From the
Single chain urokinase-type plasminogen activator or
pro-urokinase (pro-UK) has been reported to have a significant
intrinsic amidolytic and plasminogen activator activity, estimated to
be about 0.2-0.6% that of two-chain urokinase (UK). However, it
has also been suggested that this reported activity is related entirely
to trace UK contaminants generated during the analytic procedures. In
an attempt to resolve this controversy, it was decided to measure the
incorporation of diisopropyl fluorophosphate (DFP) by pro-UK and UK.
Surprisingly, it was found that although >98% of the apparent
intrinsic activity of pro-UK was inhibited by 5 m
M DFP,
>97% of this activity was recoverable after exhaustive dialysis of
the preparation. This finding could not be explained by UK generation,
which was excluded. Instead, the findings indicated that DFP inhibition
of pro-UK, in contrast to UK and other serine proteases, was largely
reversible. The reaction rate of the reversible inhibition was
significantly slower than that of irreversible inhibition by DFP. When
the hydrolysis of DFP (2 m
M) during incubation (37 °C)
with or without pro-UK (20 µ
M) was compared, a >5-fold
acceleration of DFP hydrolysis in the presence of pro-UK was found,
whereas little loss of DFP occurred in the presence of UK (20
µ
M), consistent with 1:1 stoichiometry. This suggested
that pro-UK acted as a slow DFPase in the reaction, a finding
consistent with a reversible DFP-enzyme reaction. It was concluded that
pro-UK has a distinct and measurable intrinsic catalytic activity,
which is qualitatively unique and thereby distinguishable from that of
UK as well as other serine proteases.
Single-chain urokinase-type plasminogen activator behaves like a
zymogen in plasma, since it is inert at physiological concentrations
and does not form SDS stable complexes with plasma inhibitors
(1) . Since it is the precursor of two-chain urokinase
(UK),(
However, relative to other
zymogen precursors of serine proteases, pro-UK has been found by
several studies to have a much higher intrinsic activity estimated to
be 0.4%
(3, 4, 5) to 0.6%
(6) against
plasminogen and 0.1-0.2% against synthetic substrate
(1, 3, 6) of the activity of UK. This relative
activity is about 5,000-fold higher than that of most protease
zymogens. By contrast, some studies have suggested that pro-UK has no
measurable intrinsic activity
(7, 8) and that the
reported activity is related to trace UK contaminants.
The
discrepancies among these studies have not been reconciled but are
related to the technical problem involved in effectively preventing UK
generation by plasmin, the product of plasminogen activation. In order
to help resolve the controversy, it was decided to analyze the
incorporation of radiolabeled diisopropyl fluorophosphate (DFP), which
has been shown previously to be incorporated by pro-UK
(9) ,
since any incorporation by two-chain UK contaminants can be readily
identified by electrophoresis under reducing conditions.
Surprisingly, it was found that the inactivation of pro-UK by DFP
was predominantly (>97%) reversible. To our knowledge, this finding
is unique to pro-UK since the interaction between DFP and UK as well as
other serine proteases is irreversible. As a result, the findings
demonstrate that the intrinsic activity of pro-UK can be qualitatively
distinguished from that of UK contaminants.
[
Based on the findings from the above
experiment, DFP (2.0 m
M) was then incubated (37 °C) in the
presence or absence of 20 µ
M pro-UK or UK. After a 16-h
incubation, 100-µl aliquots were removed from the reaction mixture
and incubated (37 °C) with 100 µl of plasmin (100 n
M)
for 30 min. 50 µl of the incubation mixture was then removed and
added to 50 µl of S2251 (3.0 m
M) to measure residual
plasmin activity. S2251 is insensitive to UK or pro-UK.
[
These
findings are consistent with a reversible inhibition of pro-UK by DFP.
Although a DFP-enzyme complex was formed in the active site of pro-UK,
this complex appears to be unstable. By contrast, this complex has been
described as being ``indefinitely stable'' with other serine
proteases
(12) . Although this phenomenon remains to be
explained, it may be seen as representing yet another of a series of
exceptional properties of the catalytic site of pro-UK. These include
its relatively high intrinsic activity
(1, 3, 4, 5, 6) ; its
K
In conclusion,
the present findings confirm that pro-UK has a measurable intrinsic
activity, which is qualitatively distinct and readily distinguishable
from UK contaminants. To our knowledge, a reversible inactivation by
DFP has not been reported previously for any serine protease.
)
this proenzyme is commonly referred to as
pro-urokinase (pro-UK)
(2) .
H]DFP Incorporation of
Pro-UK-Highly purified recombinant pro-UK (10
µ
M) from Escherichia coli (Farmitalia Carlo Erba,
Milan, Italy), which was DFP-pretreated (5 m
M) to inactivate
trace UK contaminants, or UK (1 µ
M) was incubated (37
°C) with 0.1 m
M [
H]DFP (DuPont NEN;
maximal concentration obtainable) for 6 h with or without 0.1
m
M Glu-Gly-Arg-chloromethyl ketone in 0.1
M sodium
phosphate (pH 7.4), 0.15
M NaCl, 0.2% bovine serum albumin,
and 0.01% Tween 80. Samples were removed, and either boiled immediately
in reducing sodium dodecyl sulfate (SDS) sample buffer or boiled after
exhaustive dialysis. After 10% SDS-polyacrylamide gel electrophoresis
(PAGE) and autoradiography, the radioactive bands were cut out for
counting. The single-chain pro-UK was seen separately as a
50-kDa
band, whereas any UK contaminants were seen at
30 kDa. Evidence
that incorporation of DFP occurred at the active site serine rather
than elsewhere was obtained by using denatured (reduced and
carboxymethylated) pro-UK as a control.
Amidolytic Activity of Pro-UK in the Presence of DFP and after
Dialysis
20 µ
M pro-UK or 40 n
M UK was
incubated (37 °C) with 5 m
M DFP for 6 h. A 500-fold excess
of pro-UK was used based on the intrinsic activity of pro-UK against
S2444 being 0.2% the activity of UK
(3) . The samples were
then exhaustively dialyzed to remove DFP. Using the synthetic substrate
S2444 (final concentration of 0.75 m
M), aliquots of incubated
samples were taken and diluted by 1:2 for assay of amidolytic activity
before and after dialysis. The amidolytic activity was measured by the
increase in absorbanceover time at 410 nm against a reference wave
length of 490 nm (410/490 nm) on a microtiter plate reader (MR5000;
Dynatech Laboratory Inc., Alexandria, VA) as described previously
(10) . As a control, pro-UK (20 µ
M) without DFP was
similarly incubated and dialyzed. Protein concentrations of pro-UK
before and after dialysis were determined by measuring
A
(E280% = 1.36).
DFP Inactivation of Pro-UK and UK
Pro-UK (2.5
µ
M) or UK (5.0 n
M) was incubated with S2444 (0.75
m
M) at 37 °C in the presence of a range of concentrations
(0-10 m
M) of DFP. The amount of amidolytic activity
generated was measured by the increase in absorbance over 30 min, as
described previously
(10) .
Acceleration of DFP Hydrolysis by Pro-UK
The
spontaneous hydrolysis of DFP was determined by measuring the
amidolytic activity of plasmin remaining after its addition to DFP
samples as follows. DFP (0.2, 2.0, and 5.0 m
M) was incubated
(37 °C); at time points ranging from 0 to 48 h, 100-µl aliquots
were removed for incubation (37 °C) with 100 µl of plasmin (100
n
M) for 30 min. 50 µl of the incubation mixture was then
removed and added with 50 µl of S2251 (3.0 m
M) to measure
residual plasmin activity.
H]DFP Incorporation of
Pro-UK-Relative to the [
H]DFP
incorporation by UK (100%), incorporation by pro-UK was only 0.04
± 0.03%. Autoradiography of reduced SDS-PAGE showed essentially
a single band with a M
50,000 in the lane
corresponding to pro-UK (10 µ
M), which was readily
distinguishable from a band with a M
33,000 in
the lane corresponding to the UK (1 µ
M) control. There was
no measurable incorporation of [
H]DFP by
denatured pro-UK, indicating that incorporation of the
[
H]DFP by pro-UK was specific for the active site
serine. This was further confirmed by the fact that
[
H]DFP incorporation was largely diminished in
the presence of 0.1 m
M Glu-Gly-Arg-chloromethyl ketone, a
specific inhibitor of the active site of UK. A similar incorporation of
[
H]DFP by the active site of a plasmin-resistant
mutant pro-UK has been reported previously
(11) . However, the
trace amount of [
H]diisopropyl phosphate-pro-UK
was not very stable, because it was found that the
[
H]DFP-incorporated M
50,000
band disappeared on the reduced SDS-PAGE after the sample was
exhaustively dialyzed. By contrast, the band of
[
H]diisopropyl phosphate-UK remained.
Amidolytic Activity of Pro-UK Inhibited by DFP Is Mostly
Recovered after Dialysis (Fig. 1)
The base-line amidolytic
activity of the pro-UK preparation was 0.2% that of UK, consistent with
previous reports
(1, 3, 6) . The presence of DFP
(5.0 m
M) resulted in a loss of >98.6% of the amidolytic
activity of pro-UK and >99.99% of the activity of UK. However, after
exhaustive dialysis, 97.2% of the amidolytic activity of pro-UK was
recovered, whereas no activity was recovered from DFP-treated UK. The
possibility that this finding was related to trace UK generation due to
autoactivation of pro-UK or due to activation by a non-serine protease
contaminant was tested by autoradiography of a
I-pro-UK
probe, which allowed the detection of
0.08% UK (
40 ng)
generation from the 50-µg pro-UK sample (20 µ
M
50 µl) containing 0.5% of
I-pro-UK (
5
µCi/µg or
8000 cmp/ng). No evidence of UK generation was
found (date not shown). This was further confirmed by a control in
which 20 µ
M pro-UK was incubated in parallel and dialyzed
without DFP treatment (Fig. 1). Under these conditions no
significant UK was generated, since only 0.8% (20.00 versus 20.16 mOD/min) increase in UK activity occurred, which was
insignificant since it was much smaller than the S.D. of the
experiment. These data therefore indicate that DFP inactivation of
pro-UK, in contrast to other serine proteases, is almost completely
reversible. This conclusion is also consistent with a previously
published, but unexplained observation, that
[
H]DFP incorporation by a plasmin-resistant
mutant pro-UK was not prevented by preincubation with excess unlabeled
DFP (with subsequent hydrolysis of the unlabeled DFP)
(11) .
Figure 1:
The effect of DFP on amidolytic
activity of u-PA. All samples were assayed with S2444 in a 1:2
dilution. 1, pro-UK (20 µ
M); 2, pro-UK
(20 µ
M) in the presence of DFP (5 m
M);
3, same as (2) after dialysis; 4, UK (40
n
M); 5, UK (40 n
M) in the presence of DFP (5
m
M); 6, same as 5 after dialysis;
7, pro-UK (20 µ
M) incubated in buffer followed by
dialysis.
DFP Inactivation of Pro-UK and UK
(Fig. 2)
The amidolytic activity of UK was rapidly
inhibited by 1 m
M DFP, and this inhibition was irreversible as
is characteristic for serine proteases. By contrast, pro-UK was far
more resistant to inactivation by DFP as also observed previously
(9, 11) . Only 40% of its activity was inhibited by 1
m
M DFP, and 85% was inhibited by 5-10 m
M DFP
within 30 min of incubation. The remaining pro-UK activity was
inhibited by longer (>30 min) incubation with higher (>10
m
M) concentrations of DFP. However, as noted above, 97% of
this activity was recoverable and therefore represented a reversible
inhibition by DFP.
Figure 2:
DFP
inactivation of pro-UK () and UK (
). Pro-UK (2.5
µ
M) or UK (5.0 n
M) was incubated with S2444 (0.75
m
M) in the presence of DFP (0-10 m
M) and the
amidolytic activity measured by absorbance increase over 30
min.
Acceleration of DFP Hydrolysis by Pro-UK (Fig.
3)
Incubation (37 °C) of DFP (0.2, 2.0, and 5.0 m
M)
for 0-48 h showed that its activity, sufficient to completely
inactivate plasmin (50 n
M), was dose-dependent but was
retained for at least 6 h, followed by a progressive loss of activity.
At the highest concentration of DFP (5 m
M), activity was
preserved for 24 h, as indicated by essentially complete plasmin
inactivation (Fig. 3).
Figure 3:
Spontaneous hydrolysis of DFP over time
measured by the residual amidolytic activity of plasmin (50
n
M). DFP 0 (), 0.2 (
), 2.0 (
), and 5.0
(
) m
M was incubated (37 °C) with 100 µl of
plasmin (100 n
M) for 30 min, after which 50 µl was removed
and added to 50 µl of S2251 (3.0 m
M) for measurement of
residual plasmin activity (absorbance over 30
min).
A 16-h period of incubation was
therefore selected for the experiment with 20 µ
M pro-UK or
UK incubated with 2.0 m
M DFP. The DFP remaining was expressed
by the residual plasmin S2551 activity measured after 30-min incubation
of plasmin and DFP samples. Less residual plasmin activity indicated
more DFP remaining. When DFP was incubated without pro-UK, the residual
plasmin activity after 16 h was about 1.46 mOD/min, corresponding to
5.8% of the total added plasmin activity (25 mOD/min). However, when
pro-UK was included in the reaction mixture, 7.95 mOD/min of plasmin
activity remained, which corresponded to 31.8% of the total added
plasmin. This reflected a significantly greater loss of DFP activity in
the presence of pro-UK. When the same molar concentration of UK was
substituted for pro-UK, little effect (1.46 versus 1.47
mOD/min) on DFP inactivation of plasmin was observed, consistent with a
1:1 stoichiometric interaction. The findings indicate that hydrolysis
of DFP is accelerated by pro-UK and suggest that pro-UK acts as a slow
DFPase, which catalyzed the reaction. Therefore, the interaction of DFP
with pro-UK, in contrast to UK, is non-stoichiometric.
, which is significantly lower than that
of UK
(13, 14) indicative of a better formed substrate
binding pocket than that of the enzyme; and the 500-fold promotion of
its intrinsic activity against native plasminogen when the latter is
bound to fibrin fragment E, giving single-chain pro-UK a catalytic
efficiency comparable to that of two-chain UK
(15) . The
structural determinants responsible for these properties of the
catalytic domain of pro-UK remain to be determined.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.