A monoclonal antibody to the epsilon-aminocaproic acid binding site on the kringle 4 region of human plasminogen that accelerates the activation of Glu1-plasminogen by urokinase.
A monoclonal antibody, 10-F-1, previously shown [V. A. Ploplis, H. S. Cummings, and F. J. Castellino (1982) Biochemistry 21, 5891-5897] to interact with a particular epsilon-aminocaproic acid (EACA)3 binding site on the kringle 4 (K4) region of human Glu1-plasminogen (Glu1-Pg), has been employed to assess the contribution of this particular EACA site toward the enhancement, by EACA and its analogs, of the urokinase (UK)-catalyzed activation of Glu1-Pg. As is the case with EACA-like compounds, the presence of antibody 10-F-1 accelerates the activation of Glu1-Pg by UK, but does not enhance the similar activation of Lys77-plasminogen. In the presence of concentrations of antibody 10-F-1 which saturate its binding site on Glu1-Pg, the Km of Glu1-Pg activation by UK is raised from 1.4 +/- 0.2 microM, a value obtained in the absence of antibody, to 17.0 +/- 2.0 microM. On the other hand, the kcat for this activation, 0.038 +/- 0.005 s-1, is elevated to 2.45 +/- 0.2 s-1 at saturating concentrations of antibody 10-F-1. The kcat/Km for activation under these conditions is 0.027 s-1 microM-1 in the absence of antibody, and 0.144 s-1 microM-1 in the presence of saturating levels of antibody 10-F-1. This demonstrates that the interaction of this antibody with its epitope results in a fivefold stimulation of the activation rate of Glu1-Pg by UK. The availability of antibody 10-F-1 allows for a specific means of probing the function of one of the four to five thermodynamically equivalent weak EACA sites on human plasminogen. From this particular study, it is concluded that the weak binding site for EACA on the K4 domain of Glu1-Pg is either in-part or in-whole responsible for the enhancing effect of EACA on human Glu1-Pg activation by UK.
Cummings
H S
HS
Castellino
F J
FJ
D000614:Aminocaproates / Q000378:metabolism*; D015119:Aminocaproic Acid / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D000614:Aminocaproates; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator; D015119:Aminocaproic Acid