Degree Name

Doctor of Philosophy


School of Biological Sciences


The lysine-dependent, activation-resistant, conformation of glu-plasminogen is converted to an activation susceptible conformation after binding to cell-surface lysine residues. These lysine-dependent interactions of glu-plasminogen are mediated by its lysine binding sites (LBS's). Thus, it is hypothesised that lysine residues from the cell surface plasminogen receptors compete with lysine residues from gluplasminogen for occupation of its LBS's, thereby inducing a conformational change to glu-plasminogen. Previously, an α-enolase-related molecule was identified as a cell surface plasminogen receptor. Hence, the plasminogen binding characteristics of recombinant human α-enolase (r-α-enolase) were assessed.

R-α-enolase bound glu-plasminogen in a lysine-dependent manner with an apparent Kd of 1.9 μM. This interaction [1] was dependent on the C-terminal lysine residue of r-α-enolase, [2] enhanced the activation rate of glu-plasminogen and [3] blocked α2-antiplasmin from binding glu-plasminogen.

BIACORE kinetic analysis of the interaction suggested that the dissociation of glu-plasminogen from r-α-enolase was mediated by at least two components with apparent dissociation rate constants of kdl=4.7x10-2 s-1 andkd2=l.6x10-3 s-1.Global analysis of the interaction suggested that it was a two-state conformational change reaction, mediated by a concentration-dependent increase in the initial association rate. Intrinsic fluorescence spectroscopy confirmed that r-α-enolase induced a more open conformation of glu-plasminogen. Thus, the gene product of human ENOl encoded an authentic plasminogen binding protein and the binding of gluplasminogen to α-enolase is mediated by an initial lysine-dependent competition reaction that results in a conformational change to the zymogen.

The binding of glu-plasminogen to the metastatic breast cancer cell line MDA-MB-231 in this study was: [1] lysine-dependent [2] low affinity (Kd = 1.8 μM), but high capacity (5.0x107 sites/cells) and [3] dependent on the viability status of the cells. Multiple plasminogen binding proteins may be responsible for localising glu-plasminogen to the cell surface. The MDA-MB-231 cells were capable of generating large amounts of plasmin compared to the non-metastatic breast cancer cell lines which did not have a high plasminogen binding capacity.

Therefore, glu-plasminogen binds to the cell surface by a lysine-competitive, two-step binding event that results in a more open, activation-susceptible conformation. This competitive-lysine reaction mechanism of glu-plasminogen explains the relationship between binding, conformation and activation of gluplasminogen.