Abstract
Objective: Plasminogen binds with apparent low affinity to cell-surface receptors via its lysine binding sites. This enhances/stabilizes the activation-susceptible conformation. However, it is not known whether this lysine-mediated conformational change of plasminogen may affect its subsequent dissociation rate and hence its stability at the cell surface. Therefore, we sought to determine the relationship between the lysine-dependent conformation of plasminogen and its dissociation rate from its receptor. Design: BIACORE experiments were used to determine the kinetics of the interaction of glu-plasminogen with its receptor α-enolase. Intrinsic and extrinsic fluorescence spectroscopy were utilized to confirm if α-enolase induced a conformational change to glu-plasminogen as predicted by analyses of the BIACORE data. Results: The dissociation of glu-plasminogen from α-enolase was mediated by at least two components with apparent dissociation rate constants of kd1=4.7 × 10-2s-1 and kd2=1.6 × 10-3s-1. This second slower dissociation event reflects an increase in the stability of the complex. Global analysis of the interaction suggested a two-state conformational change reaction, mediated by a concentration-dependent increase in the initial association rate constant. The apparent Kd predicted by this analysis was 1 μM. Fluorescence spectroscopy confirmed that α-enolase induced a more open conformation of glu-plasminogen. Conclusions: These results provide direct evidence that the binding of glu-plasminogen to α-enolase is not simply a low-affinity interaction, but involves a multivalent, competition binding reaction that is associated with a glu-plasminogen conformational change. This mechanism is compatible with the structure of glu-plasminogen. This has implications for the stability of binding and activation of glu-plasminogen at the cell surface.
Original language | English |
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Pages (from-to) | 327-336 |
Number of pages | 10 |
Journal | Fibrinolysis and Proteolysis |
Volume | 14 |
Issue number | 6 |
Publication status | Published - 2000 |
Externally published | Yes |