Domain structure of human 72-kDa gelatinase/Type IV collagenase: Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions

摘要: The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play role in metastasis and angiogenesis, forms noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), potent enzyme activity. To define regions gelatinase responsible for TIMP-2 binding, series NH2- COOH-terminal deletions were constructed using polymerase chain reaction technique. full-length truncated enzymes expressed recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two ending at residues 425 (delta 426-631) 454 455-631) purified. Like gelatinase, both COOH-terminally activated organomercurial digested gelatin native collagen type IV. In contrast enzyme, delta 426-631 455-631 less sensitive inhibition requiring 10 mol TIMP-2/mol achieve maximal enzymatic but not latent proteins formed only when excess molar concentrations used. We also 205-amino acid fragment, 1-426, found that it binds TIMP-2. addition, version lacking NH2-terminal 78 amino acids 1-78) proenzyme retained ability bind These studies demonstrate gelatinases domain retain full activity acquire reduced sensitivity inhibition. data suggest active site tail independently cooperatively participate binding.