Interaction of single-chain urokinase and plasminogen activator inhibitor type 1.
Urokinase (u-PA) is synthesized and secreted as a single-chain polypeptide (single-chain u-PA, scu-PA), which has such little enzymatic activity in solution that it has been considered essentially enzymatically inert. We found that plasminogen activator inhibitor type 1 (PAI-1), the major PAI in plasma, demonstrated concentration-dependent inhibition of this solution-phase scu-PA enzymatic activity. 125I-scu-PA formed complexes with PAI-1 in a concentration- and time-dependent manner, as detected by SDS-polyacrylamide gel electrophoresis under reducing conditions. Among a given population of scu-PA molecules, all measurable enzymatic activity was inhibited by a 10-fold molar excess of PAI-1. However, at this stoichiometry, only a minority of 125I-scu-PA molecules formed SDS-stable complexes with PAI-1 (i.e. complexes that formed a covalent bond upon denaturation), even though the uncomplexed PAI-1 molecules remained competent to inhibit u-PA enzymatic activity. Neither the extent nor the time course of complex formation was altered by using PAI-1 that had been pre-incubated with native human vitronectin, compared with native PAI-1 alone. 125I-scu-PA.PAI-1 complexes that would form a covalent bond if denatured were reversible and existed in equilibrium with either non-complexed or loosely complexed reactants. These data suggest that scu-PA has more enzyme-like properties than previously appreciated and raises the possibility that it resembles single-chain tissue type-plasminogen activator in lacking a complete zymogen conformation.
Manchanda
N
N
Department of Medicine, University of Wisconsin, Madison, USA.
Schwartz
B S
BS
D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019096:Vitronectin
D006023:Glycoproteins; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D019096:Vitronectin; D014568:Urokinase-Type Plasminogen Activator