Lys-Pro-Val-Glu · Nva-Trp-Arg-Glu-(EDANS)-Ala-Lys-NH2, · = cleavage site). For its use in biological media, cleavage of TNO003 by enzymes other than stromelysin was effectively blocked by a proteinase inhibitor cocktail. Spiking of MMP-3 to synovial fluid resulted in an MMP-3 concentration-dependent linear increase in activity. The measured MMP-3 activity was not affected by the addition of MMP-13, even in a 5-fold excess over MMP-3. Synovial fluid from rheumatoid arthritis patients demonstrated 100-fold higher levels of active stromelysin than control synovial fluids. Chemicals/CAS: Biological Markers; Matrix Metalloproteinase 3, EC 3.4.24.17