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The in vitro activity of ADAM‐10 is inhibited by TIMP‐1 and TIMP‐3
Author(s) -
Amour Augustin,
Knight C.Graham,
Webster Ailsa,
Slocombe Patrick M.,
Stephens Paul E.,
Knäuper Vera,
Docherty Andrew J.P.,
Murphy Gillian
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)01528-3
Subject(s) - in vitro , matrix metalloproteinase , chemistry , fusion protein , recombinant dna , myelin basic protein , cleavage (geology) , peptide , biochemistry , microbiology and biotechnology , myelin , biology , paleontology , neuroscience , fracture (geology) , central nervous system , gene
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.