ENHANCEMENT OF THE ENZYMATIC-ACTIVITY OF SINGLE-CHAIN UROKINASE PLASMINOGEN-ACTIVATOR BY SOLUBLE UROKINASE RECEPTOR

Single-chain urokinase (scuPA), the unique form of urokinase secreted by cells, is converted to an active two-chain molecule through the cle avage of a single peptide bond by plasmin and other specific proteinas es. Although scuPA may express limited enzymatic activity, its contrib ution to plasminogen activation on cell surfaces remains uncertain. Fu rther, although it is well known that scuPA binds to a specific extrac ellular urokinase type plasminogen activator receptor, the effect of t his interaction on the enzymatic activity of scuPA has not been descri bed. In the present paper we report that the binding of scuPA to cellu lar and to recombinant soluble urokinase-type plasminogen activator re ceptors (suPAR) increases its catalytic activity as measured by the cl eavage of a urokinase-specific chromogenic substrate. suPAR increased the V-max of scuPA 5-fold with little change in its K-m. suPAR also st imulated the plasminogen activator activity of scuPA by decreasing its K-m for Glu-plasminogen from 1.15 mu M to 0.022 mu M and by increasin g the k(cat) of this reaction from 0.0015 to 0.022 s(-1). Preincubatio n of scuPA with suPAR also enhances its susceptibility to inhibition b y plasminogen activator inhibitor type 1, consistent with exposure of its catalytic site. The activity of scuPA bound to suPAR is not accomp anied by cleavage of scuPA, which continues to migrate as a single ban d in SDS-polyacrylamide gel electrophoresis under reducing conditions. Moreover, suPAR increases the plasminogen activator activity of a pla smin-insensitive variant, scuPA (scuPA-Glu(158)), as well. Enhancement of scuPA activity by suPAR is both prevented and reversed by its amin oterminal fragment (amino acids 1-135), which competes for receptor bi nding, suggesting that continued binding to the receptor is required f or expression of scuPA's enzymatic activity. Thus, our data suggest th at scuPA may undergo a reversible transformation between a la tent and an active state. The urokinase receptor may induce or stabilize scuPA in its active conformation, thereby contributing to the initiation of plasminogen activation on cell surfaces.