(Received for publication, April 25, 1995)
From the
Single-chain urokinase (scuPA), the unique form of urokinase
secreted by cells, is converted to an active two-chain molecule through
the cleavage of a single peptide bond by plasmin and other specific
proteinases. Although scuPA may express limited enzymatic activity, its
contribution to plasminogen activation on cell surfaces remains
uncertain. Further, although it is well known that scuPA binds to a
specific extracellular urokinase-type plasminogen activator receptor,
the effect of this interaction on the enzymatic activity of scuPA has
not been described. In the present paper we report that the binding of
scuPA to cellular and to recombinant soluble urokinase-type plasminogen
activator receptors (suPAR) increases its catalytic activity as
measured by the cleavage of a urokinase-specific chromogenic substrate.
suPAR increased the V
Plasmin has been implicated in a variety of physiologic and
pathophysiologic processes by virtue of its capacity to proteolyze
diverse substrates. Plasmin, which is formed from plasminogen by
cleavage of the Arg The cell surface may regulate these initial steps by
providing specific binding sites for urokinase and plasminogen. Several
groups have reported that the binding of scuPA to uPAR in the presence
of plasminogen markedly accelerates plasmin
formation(3, 4, 5) . Plasmin formation is
also accelerated in systems where the scuPA has been linked directly to
the cell surface by a glycosyl phosphatidylinositol anchor (6) or when complexed with plasminogen by specific monoclonal
anti-uPA antibodies(7) . Based on these data, it has been
postulated that the enhancement of plasmin generation may be a
consequence of the increased local concentration of single-chain
urokinase (scuPA) and plasminogen on cell surfaces relative to
plasma(6) . However, it is also possible that cell surface
receptors may modulate the induction or stability of critical
intermediates in plasmin formation, which have been described to date
only in fluid phase systems. For example, there is evidence to suggest
that the limited plasminogen activator activity of scuPA (8, 9) may be stimulated when lysine or fibrin binds
to its substrate, Glu-plasminogen(10) . In addition, a
transitional state of scuPA has been described to occur after it has
been cleaved by plasmin or kallikrein in which the molecule has greater
catalytic efficacy against Glu-plasminogen than does
tcuPA(11) . Despite these observations, soluble scuPA is
generally considered to contribute little to plasminogen activator
activity relative to tcuPA in most settings. However, there may be
specific circumstances in which the intrinsic enzymatic activity of
scuPA is enhanced. In this study, we asked whether the binding of scuPA
to its receptor (uPAR) is one such situation. Our studies indicate that
binding of scuPA to soluble uPAR is sufficient to expose the catalytic
site of scuPA and cause an approximately 50-fold reduction in its K
The
amidolytic activity induced by the binding of scuPA to its receptor was
also determined on cell surfaces. First trimester human trophoblastic
cells were obtained and characterized as described
previously(16) . Confluent cultures grown in 48-well tissue
culture plates (Falcon) were washed twice with phosphate-buffered
saline, incubated for 5 min with glycine buffer, pH 3, to elute
endogenous uPA, and washed two additional times. The cells were then
incubated with phosphate-buffered saline containing 300 µM
Spectrozyme UK and 10 mM aprotinin in the presence and the
absence of 10 nM scuPA for 3 h at 37 °C, and the A
Kinetic analysis of plasminogen activation
was performed as described (17) . Briefly, 10 nM scuPA
in the presence and the absence of an equimolar concentration of suPAR
was incubated with increasing concentrations of Glu-plasminogen and 500
µM amidolytic substrate of plasmin in phosphate-buffered
saline at room temperature, and the A
Soluble uPAR augments the enzymatic activity of scuPA, as
measured by the cleavage of the chromogenic substrate Spectrozyme UK (Fig. 1). The expression of enzymatic activity by scuPA in the
presence of its receptor followed a time-dependent pattern in which
three phases could be discerned (Fig. 1, inset). When
suPAR, scuPA, and the chromogenic substrate were added
contemporaneously, no amidolytic activity was detectable for the first
19 min, after which the enzymatic activity was stimulated approximately
220% compared with scuPA alone. Beginning at approximately 50 min, an
additional 2-fold increase in the velocity of the reaction became
evident.
Figure 1:
suPAR
stimulates the amidolytic activity of scuPA. 60 nM scuPA was
incubated with (
We hypothesized that this lag phase reflects the time
required for scuPA within the suPAR
Figure 2:
Preincubation of scuPA with suPAR
stimulates its amidolytic activity. 60 nM scuPA was
preincubated with (
Figure 3:
Kinetics of scuPA activation by suPAR. A, 60 nM scuPA was preincubated with (
We then examined whether suPAR also
stimulated the plasminogen activator activity of scuPA using its
physiologic substrates Glu- and Lys-plasminogen. Kinetic analysis of
plasminogen activator activity shows that the interaction of scuPA with
suPAR decreases its K
Figure 4:
Effect of suPAR on the kinetics of
Glu-plasminogen activation by scuPA. A, 10 nM scuPA
was incubated with varying concentrations of Glu-plasminogen
(0.1-0.65 µM). The initial rates of plasmin
generation, determined as described under ``Experimental
Procedures,'' were plotted against plasminogen concentration as a
double-reciprocal plot. B, 10 nM scuPA was incubated
with equimolar concentrations of suPAR and varying concentrations of
Glu-plasminogen (0.004-0.030 µM), and the data were
analyzed as above. In both panels, the results of an experiment
representative of two so performed are
shown.
Figure 5:
suPAR
stimulates the plasminogen activator activity of scuPA. A,
cleavage of Glu-plasminogen by suPAR
The next series of experiments was
performed to determine whether this stimulation of scuPA activity by
suPAR was indeed dependent on persistent binding to its receptor,
required only initial contact between scuPA and suPAR, or was the
result of its conversion to two-chain urokinase by enzymes such as
plasmin. First, we examined the effect of ATF, which competes for
receptor binding but has no enzymatic activity. The results shown in Fig. 6indicate that the presence of ATF during the preincubation
step totally prevented the stimulatory effect of suPAR on the
amidolytic activity of scuPA. Second, in contrast,
Figure 6:
ATF inhibits the stimulation of scuPA
amidolytic activity by suPAR. 60 nM scuPA was incubated for 50
min alone (open bar), with equimolar concentrations of suPAR (bar with dots), with suPAR and 6.6-fold molar excess ATF (bar with squares), or with suPAR and 200 nM
Figure 7:
Migration of scuPA in the absence and the
presence of suPAR on SDS-polyacrylamide gel electrophoresis. 200 nM scuPA was incubated with 200 nM suPAR for 1 h (A) and for 24 h (B). Lanes 1, molecular
mass markers (fourth band from top corresponds to
protein, M
Figure 8:
The stimulation of scuPA amidolytic
activity is reversible by ATF. Preformed suPAR
Figure 9:
The
stimulation of scuPA plasminogen activator activity is reversible by
ATF. Preformed suPAR
We then asked whether
binding of scuPA to cellular uPA receptors stimulated plasminogen
activator activity as well. In these experiments 10 µM aprotinin was added to prevent endogenous serine protease activity
that might be directed at either scuPA itself or its substrate. In the
absence of added scuPA, no amidolytic activity was detected. Addition
of scuPA to the cells stimulated its PA activity approximately 6-fold
(not shown). This stimulation was almost completely (>90%) inhibited
by the addition of ATF, showing that binding of scuPA to cellular uPAR
was required. It has been reported that PAI-1 shows an approximate
40% reduction in its capacity to inhibit tcuPA bound to its cellular
receptor compared with the free enzyme(19) . Therefore, we
examined the capacity of PAI-1 to inhibit the intrinsic activity
expressed by scuPA and scuPA bound to suPAR. As shown in Fig. 10, the amidolytic activity of suPAR
Figure 10:
Inhibition of scuPA and scuPA
The results of this study indicate that the interaction of
scuPA with its receptor stimulates its amidolytic activity using a
direct assay of urokinase activity and an indirect assay that depends
upon plasminogen activation. The requirement for the physical
interaction between scuPA and its receptor is evidenced by the lag time
before enzymatic activity was observed, by the fact that the lag time
was abolished when scuPA was preincubated with suPAR, and by the fact
that stimulation of the enzymatic activity was completely abolished by
ATF. Preincubation of scuPA with soluble, recombinant uPAR increased
the V The generation of enzymatic activity
was not due to its conversion to two-chain urokinase and was thus
intrinsic to the scuPA molecule itself. This conclusion is supported by
several observations. First, scuPA continued to migrate as a
single-chain molecule in SDS-polyacrylamide gel electrophoresis.
Second, ATF both prevented and reversed the stimulatory effect of suPAR
even after stimulation had begun. Third, suPAR stimulated the
plasminogen activator activity of a variant scuPA molecule that is
unable to be cleaved by plasmin. The kinetic parameters of the
resultant suPAR Taken together, the
data are most compatible with a model in which scuPA bound to its
receptor undergoes a conformational change that exposes its catalytic
site to substrate. This hypothesis is supported further by the
increased susceptibility of suPAR The observation that
the interaction of scuPA with its receptor enhances its enzymatic
activity is compatible with previous observations concerning the role
of uPAR in accelerating the rate of plasmin formation on cell surfaces.
The effect of uPAR has generally been attributed to its role in
increasing the local concentrations of scuPA (or tcuPA) and
plasminogen, although it has been reported that the rate of plasminogen
activation by a glycolipid-anchored tcuPA molecule is retarded relative
to its fluid phase counterpart(6) . This observation raises the
possibility that the receptor activates scuPA, as suggested
previously(5) . The results of this study make it likely that
the binding of scuPA to its receptor increases plasmin generation above
that which can be attributed to a concentration effect alone. Thus, the
initiation of plasminogen activator activity attendant on the binding
of scuPA to its receptor may comprise an important initial step in
plasmin formation and the subsequent generation of tcuPA on cell
surfaces.
of scuPA 5-fold with
little change in its K
. suPAR also
stimulated the plasminogen activator activity of scuPA by decreasing
its K
for Glu-plasminogen from 1.15
µM to 0.022 µM and by increasing the k
of this reaction from 0.0015 to 0.022
s
. Preincubation of scuPA with suPAR also enhances
its susceptibility to inhibition by plasminogen activator inhibitor
type 1, consistent with exposure of its catalytic site. The activity of
scuPA bound to suPAR is not accompanied by cleavage of scuPA, which
continues to migrate as a single band in SDS-polyacrylamide gel
electrophoresis under reducing conditions. Moreover, suPAR increases
the plasminogen activator activity of a plasmin-insensitive variant,
scuPA (scuPA-Glu
), as well. Enhancement of scuPA activity
by suPAR is both prevented and reversed by its amino-terminal fragment
(amino acids 1-135), which competes for receptor binding,
suggesting that continued binding to the receptor is required for
expression of scuPA's enzymatic activity. Thus, our data suggest
that scuPA may undergo a reversible transformation between a latent and
an active state. The urokinase receptor may induce or stabilize scuPA
in its active conformation, thereby contributing to the initiation of
plasminogen activation on cell surfaces.
-Val
bond by
two-chain urokinase (tcuPA)
(
)and other
plasminogen activators(1) , in turn cleaves the
Lys
-Ile
bond in scuPA to form
tcuPA(2) . Although the capacity of plasmin and tcuPA to
mutually reinforce the production of each other is well established,
the initial steps in the conversion of plasminogen to plasmin are less
clear.
and an approximately 850-fold increase
in its catalytic efficiency.
Materials
scuPA and its amino-terminal fragment
(amino acids 1-132) and recombinant soluble uPA receptor were
prepared and isolated as
described(4, 12, 13) . PAI-1 was a gift of
Dr. Christopher Reilly (Merck Sharpe and Dohme) and was reactivated
with guanidine(4) . The chromogenic substrate of urokinase,
Spectrozyme UK
(carbobenzoxy-L--glutamyl-(
-t-butoxyl)-glycyl-arginine-p-nitroanilide
diacetate salt), Glu-plasminogen, Lys-plasminogen, the chromogenic
substrate of plasmin, Spectrozyme PL (H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide
diacetate salt), plasmin, and high molecular weight two-chain urokinase
were provided by American Diagnostica Inc. (Greenwich, CT).
-Antiplasmin was obtained from Sigma.
Variant scuPA
The cDNA for scuPA-Glu (kindly supplied by Dr. H. R. Lijnen, Leuven, Belgium) (14) was ligated into the HindIII site of pcDNA 3
(Invitrogen) and amplified in DH5 cells, and its nucleotide sequence
was verified (Sequenase, U. S. Biochemical Corp.). Transient
transfection in COS-7 cells was performed using DEAE-dextran. Cell
supernatants were collected in Dulbecco's modified Eagle's
medium containing 2 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, and 10 mM benzamidine.
scuPA-Glu
was isolated by affinity chromatography using a
monoclonal antibody directed at its kringle immobilized on Sepharose
(Pharmacia Biotech Inc.). scuPA-Glu
migrated as a single
55-kDa protein on a SDS-polyacrylamide gel under reducing conditions
before and after incubation with plasmin-Sepharose, whereas native
scuPA migrated as a 33- and a 20-kDa protein after the same exposure to
plasmin. No scuPA was detected in supernatants from COS cells
transfected with vector alone. Purified scuPA-Glu
was
then incubated sequentially with plasmin-Sepharose, diisopropyl
fluorophosphate, and Glu-Gly-Arg-chloromethyl ketone prior to use as
described (5) to eliminate any native scuPA that might have
been present but have gone undetected.
Measurement of scuPA Amidolytic Activity
The
amidolytic activity of scuPA was monitored by the release of p-nitroaniline from Spectrozyme UK, measured by the rate of
change in light absorbance at 405 nm. The reaction mixture contained 60
nM scuPA and 58 µM Spectrozyme UK in
phosphate-buffered saline, pH 7.4. In some experiments equimolar
concentrations of suPAR (60 nM) were added simultaneously with
scuPA and the chromogenic substrate, whereas in others scuPA and suPAR
were preincubated for various times before the chromogenic substrate
was added. The reaction was monitored continuously for the indicated
times, and the reaction rate (V) and the K
were calculated from the linear
portions of the activity curves as described(15) .
was determined. In other experiments, the
incubation was performed in an identical manner in the presence of
scuPA and ATF (100 nM).
Measurement of Plasminogen Activator Activity
The
plasminogen activator activity of scuPA and scuPA-Glu were measured in the presence of plasminogen using the plasmin
substrate Spectrozyme PL. 2 nM scuPA or 1 nM scuPA-Glu
were preincubated with or without
equimolar concentrations of suPAR for 50 min. 1 µM Glu- or
Lys-plasminogen and 500 µM chromogenic substrate were
added in phosphate-buffered saline, pH 7.4, for various periods of
time, and the reaction was monitored at 405 nm. A high concentration of
the chromogenic substrate (1724-fold higher than the K
) was used to minimize activation of
scuPA by plasmin and thereby isolate the stimulatory effect of suPAR.
In one set of experiments, scuPA alone or scuPA and suPAR were
preincubated for 50 min before plasminogen and the chromogenic
substrate were added. In another set of experiments, 10-fold molar
excess ATF was added to preformed suPAR
scuPA complexes
immediately prior to or 4 min after adding plasminogen and the
chromogenic substrate.
was
measured continuously over time. The A
generated in the absence of added scuPA at each time point
determined in parallel was subtracted. The concentration of plasmin
generated during each interval was calculated from the rate of change
in A
using a standard curve made with known
concentrations of plasmin. The kinetic parameters of plasmin generation
were then determined using a double-reciprocal plot of the rates of
plasmin generation versus plasminogen
concentration(17) .
Effect of PAI-1, ATF, and
In some experiments,
400-600 nM ATF or 200 nM
-Antiplasmin
-antiplasmin were added to scuPA alone or to preformed
suPAR
scuPA complexes during the preincubation step. To test the
reversibility of the reaction, scuPA and suPAR were preincubated for 50
min as above and 10-fold molar excess ATF was then added for 0-60
min before the amidolytic activity was measured. In other experiments,
120 nM PAI-1 was incubated with scuPA or with preformed
suPAR
scuPA complexes for 5 min before the chromogenic substrate
was added.
Gel Electrophoresis
Samples of 200 nM
scuPA alone, 40 nM suPAR alone, or 200 nM scuPA
preincubated with equimolar concentrations of suPAR for either 60 min
or 24 h at 37 °C were mixed with equal volumes of 10% SDS and 10%
2-mercaptoethanol. The mixture was heated at 100 °C for 5 min.
20-µl aliquots were then applied to a 10% SDS/12% polyacrylamide
gel(18) , and the protein bands were identified by staining
with Coomassie blue.
) or without (⊡) equimolar concentrations
of suPAR and the urokinase chromogenic substrate Spectrozyme UK for 100
min in phosphate-buffered saline, pH 7.4, and the chromogenic activity (A
) was monitored. suPAR alone had no
activity (
). The mean ± S.D. of three separate experiments
is shown. Inset, the triphasic pattern of the amidolytic
activity of the suPAR
scuPA complexes is
illustrated.
scuPA complex to undergo
structural isomerization to a more active form. To examine this
possibility, scuPA was preincubated with suPAR for 50 min prior to
adding chromogenic substrate. Under these conditions, the amidolytic
activity was evident immediately (Fig. 2), and the velocity of
the reaction was similar to the third phase of activity seen when the
preincubation step was omitted (Fig. 2, inset). The
third phase of activity was linear and stable over time, because
incubating scuPA with suPAR in the presence and the absence of
substrate for longer periods did not enhance its activity further. The
enzyme kinetic properties were then studied in greater detail under the
conditions of preincubation. The V
of scuPA
preincubated with suPAR for the chromogenic substrate increased more
than 5-fold, whereas the K
increased only
30% compared with the activity of scuPA alone (Fig. 3, A and B).
) or without (⊡) equimolar
concentrations of suPAR for 50 min before the addition of the urokinase
chromogenic substrate as described in the legend to Fig. 1. The
mean ± S.D. of three separate experiments is shown. Inset, the third phase of the amidolytic activity of
suPAR
scuPA complexes shown in Fig. 1(top curve)
is compared with the activity measured after preincubation of scuPA
with suPAR (Fig. 2, bottom
curve).
) or
without (⊡) equimolar concentrations of suPAR for 50 min before
increasing concentrations of the urokinase chromogenic substrate were
added. B, analysis of the data presented in A using a
double-reciprocal plot. The results from one of two such experiments
are shown.
toward
Glu-plasminogen from 1.15 to 0.022 µM and increases the k
from 0.0015 to 0.022 s
.
This corresponds to an increase in enzyme efficiency (k
/K
) from 0.0013
to 1.1 µM
s
(Fig. 4). At approximate physiologic concentrations of
scuPA (2 nM) and Glu-plasminogen (1 µM), suPAR
increased the PA activity 79.3-fold at 16.5 min compared with scuPA
alone (Fig. 5A). Lys-plasminogen was cleaved more
efficiently by scuPA than was Glu-plasminogen (Fig. 5B), as expected(10) . However, the
plasminogen activator activity of scuPA for Lys-plasminogen was
enhanced 8.9-fold following preincubation with suPAR under the same
experimental conditions described for Glu-plasminogen (Fig. 5B).
scuPA complexes. 2 nM scuPA was preincubated for 50 min in the absence (
) or the
presence (⊡) of equimolar concentrations of suPAR. 1
µM Glu-plasminogen and 500 µM plasmin
chromogenic substrate Spectrozyme PL were added for the indicated
times, and the A
was monitored. The mean
± S.D. of five separate experiments is shown. B, the
experiment was performed as described in A except that 1
µM Lys-plasminogen was used as the substrate instead of
Glu-plasminogen. The mean ± S.D. of three separate experiments
is shown.
-antiplasmin had no inhibitory effect, excluding
contamination of either preparation by plasmin. Third, the expression
of amidolytic activity was not due to the conversion of scuPA to tcuPA
by another enzyme (Fig. 7A). Rather, scuPA remained as
a single-chain molecule throughout the experiment, and no two-chain uPA
was detected even when incubations were continued for as long as 24 h (Fig. 7B). Fourth, the enhancement of the amidolytic
activity of scuPA by suPAR was totally reversible, because it was
abolished by ATF even when added 50 min after suPAR
scuPA
complexes had been allowed to form (Fig. 8). ATF also abolished
the stimulatory effect of suPAR on scuPA activity using Glu-plasminogen
as the substrate (Fig. 9). Fifth, to address the possibility
that ATF might have inhibited scuPA activation to tcuPA by an unknown
mechanism, we added ATF after the stimulatory effect of suPAR had
begun. When ATF was added to suPAR
scuPA complexes 20 min after
the addition of the urokinase chromogenic substrate, the amidolytic
activity was suppressed to the level seen with scuPA alone (Fig. 8). Further, when ATF was added 4 min after the addition
of suPAR
scuPA to Glu-plasminogen at a point when plasmin
formation had already begun, the cleavage of the chromogenic substrate
of plasmin became linear over time. The latter result is consistent
with ATF preventing further stimulation of plasmin formation by
suPAR
scuPA. In contrast, under the same experimental conditions,
neither ATF, suPAR, nor a combination of ATF and suPAR affected the
amidolytic or PA activity of two-chain urokinase or the enzymatic
activity of plasmin over 1 h (not shown), indicating that the
inhibition of scuPA activity described above was due to competition for
binding to suPAR. Finally, to further exclude any possible contribution
of plasmin-mediated conversion of scuPA to tcuPA, we analyzed the
capacity of suPAR to augment the PA activity of a variant scuPA
(scuPA-Glu
) that cannot be cleaved by
plasmin(5, 14) . suPAR stimulated the PA activity of
the mutant scuPA approximately 115-fold in the presence of 1
mM Glu-plasminogen (not shown).
-antiplasmin (hatched bar). Each mixture
was then incubated with the urokinase chromogenic substrate for an
additional 40 min, and the A
was determined.
The mean ± S.D. of three separate experiments is
shown.
= 46 kDa); lanes 2,
suPAR alone; lane 3, in A, suPAR and scuPA incubated
for 60 min; lane 3 in B, suPAR and scuPA incubated
for 24 h; lanes 4, scuPA alone.
scuPA complexes
were incubated with the urokinase chromogenic substrate in the absence
(
) or in the presence of 10-fold molar excess ATF added either
coincident with (
) or 20 min after (
) the addition of
substrate. The amidolytic activity seen when ATF and the chromogenic
were added to suPAR
scuPA complexes at the same time cannot be
distinguished from the activity of scuPA alone (not shown). The mean
± S.D. of three separate experiments is
shown.
scuPA complexes were incubated with
Glu-plasminogen as described in Fig. 5in the absence of ATF
(⊡) or in the presence of 10-fold molar excess ATF added either
coincident with (
) or 4 min after (
) the addition of
Glu-plasminogen. The plasminogen activator activity seen when ATF and
the Glu-plasminogen were added to suPAR
scuPA complexes at the
same time cannot be distinguished from the activity of scuPA alone (not
shown). The mean ± S.D. of three separate experiments is
shown.
scuPA complexes
was totally inhibited by a 3-fold molar excess of PAI-1, whereas scuPA
was virtually insensitive to inhibition under the same conditions.
suPAR
by PAI-1. The amidolytic activity of scuPA and scuPA
suPAR were
determined as in Fig. 5in the presence or the absence of a
5-min incubation with 120 nM PAI-1. Black bar, scuPA
alone; hatched bar, scuPA preincubated with PAI-1; shaded
bar, scuPA preincubated with suPAR for 50 min; white bar,
scuPA preincubated with suPAR for 50 min and an additional 5 min with
PAI-1 (not evident). The mean ± S.D. of three separate
experiments is shown.
of the reaction on its chromogenic
substrate approximately 500% with minimal change in the K
, compatible with an increase in the
total amount of enzymatic activity. Further, suPAR increased the
catalytic efficiency of scuPA approximately 850-fold when the cleavage
of Glu-plasminogen was studied.
scuPA complexes described here differ from those
reported for high molecular weight uPA, low molecular weight
uPA(20) , and scuPA itself, suggesting the formation of a
previously undescribed activity state of scuPA.
scuPA complexes to inhibition by
PAI-1 compared with scuPA alone. Thus, scuPA may be better thought of
as a regulatory enzyme rather than solely as a proenzyme. It is likely
that the increase in enzymatic activity is induced by binding to suPAR
as a result of a conformational change in scuPA. In support of this
concept, we have observed that preincubation of scuPA with suPAR also
promotes its binding to vitronectin, that this binding of
suPAR
scuPA complexes is no longer inhibitable by scuPA alone, and
that the binding of scuPA to
-macroglobulin
receptor/lipoprotein receptor-related protein is inhibited by suPAR as
well (manuscript submitted). Our data do not exclude the possibility
that soluble scuPA undergoes a reversible transition between an active
and an inactive state and that the receptor stabilizes the active
conformation as proposed (5) or, theoretically, binds
preferentially to the enzymatically active form of the molecule. Thus,
the presence of suPAR may change the equilibrium of the reaction
favoring the generation of scuPA molecules that are in the active
conformation. However, the relationship of the conformation of scuPA
bound to its receptor to the intermediates that develop during its
conversion to tcuPA (11) remains to be determined. In either
case, it is clear that the transformation of scuPA to an active enzyme
is a reversible process dependent on continued binding to suPAR,
because the addition of ATF, even after scuPA's enzymatic
activity had already been stimulated, abrogated any subsequent
receptor-dependent augmentation. The physiologic relevance of this
reaction is supported both by the fact that the cleavage of
Glu-plasminogen by single-chain urokinase was stimulated by suPAR as
well by the fact that binding of scuPA to cellular uPA receptors
stimulated its intrinsic catalytic activity.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.