The control of the urokinase-catalyzed activation of human glutamic acid 1-plasminogen by positive and negative effectors.
The urokinase-catalyzed activation of human Glu1-plasminogen (Glu1Pg) has been found to be inhibited by monovalent anions in the following order of effectiveness: I- greater than SCN- greater than Cl- greater than IO3- greater than HCOO- greater than F- greater than OAc-. The inhibition is reversed by epsilon-aminocaproic acid, with its effectiveness in this capacity generally inversely proportional to the strength of the binding of the anion. The physical basis for the anion inhibition and epsilon-aminocaproic acid stimulation lies in the ability of these effectors to cause measurable opposite alterations in the conformation of Glu1Pg, which are revealed through study of the sedimentation velocity of the protein under various conditions. The kinetic mechanism of the chloride inhibition of Glu1Pg activation has been examined in detail. It has been found that the Glu1Pg.Cl complex serves as an alternate substrate to Glu1Pg for urokinase, with a greatly increased Km (25 +/- 3 and 2.2 +/- 0.3 microM, respectively) for activation. The kcat for the urokinase.Glu1Pg.Cl complex is approximately the same as that for urokinase.Glu1Pg (1.6 +/- 0.2 - 2.0 +/- 0.2/s). Similarly, the stimulation by epsilon-aminocaproic acid also results from effects on the Km of the activation, which is reduced to 1.8 +/- 0.2 microM for the Glu1Pg.Cl.epsilon-aminocaproic acid complex. The kcat for the urokinase.Glu1Pg.Cl.epsilon-aminocaproic acid of 2.4 +/- 0.3/s complex is not greatly different from that for urokinase.Glu1Pg.Cl. Nuclear magnetic resonance studies of the Glu1Pg-induced line broadening of the 35Cl- spectra in the presence and absence of epsilon-aminocaproic acid suggest that Cl- and epsilon-aminocaproic acid simultaneously bind to the protein and that each of these effectors displays its effects through separate binding sites.
Urano
T
T
Department of Chemistry, University of Notre Dame, Indiana 46556.
Sator de Serrano
V
V
Chibber
B A
BA
Castellino
F J
FJ
D000838:Anions; D006801:Humans; D007700:Kinetics; D009994:Osmolar Concentration; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators; D012492:Salts; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D000838:Anions; D012492:Salts; D010958:Plasminogen; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator