TIME-RESOLVED POLARIZED FLUORESCENCE SPECTROSCOPY STUDIES OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-1 - CONFORMATIONAL-CHANGES OF THE REACTIVE CENTER UPON INTERACTIONS WITH TARGET PROTEASES, VITRONECTIN AND HEPARIN

Plasminogen activator inhibitor type 1 (PAI-1) is an important physiol ogical inhibitor of the plasminogen activator system. To investigate t he structure-functional aspects of this inhibitor, we have taken advan tage of the lack of cysteine residues in the PAI-1 molecule and substi tuted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluo rescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, dacenyl-3-propionyl)-N-(iodoacetyl)et hylenediamine (BDYIA). The labeled mutants were found to reveal bioche mical characteristics very similar to those of wild type PAI-1. Time-r esolved fluorescence spectroscopy was used to examine orientational fr eedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex-formation with target proteases, tissue type plasminogen acti vator (tPA) and urokinase type plasminogen activator (uPA), caused dec reased orientational freedom of BDYIA in the P3 position, while the or ientational freedom of BDYIA in position P1' increased to a level simi lar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, co mplex formation with modified anhydro-uPA, which is unable to cleave i ts substrate, largely restricted the orientational freedom of BDYIA pr obe in the P1' position. Together, these findings suggest that the P1- P1' bond of the BDYIA-labeled PAI-I mutants is cleaved in the native c omplex with PAs. Since vitronectin and heparin interact with PAI-1, th eir influence on the orientational freedom of BDYIA in the reactive ce nter of the PAI-1 molecule was also studied. The fluorescence anisotro py suggests that interactions with vitronectin and heparin induce conf ormational changes in the reactive center, indicating that PAI-1 has a mobile reactive center loop which is conformationally linked to both the vitronectin and heparin binding sites.