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

FEBS Letters

van El, Benno¹  Ronday, H.Karel¹  Beekman, Bob¹  Drijfhout, Jan Wouter²  TeKoppele, Johan M¹ 

[1] Gaubius Laboratory, TNO Prevention and Health, Department of Vascular and Connective Tissue Research, P.O. Box 2215, 2301 CE Leiden, The Netherlands;Department of Immunohaematology and Bloodbank, Leiden University Hospital, P.O. Box 9600, 2300 RC Leiden, The Netherlands

关键词: Fluorogenic substrate;    Matrix metalloproteinase;    MMP-3;    Rheumatoid arthritis;    Stromelysin;    Synovial fluid;    BB94/RS47;    112;    [4-(N-hydroxyamino)-2R-isobutyl-3S-(thiophen-2-ylthiomethyl)-succinyl]-l-phenylalanine-N-methylamide;    Dabcyl;    4-((4-dimethylamino)phenylazo)benzoyl;    Dnp;    2;    4-dinitrophenyl;    EDANS;    5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid;    Mca;    (7-methoxycoumarin-4-yl)acetyl;    MMP;    matrix metalloproteinase;    Nva;    norvaline;    RA-SF;    synovial fluid of rheumatoid arthritis patients;    RFU;    relative fluorescence units;    RP-HPLC;    reversed phase-high performance liquid chromatography;    SC44;    463;    N-[3-(-N′-hydroxycarboxamido)2-(2-methylpropyl)propanoyl]-O-methyl-l-tyrosine-N-methylamide;    SF;    synovial fluid;    TOF-MALDI;    time-of-flight matrix-assisted laser-desorption ionization;   

DOI  :  10.1016/S0014-5793(97)01371-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown