The in vitro activity of ADAM‐10 is inhibited by TIMP‐1 and TIMP‐3

FEBS Letters

Webster, Ailsa³  Stephens, Paul E.³  Amour, Augustin¹  Murphy, Gillian¹  Docherty, Andrew J.P.³  Knight, C.Graham²  Knäuper, Vera¹  Slocombe, Patrick M.³ 

[1] School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK;Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK;Celltech Group plc, 216 Bath Road, Slough SL1 4EN, UK

关键词: Metalloproteinase;    Disintegrin metalloproteinase;    Tissue inhibitor of metalloproteinase;    DMSO;    dimethylsulfoxide;    HB-EGF;    heparin-binding epidermal growth factor;    HPLC;    high performance liquid chromatography;    K i;    inhibition constant;    MBP;    myelin basic protein;    MMP;    matrix metalloproteinase;    MMP-1;    collagenase-1;    MMP-2;    gelatinase A;    MMP-3;    stromelysin-1;    MMPI;    MMP inhibitor;    NS0;    non-secreter zero;    SDS–PAGE;    sodium dodecyl sulfate–polyacrylamide gel electrophoresis;    TACE;    tumour necrosis factor-α converting enzyme;    TFA;    trifluoroacetic acid;    TIMP;    tissue inhibitor of metalloproteinases;   

DOI  :  10.1016/S0014-5793(00)01528-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described ‘metallosheddase’ cleavage sites of tumour necrosis factor α, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.

【 授权许可】

Unknown