THE HUMAN ENO1 GENE-PRODUCT (RECOMBINANT HUMAN ALPHA-ENOLASE) DISPLAYS CHARACTERISTICS REQUIRED FOR A PLASMINOGEN BINDING-PROTEIN

Plasminogen binds with low affinity in a lysine-dependent manner to ma ny cell types. Previously, a 54 kDa plasminogen receptor found on the surface of U-937 cells was identified as an cu-enolase-like molecule. The aims of this study were to determine whether recombinant alpha-eno lase (r-alpha-enolase), encoded by ENO1, was a plasminogen binding pro tein and to generate polyclonal antibodies against this antigen. Plasm inogen specifically bound r-alpha-enolase with a K-d 1.9 mu M and appr oached saturation at 10 mu M Lysine-dependent plasminogen binding to r -alpha-enolase was demonstrated by a greater than 80% inhibition of bi nding by the lysine analogues epsilon-amino caproic acid and tranexami c acid, whilst only 14% inhibition occurred with the arginine analogue benzamidine. Removal of the C-terminal lysine residue of r-alpha-enol ase with carboxypeptidase B significantly reduced its plasminogen bind ing capacity, suggesting that binding required C-terminal lysine resid ue of r-alpha-enolase. Binding to r-alpha-enolase enhanced the activat ion rate of plasminogen by urokinase but prevented alpha 2-antiplasmin from binding plasminogen. Taken together, these data suggest that the gene product of human ENO1 encodes an authentic plasminogen binding p rotein.