CHARACTERIZATION OF THE BINDING OF UROKINASE-TYPE PLASMINOGEN-ACTIVATOR (U-PA) TO PLASMINOGEN, TO PLASMINOGEN-ACTIVATOR INHIBITOR-1 AND TO THE U-PA RECEPTOR
Hr. Lijnen et al., CHARACTERIZATION OF THE BINDING OF UROKINASE-TYPE PLASMINOGEN-ACTIVATOR (U-PA) TO PLASMINOGEN, TO PLASMINOGEN-ACTIVATOR INHIBITOR-1 AND TO THE U-PA RECEPTOR, European journal of biochemistry, 224(2), 1994, pp. 567-574
Binding parameters [association-rate (k(ass)) and dissociation-rate (k
(diss)) constants, and affinity constants (K-A = k(ass)/k(diss))] for
the interaction between urokinase-type plasminogen activator (u-PA) an
d its substrate plasminogen, its inhibitor plasminogen activator inhib
itor-1 (PAI-1) and its receptor (u-PAR), were determined by real-time
biospecific interaction analysis (BIA). The K-A values for the binding
of [S741A]recombinant plasminogen (plasminogen with N-terminal Glu an
d with the active site Ser741 mutagenized to Ala) or of active site-bl
ocked plasmin (D-ValPheLysCH(2)-plasmin) to the 54-kDa or 32-kDa molec
ular forms of recombinant single-chain u-PA (rscu-PA) ranged between 0
.57X10(6)M(-1) and 1.7X10(6)M(-1), compared to 14-22X10(6) M(-1) for b
inding to the corresponding active site-blocked recombinant two-chain
u-PA (rtcu-PA) moieties. K-A values for binding of these plasmin(ogen)
moieties to [Ser356deHAla]rtcu-PA (rtcu-PA with the active site Ser35
6 converted to dehydroAla) were 81X10(6)M(-1) and 670X10(6) M(-1), res
pectively. Binding of active site-blocked LMM-plasmin (a low-molecular
-mass plasmin derivative lacking kringles 1-4) and of the plasmin B ch
ain to [Ser356deHAla]rtcu-PA occurred with K-A values of 3.7X10(6)M(-1
) and 0.33X10(6) M(-1), compared to 670X10(6) M(-1) for the binding of
intact D-ValPheLysCH(2)-plasmin to [Ser356deHAla]rtcu-PA. The K-A val
ues for binding of latent PAI-1 to 54-kDa or 32-kDa molecular forms of
rscu-PA and rtcu-PA were in the range 0.34-2.1X10(6) M(-1). Reactivat
ed PAI-1 bound to 54-kDa and 32-kDa rtcu-PA moieties with K-A values o
f 26x10(6)M(-1) and 28x10(6) M(-1), compared to 0.77 X10(6)M(-1) and 3
.2X10(6) M(-1) for binding to the corresponding single-chain u-PA spec
ies, and 450X10(6) M(-1) for binding to [Ser356deHAla]rtcu-PA. K-A val
ues 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 (K-A of 4.6-8.
5x10(9) M(-1)), whereas no significant binding of 32-kDa u-PA moieties
was observed (K-A less than or equal to 0.2X10(6) M(-1)). Binding of
the covalent rtcu-PA/PAI-1 complex to u-PAR was indistinguishable from
that of rtcu-PA. These results indicate that plasminogen and plasmin
bind with higher affinity to two-chain u-PA than to single-chain u-PA
moieties, whereas binding requires the presence of both the A and the
B chain; reactivated PAI-1 binds to rtcu-PA moieties with a higher aff
inity than latent PAI-1, but has a very low affinity for rscu-PA; 54-k
Da u-PA moieties bind in a 1:1 stoichiometry to u-PAR with high affini
ty which does not require the u-PA active site; and the binding sites
of u-PA for PAI-1 and for plasminogen are within the 32-kDa u-PA moiet
y (Leu144-Leu411), they do not overlap and are fully accessible when 5
4-kDa u-PA is bound to its receptor.