Structural and functional characterization of the inhibition of urokinase by alpha 2-macroglobulin.
We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.
D L
DL
Straight
M A
MA
Hassett
P A
PA
McKee
D000936:Antigen-Antibody Complex; D000990:Antithrombin III / Q000494:pharmacology; D001665:Binding Sites; D004789:Enzyme Activation; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007074:Immunoglobulin G; D007700:Kinetics; D046911:Macromolecular Substances; D010958:Plasminogen / Q000302:isolation & purification; D011485:Protein Binding; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*; D000511:alpha-Macroglobulins / Q000378:metabolism / Q000494:pharmacology*
D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D000511:alpha-Macroglobulins; D000990:Antithrombin III; D010958:Plasminogen; D006493:Heparin; D014568:Urokinase-Type Plasminogen Activator