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

A recombinant soluble form of the catalytic domain of human ADAM-10 was exp ressed as an Fc fusion protein from myeloma cells. The ADAM-10,vas catalyti cally active, cleaving myelin basic protein and peptides based on the previ ously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay mas used to demonstrate that hydroxamate inhibitors of ma trix 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 Ne obtained approximate apparent inhibition constants of 0.1 n M (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could ther efore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays. (C) 2000 Federation of E uropean Biochemical Societies.