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

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....

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Bibliographic Details
Published in:FEBS letters 1997-12, Vol.418 (3), p.305-309
Main Authors: Beekman, Bob, van El, Benno, Drijfhout, Jan Wouter, Ronday, H.Karel, TeKoppele, Johan M
Format: Article
Language:English
Subjects:
(7-methoxycoumarin-4-yl)acetyl
2,4-dinitrophenyl
4-((4-dimethylamino)phenylazo)benzoyl
5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid
[4-(N-hydroxyamino)-2R-isobutyl-3S-(thiophen-2-ylthiomethyl)-succinyl]-l-phenylalanine-N-methylamide
Arthritis, Rheumatoid - metabolism
BB94/RS47,112
Biological Assay
Biomarkers
Dabcyl
Dnp
EDANS
Fluorescence
Fluorogenic substrate
Humans
Matrix metalloproteinase
Matrix Metalloproteinase 3 - metabolism
Mca
MMP
MMP-3
N-[3-(-N′-hydroxycarboxamido)2-(2-methylpropyl)propanoyl]-O-methyl-l-tyrosine-N-methylamide
norvaline
Nva
RA-SF
relative fluorescence units
reversed phase-high performance liquid chromatography
RFU
Rheumatoid arthritis
RP-HPLC
SC44,463
Stromelysin
Synovial fluid
Synovial Fluid - metabolism
synovial fluid of rheumatoid arthritis patients
time-of-flight matrix-assisted laser-desorption ionization
TOF-MALDI
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Summary: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-NH 2, ♦=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.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(97)01371-9