Allosteric regulation of tPA-mediated plasminogen activation by a modifier mechanism: evidence for a binding site for plasminogen on the tPA A-chain.
We studied the mechanism responsible for nonlinear double reciprocal plots for tissue type plasminogen activator (tPA)-mediated plasminogen activation reported previously by several groups. We found nonlinear Eadie-Scatchard plots for Glu-plasminogen activation by recombinant single-chain tPA confirming a non-Michaelis-Menten behavior of tPA. In order to characterize this mechanism, enzyme kinetic studies with truncated substrates (Lys- and miniplasminogen) and modified or truncated enzymes (two-chain tPA and tPA B-chain) were performed. Thereby it could be excluded that product-mediated modifications of the enzyme or the substrate are responsible for the nonlinear plots. Linear plots, i.e., Michaelis-Menten kinetics, were only found when tPA B-chain was used as a plasminogen activator, indicating that the tPA A-chain should be responsible for the non-Michaelis-Menten behavior. Binding studies of plasminogen to immobilized tPA A-chain in fact demonstrated a saturable binding of Glu- and miniplasminogen to the A-chain of tPA with a KD approximately 0.1 microM and one binding site per molecule of tPA A-chain. These data suggested a modifier mechanism responsible for the nonlinear plots whereby the substrate plasminogen itself could function as a modifier. When such a mechanism was included into a model for tPA-mediated plasminogen activation, the experimentally obtained data could be fitted into such a model by nonlinear regression analysis with resulting p-values of less than 0.001.
Geppert
A G
AG
Department of Medical Physiology, University of Vienna, Austria.
Binder
B R
BR
D000494:Allosteric Regulation; D001665:Binding Sites; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D010446:Peptide Fragments / Q000378:metabolism; D010958:Plasminogen / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; C018998:lysyl-plasminogen; D007531:Isoflurophate; D010958:Plasminogen; D010959:Tissue Plasminogen Activator