title: Analysis of plasmin binding and urokinase activation of plasminogen bound to the Heymann nephritis autoantigen, gp330.
abstract: Previously, we demonstrated that the Heymann nephritis autoantigen, gp330, can serve as a receptor site for plasminogen. This binding was not significantly inhibited by the lysine analogue epsilon-amino caproic acid (EACA), indicating that plasminogen binding was not just through lysine binding sites as suggested for other plasminogen binding sites. We now report that once plasminogen is bound to gp330, it can be converted to its active form of plasmin by urokinase. This conversion of plasminogen to plasmin proceeds at a faster rate when plasminogen is first prebound to gp330. Although there is a proportional increase in the Vmax of the urokinase-catalyzed reaction with increasing gp330 concentrations, no change in Km was observed. Once activated, plasmin remains bound to gp330 in an active state capable of cleaving the chromogenic tripeptide, S-2251. The binding of plasmin to gp330 did not significantly change its enzymatic activity; however, gp330 did have a stabilizing effect on plasmin activity at 37 degrees C. While bound to gp330, plasmin is protected from inactivation by its natural inhibitor alpha 2-antiplasmin. The binding of plasmin to gp330 as analyzed by ELISA was shown to be time dependent, reversible, saturable, and specific for gp330. Inhibition of binding of both plasminogen and plasmin to gp330 by benzamidine was similar, although EACA inhibited the binding of plasmin to gp330 slightly more than the binding of plasminogen to gp330. These results indicate that the binding of plasminogen to gp330 serves as an effective means of increasing the rate of plasmin production on the glomerular and tubular epithelial cell surface while protecting the active plasmin from natural inhibitors.
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D026541:Heymann Nephritis Antigenic Complex; D066298:In Vitro Techniques; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000494:pharmacology
chemical_list: D026541:Heymann Nephritis Antigenic Complex; D008562:Membrane Glycoproteins; D000511:alpha-Macroglobulins; C024668:plasmin-alpha(2)-macroglobulin complex; D010958:Plasminogen; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator