Interaction of urokinase with alpha2-macroglobulin investigated by isoelectric focusing. Evidence for non-specific dissociable binding.

The binding of urokinase to human alpha2M (alpha2-macroglobulin) was investigated in comparison with the formation of the equimolar trypsin-alpha2M complex. Experiments were performed by molecular-sieving on Sephadex G-200, subunit conversion by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis after reduction and isoelectric focusing in linear sucrose gradients with ampholytes pH 3.5-10.0. Urokinase activity was determined with alpha-N-acetyl-L-lysine methyl ester and by activation of plasminogen on unheated fibrin plates. alpha2M was determined by single radial immunodiffusion. alpha2M was capable of binding some urokinase by a non-specific type of attachment that could be disrupted by isoelectric focusing but not by gel filtration. The pI of the undissociated trypsin-alpha2M complex was 6.0, and differed from that of the pure alpha2M (5.2-5.4). Likewise the pI of the immunoreactive alpha2M was 5.2 after exposure to urokinase, whereas the dissociated urokinase focused at pI 10.2. This indicated lack of true inhibitor-complex formation, which was also sustained by total absence of subunit conversion. The results are in agreement with our previous findings with pancreatic and urinary kallikreins.