From the Cardiovascular Biology Laboratory, Harvard School of
Public Health and the § Massachusetts General Hospital,
Boston, Massachusetts 02114 and the Graduate Department
of Biochemistry, Brandeis University,
Waltham, Massachusetts 02453
Received for publication, October 11, 2000
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ABSTRACT |
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The therapeutic properties of plasminogen
activators are dictated by their mechanism of action. Unlike
staphylokinase, a single domain protein, streptokinase, a 3-domain
( Cleavage of the zymogen plasminogen
(Pg)1 to the active enzyme
plasmin by Pg activators is the first step in fibrinolysis, the process
of blood clot dissolution. Two bacterial Pg activators, streptokinase
(SK) and staphylokinase (SAK), initiate fibrinolysis in humans by
forming a cofactor-enzyme complex with plasmin (1-4). In this complex,
SK or SAK serves as a cofactor that redirects the substrate specificity
of plasmin from the cleavage of fibrin to the cleavage of Pg. Despite
their similarities, SAK and SK have fundamentally different mechanisms.
SK binds with significantly greater affinity to plasmin than SAK (5).
The SAK·plasmin complex, but not the SK·plasmin complex is
inactivated by The mechanism by which SK nonproteolytically generates an active site
in the zymogen Pg (Pg*) is controversial (10-12). Clues to this
mechanism have come from comparisons of the structures of SK and SAK
(13). SK has three sequential domains of roughly equal sizes ( Novel insights into the structural elements required for a
staphylokinase versus a streptokinase mechanism of Pg
activation may come from an analysis of a recently isolated
Streptococcus uberis Pg activator (SUPA, also known as PauA)
(15). SUPA is produced by a streptococcus that causes mastitis in cows
and has no significant nucleotide or genomic homology to streptokinase or staphylokinase (16, 17). SUPA has a putative two-domain structure (29 kDa) that is intermediate between the one-domain structure of SAK (16 kDa) and the three-domain structure of SK (47 kDa). SUPA has been considered a new class of bacterial Pg activator
that may act through a unique mechanism (16, 17) with Pg activation
kinetics similar to streptokinase and a staphylokinase-like susceptibility to inhibition by Protein and Reagents--
-Bovine (b) Pg was prepared from fresh
citrated b-plasma by affinity chromatography with lysine-substituted
Sepharose and pretreated with aprotinin-agarose to remove contaminating
plasmin (19). The purified b-Pg was assessed by 10% SDS-PAGE and
active site titration after activation by urokinase (9 nM,
1 h at 37 °C, in 50 mM Tris buffer, 0.15 M NaCl, 20% glycerol) as described (5). The b-Pg contained
less than 0.5% plasmin.
Cloning, Expression, and Purification of Recombinant (r)
Proteins--
The SK gene was cloned from Streptococcus
equisimilis, expressed in bacteria as a MBP fusion protein via the
pMalc vector (New England Biolabs, Beverly, MA), and purified as
described in detail (5). The
Eleven strains of Streptococcus uberis were tested for their
ability to activate b-Pg. Genomic DNA was isolated as described (20).
Polymerase chain reaction was performed with primers containing EcoRV and HindIII
5'-gatatcaccggttaygaywsngaytaytaygc and
tctagattaaggtttataacttttyttngtdatnarrtayttytc. The
amplified DNA from SUPA clone 70.2 was sequenced on both strands and
ligated into the pMal-c vector (New England Biolabs) at the StuI site for expression as a fusion polypeptide with
maltose-binding protein (MBP). Cloning at this site permits specific
cleavage between MBP and Ile1 of SUPA, yielding intact SUPA
with its native NH2 terminus (12). The MBP-rSUPA fusion
protein was absorbed on DEAE Affi-Gel Blue-agarose and eluted between
110 and 150 mM NaCl by a linear gradient of 0 to 200 mM NaCl in 10 mM phosphate buffer, pH 7.2. The
rSUPA was cleaved from MBP by treatment with Factor Xa (New England Biolabs) for 24 h at room temperature in 200 mM
Tris-HCl buffer, pH 8.0, 100 mM NaCl, 2 mM
CaCl2. The SUPA cDNA 70.2 was also subcloned into
pProEX HTb expression vector (Life Technologies) using BamHI and PstI restriction enzymes. This resulted in the
expression of SUPA with a short N-terminal fusion peptide containing a
His6-tag. The His6-SUPA protein was absorbed on
Ni-NTA affinity resin and eluted by 20 mM Tris-HCl, pH 8.5 (at 4 °C), with 100 mM KCl, 5 mM
2-mercaptoethanol, 10% glycerol, and 100 mM imidazole. The eluted sample was analyzed by SDS-PAGE and dialyzed against assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH
7.4).
Active Site Titration--
The molar quantity of active sites
generated by rSUPA in b-Pg was determined by active site titration with
the fluorogenic substrate 4-methylumbelliferyl
p-guanidinobenzoate (MUGB, Sigma) as described (5, 21).
Kinetic Assays--
The amidase kinetic parameters of human
plasmin (h-plasmin), b-plasmin, and the activator complexes were
studied with
H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride (S2251, Chromogenix, Sweden) as previously described (22). The h-plasmin·rSK and b-plasmin·rSUPA complexes were prepared by mixing 10 nM h-Pg ( Steady-state Pg Activation Kinetic Parameters--
The kinetics
of Pg activation by either free rSK and rSUPA or activator complexes
were studied as described (23). Stoichiometric activator complexes were
formed by mixing h-Pg ( Continuous Assay for Determination of the Rates of Active Site
Generation in Pg-SK Complexes--
The first-order rate constant for
the appearance of an amidolytic active center in the hPg·SK complex
was determined as described by Chibber and Castellino (24) at 4 °C,
in a final volume of 0.3 ml, with a low ionic strength buffer
consisting of 10 mM Hepes/NaOH, pH 7.4. Then h-Pg (100 nM; Binding Assays--
Wells of microtiter plate were coated with
50 µl of b-Pg (5 µg/ml). The wells were washed and nonspecific
protein-binding sites were blocked with 1% bovine serum albumin. After
that 50 µl of rSUPA (50 µg/ml) were added for 1 h. After
washing 50 µl of SK Influence of SUPA on the Bovine
Plasmin--
Primary Structure of SUPA--
The lysates of 11 b-isolates of
S. uberis were tested for their ability to activate b-Pg.
The SUPA gene was cloned from the S. uberis isolate (number
70) that showed the greatest Pg activation. The deduced amino acid
sequence predicted a mature peptide mass of 30.7 kDa
(GenBankTM accession number AF283574). In pairwise
alignments by ClustalW (Table I), the
putative Active Site Generation--
A critical distinction between a SAK
and SK mechanism is the ability of SK to generate an active site in Pg
under conditions in which proteolysis is blocked. Consequently, the
ability of SUPA to generate an active site generation in b-Pg was
studied in the presence of a titrant (MUBG) that rapidly acylates the active site of plasmin, as originally described by McClintock and Bell
(7). Under these conditions trace plasmin is rapidly inactivated and
SK, but not SAK, can generate an active site in Pg. When b-Pg was added
to the active site titrant only minimal numbers of preformed active
sites were detected (Fig. 2A)
indicating that only trace quantities of plasmin were present in this
preparation (21). When b-Pg was preincubated with MUGB for 5 min (to
inhibit trace amounts of plasmin), followed by the addition of
His6-rSUPA or MBP-rSUPA minimal active site generation
occurred (~5-12% of expected). However, when rSUPA was added 5 min
after b-Pg to the cuvette, there was rapid and complete active site
formation. Examination of the reaction between SUPA and b-Pg by
SDS-PAGE showed that active site generation occurred in the b-Pg
molecule without proteolytic generation of plasmin (Fig.
2B). Thus SUPA, like SK but not SAK (3), nonproteolytically
generated an active site in b-Pg. However, rSUPA was unable to generate
an active site in h-Pg and rSK could not generate one in b-Pg (Fig.
4B and not shown).
Temperature Dependence and Rate of Active Site Formation--
When
compared with rSK and h-Pg under similar conditions, active site
generation in b-Pg by rSUPA was slower (Fig. 2A) and rSUPA
also required a higher reaction temperature (19 °C) than SK
(4 °C) for any active site generation to be detected (not shown). Similarly, when rSUPA was added to b-Pg it generated an amidolytic active site with a rate constant of 0.18 min Influence of rSUPA on the b-Plasmin/ Steady State Amidase Parameters and Kinetics of Pg
Activation--
In amidolysis assays h-plasmin and h-plasmin-SK showed
similar catalytic parameters (Table II).
However, the catalytic efficiency (kcat/Km) of b-plasmin-rSUPA
was 3.6-fold less than free b-plasmin. This difference was largely due
to a 2.8-fold increase in the Km for amidolysis when
rSUPA was bound to plasmin. The b-plasmin·SUPA complex was 2.9-fold
less efficient (kcat/Km) in
amidolysis than the h-plasmin·SK complex because of a slightly higher
Km and lower kcat.
The b-Pg·rSUPA complex and free rSUPA showed similar kinetic
parameters for Pg activation (Table III).
The h-Pg·SK complex was slightly more catalytically efficient
(2-fold) as an activator of h-Pg than the b-plasmin·rSUPA complex was
as an activator of b-Pg, largely because of a lower
Km (1.7-fold). The b-plasmin·SUPA complex also
activated h-Pg, but was 7.9-fold less efficient than h-Pg-rSK, chiefly
because of a 25-fold lower kcat.
The unique amino acid and genomic structure of SUPA had suggested
that it activated Pg through a different mechanism (16, 17) with
properties typical of both SAK and SK (18). However, in the presence of
plasmin, SAK and SK mechanisms are indistinguishable (12). In the
present experiments, active site generation by SUPA was examined under
classical experimental conditions where excess acylating agent was
present to inhibit trace amounts of plasmin (7, 8). These studies
indicate that SUPA nonproteolytically activates b-Pg through a SK- and
not a SAK-type of mechanism. Moreover, like the SK activator complex,
and unlike the SAK·plasmin complex, the SUPA activator complex
resists inactivation by The b-plasmin·SUPA complex activates b-Pg with kinetic parameters
that resemble (within a factor of 3) the parameters of Pg·SK for h-Pg
activation. When compared with the h-plasmin·SK complex, the
b-plasmin·SUPA complex showed comparable kinetics for amidolysis with
a slightly higher Km (1.8-fold) and lower
kcat (1.7-fold). Although SUPA could not
generate an active site in h-Pg, the b-plasmin·SUPA complex cleaved
h-Pg, albeit with a 3.0-fold lower Km and an
25.1-fold lower kcat. This paralleled the
observation that SK cannot generate an active site in b-Pg, although
the hPg·SK complex can efficiently activate b-Pg substrate. Thus the
process of active site formation in Pg by SK molecules appears
species-specific. However, the substrate-cofactor function is conserved
because h-plasmin·SK (23, 30) and b-plasmin·SUPA complexes (this
study) can cleave Pg substrates from other species, albeit with
different efficiencies. The fact that the b-plasmin·SUPA complex can
cleave h-Pg substrate indicates that a The mechanism by which SK nonproteolytically generates an active site
in Pg has been perplexing since its original description 30 years ago
(7, 8). Pg remains a zymogen until its activation domain is
productively rearranged by a salt bridge interaction between the amino
group of the cleaved, neo-NH2 terminus of plasmin (Val562) and carboxylate group of Asp740. In
the contact activation hypothesis, SK generates this active site
through binding of the What role does the ,
, and
) molecule, nonproteolytically activates human
(h)-plasminogen and protects plasmin from inactivation by
2-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase
domain, we
examined the mechanism of action of a novel two-domain (
,
) Streptococcus uberis plasminogen activator (SUPA). Under
conditions that quench trace plasmin, SUPA nonproteolytically generated
an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by
2-antiplasmin.
Still, the lag phase in active site generation and plasminogen
activation by SUPA was at least 5-fold longer than that of
streptokinase. Recombinant streptokinase
-domain bound to the
b-plasminogen·SUPA complex and significantly reduced these lag
phases. The SUPA-b·plasmin complex activated b-plasminogen with
kinetic parameters comparable to those of streptokinase for
h-plasminogen. The SUPA-b·plasmin complex also activated
h-plasminogen but with a lower kcat (25-fold) and kcat/Km (7.9-fold) than
SK. We conclude that a
-domain is not required for a
streptokinase-like activation of b-plasminogen. However, the
streptokinase
-domain enhances the rates of active site formation in
b-plasminogen and this enhancing effect may be required for
efficient activation of plasminogen from other species.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-antiplasmin, the serine protease
inhibitor of plasmin (2, 6). Among all of the Pg activators, SK has the
unique ability to bind and nonproteolytically generate an active site
in the Pg zymogen (i.e. convert Pg to Pg*) (7-9).
,
,
and
) linked by flexible loops (11). The SK
-domain binds near
the autolysis loop of plasmin (11). On the basis of studies with
recombinant (r) fragments Young et al. (10) concluded that
the carboxyl terminus of SK (including all of
-domain) induced a
conformational change in the active center of Pg leading to
nonproteolytic activation of the molecule, a process that has been
dubbed "binding or contact activation (13)." By analogy to the
activation of tissue Pg activator, which has significantly greater
activity as a zymogen than Pg, it has been suggested that the binding
of
-domain to the autolysis loop of Pg induces the formation of a
critical intramolecular salt bridge between Lys698 and
Asp740 of Pg that productively structures the activation
domain (11). Another hypothesis ("molecular sexuality") states that
SK nonproteolytically activates Pg when the NH2-terminal
isoleucine-1 (Ile1) of the
-domain forms a salt bridge
with Asp740 of Pg (12). In this activation sequence, which
is typical of the zymogens of the chymotrypsinogen family, salt bridge
formation productively restructures the latent activation domain (14). Either mechanism, "
-domain contact activation" or molecular
sexuality, is plausible given the current structural information
for the microplasmin·SK complex (13).
2-antiplasmin (18). In
this study we have investigated the properties of the activator complex formed by SUPA and bovine Pg to determine the structural elements required for nonproteolytic active site generation and for
rendering the activator complex resistant to inhibitors such as
2-antiplasmin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-domain of SK (residues 293-414) was
cloned using the full-length cDNA of SK and the corresponding
primers (5' sense primer catcagctgttcaccatcaaatacgttg,
PvuII; 3' antisense primer gcctgcagtcattatttgtcgttagg,
PstI). After double-stranded DNA sequencing it was ligated
into pMALc vector at StuI and PstI sites. The SK
-domain fusion protein was purified by affinity chromatography on an
amylose resin (New England Biolabs) and its purity was assessed by
SDS-PAGE.
95% Glu-Pg; Chromogenix, Sweden)
or b-Pg with 20 nM rSK or rSUPA for 5 min at 37 °C. The
h-plasmin·rSUPA complex was formed by mixing 10 nM
h-plasmin (Sigma) and 20 nM rSUPA for 10 min on ice. The
enzymes were transferred to a thermostatically regulated (37 °C)
quartz cuvette containing assay buffer (50 mM Tris-HCl, 100 mM NaCl, pH7.4) and various concentrations of S2251 (80-800 µM) in a total volume of 300 µl and the change
in absorbance was monitored at 405 nm for 5 min in a Cary 100-Bio
spectrophotometer. Less than 10% of
H-D-Val-Leu-Lys-nitroanilide was consumed during the course
of the reaction. The data were plotted as V/S and analyzed by
hyperbolic curve fitting with the Sigma Plot program. An
1 M at 405 nm of 10,000 was used for
p-nitroanilide.
95% Glu-Pg; Chromogenix, Sweden) and rSK or
b-Pg and rSUPA on ice for 30 min. The stoichiometric h-plasmin·rSUPA
activator complex was formed at 37 °C for 5 min. Activators were
added to a quartz cuvette containing assay buffer, 0.5 mM
S2251, and Pg (50-900 nM) in a total volume of 300 µl at 37 °C as described. Initial reaction rates were obtained from the
first 300 s by plotting
A405/min2, and the apparent
Michaelis and catalytic rate constants were calculated by fitting the
data to a hyperbolic curve as described (23) using the Sigma Plot program.
95% Glu-Pg; Chromogenix, Sweden) or b-Pg (300 nM;
0.5% plasmin) was added to a cuvette in the presence of 0.5 mM S2251. After incubation for 5 min at 4 °C,
native SK (100 nM, Sigma) or rSUPA (300 nM) was
added, and the release of p-nitroanilide was monitored. The
rate constant for active site appearance (k) was measured as
described (24) by the equation: [P] = K[Pg-SK]ot-K[Pg-SK]o/k
as described where K = kcat[S]/(Km + [S]) and
was essentially constant because substrate depletion was
10%.
-domain (0-50 µg/ml) or MPB (0-50 µg/ml)
were added. After a 1-h incubation and washing, anti-MBP monoclonal
antibody was added for 1 h. After washing bound antibody was
detected by 125I-goat anti-mouse antibody (50,000 cpm)
followed by
-counting.
2-Antiplasmin reaction kinetic
measurements were carried out in a 1-ml cuvette in filtered, dust-free
assay buffer (50 mM Tris, 100 mM NaCl, pH 7.4)
at 37 °C using a thermostatted Cary 100-Bio spectrophotometer. The
rate constant k1 of the reaction between
b-plasmin and
2-antiplasmin was measured as described (6). Briefly b-plasmin (14 nM) was added to cuvettes
containing S2251 (500 nM), SUPA (0-500 nM),
and the change in absorbance at 405 nm recorded for 90 s prior to
the addition of human
2-antiplasmin (Calbiochem; 90 nM). The residual enzyme activity was measured by the first
derivative (dA/dt) of the curve before 60% of the enzyme was
inactivated. The apparent rate constant
(k1, app) of the inhibition of the enzyme
activity by
2-antiplasmin in the presence of various
concentrations of SUPA was calculated from the classical second-order
rate equation as described (6).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-domain of SUPA showed 11.9% identity with SAK, 30.9%
with SK
, 10.3% with SUPA
, 16.7% with SK
, and 9.5% with
SK
(25). The SUPA
-domain showed 11.9% identity with SK
,
15.6% with SAK, 27.4% with SK
, and 12.2% with SK
. By
comparison, SAK showed 17.7% identity with SK
, 14% with SK
,
and 20.6% with SK
. In contrast to previous reports, a comparison
of the three sequences by ClustalW (Fig.
1) showed that SUPA contained an
Ile-Thr-Gly terminus which is similar to the Ile-Ala-Gly sequence that
has been suggested to mediate nonproteolytic activation of Pg by SK
(12, 16, 17). In the crystal structure SK
domain residues
Glu39 and Glu134 form salt bridges with
Arg719 of h-plasmin (which is also conserved in b-Pg) (11,
13). In the ClustalW alignment these residues are not conserved in staphylokinase, although in an ALSCRIPT alignment SK Glu39
is conserved as Glu43 of SAK (26). In SUPA there is modest
conservation of these residues as His43 and
Glu113. The ClustalW alignment also highlights
conservation of residues in SUPA that have been shown to be important
for SK function: SK Leu42 (27) which is SUPA
Leu46; SK Lys257 (28), which is SUPA
Lys234; SK Arg248, Glu249 which are
SUPA Arg225, Gln226, and SK Lys282
which is SUPA Lys260 (29).
Pairwise sequence identities between domains of SUPA, SK, and SAK
-(1-126), SUPA
-(127-261),
SK
-(1-147), SK
-(148-283), and SK
-(284-414).
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Fig. 1.
Alignment of SAK, SUPA, and SK by
domains. A ClustalW alignment of the three proteins is shown.
Residues that are identical between SUPA and SK or SAK are
outlined in black. Residues that are similar between all
proteins in the alignment are boxed.
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Fig. 2.
Active site generation in h-Pg by SK and b-Pg
by SUPA. A, active site development detected by a
fluorescent active site titrant. Human Pg (100 nM) or b-Pg
(200 nM) was added to a cuvette containing 2 µM MUGB in filtered buffer (50 mM Tris-HCl,
0.15 M NaCl, pH 7.4) at 25 °C. After 5 min 220 nM SK, 440 nM rSUPA, 440 nM
MBP-rSUPA, 440 nM His6-rSUPA, or buffer alone
(control) was added to separate reactions. The development of
fluorescence was monitored continuously with excitation at 365 nm and
emission at 445 nm. B, reducing SDS-PAGE analysis of the
reaction between b-Pg and SUPA. Equimolar complex of b-Pg and rSUPA at
25 °C, pH 7.4, incubated in the presence of MUGB for 1 min
(lane 4), 10 min (lane 5), or 25 min (lane
6). Controls include SUPA (lane 2), b-Pg (lane
3), and b-plasmin (lane 7) formed by incubation for 60 min at 37 °C of a catalytic ratio of SUPA (1:30) in the absence of
MUGB.
1 while rSK
generated an active site in h-Pg with a rate of 0.55 min
1
at 4 °C (data not shown). In Pg activation studies, rSUPA showed a
greater delay or lag than rSK in the development of plasmin generation.
For example, at 37 °C the delay or lag phase before the onset of Pg
activation by rSUPA was >5-fold longer than rSK (Fig.
3). Pg activation by rSUPA was more
temperature-dependent than rSK (Fig. 3), the lag phase for
Pg activation by rSUPA lengthened twice as much as rSK for every degree
drop in reaction temperature. To determine whether the lag phase in the
onset of b-Pg activation by rSUPA was related in part to its lack of a
-domain, we examined the effects of MBP-
-domain of SK or MBP
alone on b-Pg activation by rSUPA (Fig.
4). Binding experiments confirmed that
the MBP-SK
-domain, but not MBP alone bound to the formed
SUPA·b-Pg complex (Fig. 4A). In activation experiments,
the MBP-
-domain reduced the lag phase in b-Pg activation by rSUPA by
about half (from ~10 to 5 min) while MBP or catalytic amounts of rSK
had no effect (Fig. 4B). Additional active site titration
studies were performed to determine whether this reduced "lag
phase" in Pg activation induced by the
-domain was due to an
acceleration of active site exposure in the b-Pg·rSUPA complex. When
compared with MBP as a control, the MBP-
-domain reduced the time
necessary for active site generation by rSUPA in b-Pg (from ~240 to
120 s; Fig. 4C).
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Fig. 3.
Effects of temperature on Pg activation.
A, activation of h-Pg by rSK at different temperatures.
B, activation of b-Pg by rSUPA at different temperatures.
rSK (10 nM) or rSUPA (20 nM) were added to
quartz cuvettes containing h-Pg (300 nM) or b-Pg (300 nM) with 0.5 mM S2251 in 50 mM
Tris-HCl, 0.15 M NaCl, pH 7.4, at various temperatures. The
rate of substrate cleavage was monitored by the change in absorption at
405 nm.
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Fig. 4.
Interaction of the SK
-domain with the b-Pg·rSUPA activator
complex. A, binding of
-domain to b-Pg·rSUPA
complex. To form the b-Pg·rSUPA complex, SUPA (50 µl/ml; 50 µg/ml) was added to wells coated with b-Pg (5 µg/ml) and blocked
with 1% bovine serum albumin. After a 1-h incubation and washing, 50 µl of MBP-
-domain (0-50 µg/ml) or MBP (0-50 µg/ml) was added
for 1 h. After washing the anti-MBP monoclonal antibody was added.
Bound antibody was detected by 125I-goat anti-mouse
antibody followed by
-counting. B, influence of the
domain on the lag-phase of activation of b-Pg by rSUPA. b-Pg (300 nM) in 50 mM Tris-HCl, 0.15 M NaCl,
pH 7.4, was preincubated with the MBP-
domain (300 nM),
MBP (300 nM), assay buffer or SK (20 nM) for 30 min at 25 °C. Then rSUPA (20 nM) was added and the rate
of substrate cleavage was monitored at 37 °C. C, effect
of
-domain on active site generation in b-Pg by rSUPA. The
MBP-
-domain or MBP (300 nM) was preincubated with b-Pg
(300 nM) in the presence of MUGB (2.5 µM) for
850 s prior to the addition of SUPA (450 nM). Active
site generation was monitored as described in the legend to Fig.
2.
2-Antiplasmin
Reaction--
Another property that distinguishes SK from SAK is the
resistance of the SK activator complex to inhibition by
2-antiplasmin (2, 6). The second-order rate constant
(k1) for inhibition of plasmin by
2-antiplasmin was 3.15 ± 0.61 × 105 M
1 s
1.
Increasing concentrations of rSUPA inhibited the inactivation of
b-plasmin (14 nM) by an excess of
2-antiplasmin (90 nM) in a
dose-dependent fashion, with a 50% reduction seen at a
concentration of ~30 nM rSUPA (Fig.
5).
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Fig. 5.
Influence of rSUPA on the inactivation of
b-plasmin by 2-antiplasmin
reaction. Various amounts of rSUPA (0-300 nM) were
mixed with b-plasmin (14 nM) and S2251 (500 nM)
in assay buffer (50 mM Tris-HCl, 100 mM NaCl,
pH 7.4) at 37 °C. The A405 was continuously
recorded and after 90 s human
2-antiplasmin (90 nM) was added. The residual plasmin activity was determined
at different time intervals and the apparent inhibition rate constant
(k1, app) was calculated (see "Experimental
Procedures"). The data are expressed as a percentage of the
k1 value obtained in the presence of a given
SUPA concentration versus that in the absence of rSUPA. The
data represent the mean ± S.E.
Kinetic constants for amidolysis
Kinetic constants for plasminogen activation
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-antiplasmin. These findings
indicate that for the activation of b-Pg by SUPA, a
domain is not
required for a SK-type of mechanism.
-domain per se is
also not required for substrate processing by this activator complex.
-domain to the autolysis loop of Pg which
facilitates the formation of a salt bridge between Asp740
and the counterion Lys698 of Pg. Alternatively the
molecular sexuality hypothesis suggests that the
NH2-terminal Ile1 of SK provides the counterion
for the salt bridge with Asp740 of Pg in a manner analogous
to activation of the zymogens of the chymotrypsinogen family (12).
Mutagenesis studies aimed at identifying the counterion have indicated
that Ile1 is required for nonproteolytic activation of Pg,
while Lys698 appears to be required for normal binding
interactions during the formation of the initial SK·Pg complex (12,
31). The conservation of the NH2-terminal motif
(Ile-Xxx-Gly) between SUPA and all other reported SKs underlines the
importance of this residue (12). As predicted by studies of SK,
efficient active site generation in b-Pg in the presence of an
acylating agent only occurred when the Ile1 of SUPA was
free and not when it was tethered in fusion proteins.
-domain play if SUPA can activate b-Pg
nonproteolytically, protect b-plasmin from inhibition from by
2-antiplasmin, and form an activator complex that
cleaves different species Pgs? The
-domain is found in
streptokinases isolated from humans, pigs, and horses suggesting that
there is an evolutionary pressure to maintain it which may relate to
the fact that there are species differences in the structural
requirements for Pg activation. When compared with SK, SUPA generated
an active site more slowly in b-Pg and required higher ambient
temperatures for function. Exogenous
-domain bound to the SUPA
activator complex and accelerated the process of active site formation,
suggesting that the
-domain may serve as an enhancer of these
processes, perhaps through its interactions with the autolysis loop of
b-Pg (which is 88% identical to h-Pg (32)). Although this enhancer
function is not required by b-Pg, it appears to be required by human
Pg, because a SUPA-like mutant of SK lacking the
-domain is unable
to activate human Pg.2
Analyses of the crystal structures of µPg have indicated that generation of a functional active site requires several intramolecular rearrangements that may be facilitated by interactions with the
-domain (32, 33). For example, by binding to the autolysis loop, the
-domain could promote the formation of a salt bridge between
Ile1 of SK and Asp740 of Pg by destabilizing
the hydrogen bonds between Asp740 and residues 685 and 686 that secure Pg in the zymogen conformation.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL-57314 (to G. L. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF283574.
¶ To whom correspondence should be addressed: Cardiovascular Biology Laboratory, HSPH II-127, 677 Huntington Ave., Boston, MA 02115. Tel.: 617-432-4992; Fax: 617-432-0033; E-mail: reed@cvlab.harvard.edu.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M009265200
2 I. Sazonova and G. L. Reed, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: Pg, plasminogen; b, bovine; h, human; r, recombinant; SUPA, Streptococcus uberis plasminogen activator; MBP, maltose-binding protein; SK, streptokinase from Group C streptococcus; SAK, staphylokinase; S2251, H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride; PMSF, phenylmethylsulfonyl fluoride; MUGB, 4-methylumbelliferyl p-guanidinobenzoate; PAGE, polyacrylamide gel electrophoresis.
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