©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inactivation of the Intrinsic Activity of Pro-urokinase by Diisopropyl Fluorophosphate Is Reversible (*)

Jian-ning Liu , Victor Gurewich (§)

From the (1) Vascular Research Laboratory, Institute for the Prevention of Cardiovascular Disease, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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),()this proenzyme is commonly referred to as pro-urokinase (pro-UK) (2) .

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.


MATERIALS AND METHODS

[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.

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.


RESULTS AND DISCUSSION

[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 M50,000 in the lane corresponding to pro-UK (10 µ M), which was readily distinguishable from a band with a M33,000 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 M50,000 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.

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, 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.

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.


FOOTNOTES

*
This work was supported in part by a grant-in-aid from the American Heart Association (to J. L.) and a grant from Farmitalia Carlo Erba. This study was presented in part at the XI International Congress on Fibrinolysis, June 29-July 3, 1992, Copenhagen, Denmark (Liu, J., and Gurewich, V. (1992) Fibrinolysis 6, Suppl. 2, 80 (abstr.)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Vascular Research Laboratory, Burlington Bldg., Rm. 554G, Deaconess Hospital, One Deaconess Rd., Boston, MA 02215. Tel.: 617-632-0801; Fax: 617-247-2501.

The abbreviations used are: UK, urokinase; pro-UK, pro-urokinase or single-chain urokinase-type plasminogen activator; DFP, diisopropyl fluorophosphate; PAGE, polyacrylamide gel electrophoresis; mOD, mini-absorbance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.