Characterization of the binding of urokinase-type plasminogen activator (u-PA) to plasminogen, to plasminogen-activator inhibitor-1 and to the u-PA receptor.
Binding parameters [association-rate (kass) and dissociation-rate (kdiss) constants, and affinity constants (KA = kass/kdiss)] for the interaction between urokinase-type plasminogen activator (u-PA) and its substrate plasminogen, its inhibitor plasminogen activator inhibitor-1 (PAI-1) and its receptor (u-PAR), were determined by real-time biospecific interaction analysis (BIA). The KA values for the binding of [S741A]recombinant plasminogen (plasminogen with N-terminal Glu and with the active site Ser741 mutagenized to Ala) or of active site-blocked plasmin (D-ValPheLysCH2-plasmin) to the 54-kDa or 32-kDa molecular forms of recombinant single-chain u-PA (rscu-PA) ranged between 0.57 x 10(6) M-1 and 1.7 x 10(6) M-1, compared to 14-22 x 10(6) M-1 for binding to the corresponding active site-blocked recombinant two-chain u-PA (rtcu-PA) moieties. KA values for binding of these plasmin(ogen) moieties to [Ser356deHAla]rtcu-PA (rtcu-PA with the active site Ser356 converted to dehydroAla) were 81 x 10(6) M-1 and 670 x 10(6) M-1, respectively. Binding of active site-blocked LMM-plasmin (a low-molecular-mass plasmin derivative lacking kringles 1-4) and of the plasmin B chain to [Ser356deHAla]rtcu-PA occurred with KA values of 3.7 x 10(6) M-1 and 0.33 x 10(6) M-1, compared to 670 x 10(6) M-1 for the binding of intact D-ValPheLysCH2-plasmin to [Ser356deHAla]rtcu-PA. The KA values for binding of latent PAI-1 to 54-kDa or 32-kDa molecular forms of rscu-PA and rtcu-PA were in the range 0.34-2.1 x 10(6) M-1. Reactivated PAI-1 bound to 54-kDa and 32-kDa rtcu-PA moieties with KA values of 26 x 10(6) M-1 and 28 x 10(6) M-1, compared to 0.77 x 10(6) M-1 and 3.2 x 10(6) M-1 for binding to the corresponding single-chain u-PA species, and 450 x 10(6) M-1 for binding to [Ser356deHAla]rtcu-PA. KA values for binding of plasmin(ogen) to the covalent rtcu-PA/PAI-1 complex were similar or somewhat higher than those for binding to uncomplexed rtcu-PA. Single-chain and two-chain 54-kDa u-PA moieties bound with a 1:1 stoichiometry and with very high affinity to u-PAR (KA of 4.6-8.5 x 10(9) M-1), whereas no significant binding of 32-kDa u-PA moieties was observed (KA < or = 0.2 x 10(6) M-1).(ABSTRACT TRUNCATED AT 400 WORDS)
Lijnen
H R
HR
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
De Cock
F
F
Collen
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000737:chemistry / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism*; D010960:Plasminogen Activators / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
C524898:PLAUR protein, human; D017395:Plasminogen Activator Inhibitor 1; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D010958:Plasminogen; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator