INTERACTION BETWEEN TISSUE INHIBITOR OF METALLOPROTEINASES-2 AND PROGELATINASE-A - IMMUNOREACTIVITY ANALYSES

By immunoreactivity analysis using monoclonal antibodies, we showed th at the C-terminal domain [R(415-631); R is residue] of progelatinase A [pro-matrix metalloproteinase-2 (proMMP-2); EC 3.4.24.24] affected th e immunoreactivity of a one-step sandwich enzyme immunoassay (sandwich EIA) for tissue inhibitor of metalloproteinases-2 (TIMP-2) in exactly the same way as does proMMP-2 [Fujimoto, Zhang, Iwata, Shinya, Okada and Hayakawa (1993) Clin. Chim. Acta 220, 31-45], confirming that the C-terminal domain ('tail' portion) of TIMP-2 participates in the bindi ng with the C-terminal domain of proMMP-2. We also demonstrated that n ot only the C-terminal domain but also the N-terminal domain (R(1-417) ) of proMMP-2 bound to TIMP-2 in a 1:1 molar ratio. The binding of eac h individual domain to TIMP-2, however, was weak enough that either do main could be fully replaced by proMMP-2 itself, suggesting that eithe r terminal domain binds to TIMP-2 through the same binding sites as do es proMMP-2, and also that the high-order structure of proMMP-2 allows a more stable binding to TIMP-2. We further confirmed that TIMP-2 com plexed with the N-terminal domain of proMMP-2 had fully inhibitory act ivity against the collagenolytic activity of MMP-1. We also demonstrat ed that either the interstitial collagenase-TIMP-2 complex or the gela tinase B (MMP-9)-TIMP-2 complex was able to form a ternary complex wit h proMMP-2 in a 1:1 molar ratio, clearly indicating that there are two distinct binding sites, one specific for proMMP-2 and the other for a ctive MMPs, on the TIMP-2 molecule. The C-terminal domain was able to bind to the MMP-9-TIMP-2 complex, but the binding seemed to be less st able than the binding with TIMP-2 alone. Even in the presence of a 10- fold molar excess of the N-terminal domain, ternary complex formation was not observed between the N-terminal domain and the MMP-9-TIMP-2 co mplex. These clear differences might be ascribed to some significant c onformational change(s) evoked in the TIMP-2 molecule, or hindrance of a part of the N-terminal domain binding site of TIMP-2 by complex for mation with MMP-9.