| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298; and
Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionExperimental ProceduresResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionExperimental ProceduresResultsDiscussionReferences |
|---|
and ß, each
on chromosome 16 (2, 3, 4). ß-Tryptase appears to account for tryptase
enzymatic activity and is stored in secretory granules as an active
enzyme. ß-Tryptase is secreted into the extracellular environment by
activated mast cells in parallel with histamine (5), and is ionically
bound to heparin proteoglycan, which stabilizes the active tetrameric
form of ß-tryptase in isotonic media at neutral pH (6). Heparin also
is critical to the processing of ß-tryptase precursors, being
required for the autocatalytic cleavage of ß-protryptase to
ß-pro’tryptase, and for formation of active tetramers after
conversion of ß-pro’tryptase to the mature peptide by dipeptidyl
aminopeptidase I (7). In contrast, -protryptase does not undergo
autoprocessing due to a R-3/Q-3
substitution at the pro/pro’ cleavage site. Consequently,
-protryptase appears to be secreted constitutively as an inactive
proenzyme and is the major form of tryptase found at baseline in the
blood of normal subjects (7, 8).
Because ß-tryptase is resistant to inhibition by biologic inhibitors
of serine proteases such as 1-proteinase inhibitor,
2-macroglobulin, and aprotinin (9, 10, 11, 12), regulation of
its activity may depend on regulating its association with heparin
proteoglycan. Once free of heparin, ß-tryptase rapidly dissociates
into inactive monomers in isotonic buffers at neutral pH (6, 13).
Inactivation of ß-tryptase by this mechanism involves alterations in
secondary and tertiary conformations, as reflected by circular dichroic
spectroscopy and binding of mAbs to conformational epitopes,
respectively (14). The kinetics of inactivation is first order with
respect to active enzyme (15), and is slowed by acidic pH or by higher
salt concentrations. Addition of heparin to totally inactive monomers
did not result in reactivation, suggesting that once inactive monomers
formed, the process was irreversible at neutral pH (14). However,
addition of heparin to partially inactivated enzyme appeared to restore
a portion of the activity, suggesting a transient, reversible
intermediate between active tetramer and inactive monomer (15, 16).
Indeed, evidence for a transient inactive tetrameric intermediate was
provided by analytic ultracentrifugation and circular dichroic
spectroscopy (15) as well as by fluorescence anisotropy (16)
measurements. These studies all are consistent with heparin-dependent
stabilization of ß-tryptase being the primary factor regulating the
activity of the enzyme after its release from mast cells. Once
dissociated from heparin in the extracellular environment, dilution of
the protein as it diffuses from its site of release makes reactivation
seem unlikely.
A new consideration in the regulation of ß-tryptase activity is pH. The optimal pH for ß-tryptase to process ß-protryptase to ß-pro’tryptase (8), to generate bradykinin from low m.w. kininogen (17), and to degrade fibrinogen (18) ranges from 5.5 to 6.5. The possibility of a cofactor that further augments ß-tryptase activity at acidic pH and inhibits activity at neutral pH also was hypothesized (18). The present study shows that totally inactive ß-tryptase monomers can be completely converted to active tetramers at acidic pH in the absence of heparin. Analysis of the initial kinetics of tetramer formation and reactivation suggests a mechanism whereby monomers first form dimers, dimers form inactive tetramers, and inactive tetramers convert to active tetramers. Thus, ß-tryptase can reactivate in the absence of heparin at acidic pH.
|
Experimental Procedures |
|---|
|
TopAbstractIntroductionExperimental ProceduresResultsDiscussionReferences |
|---|
HEPES, Mes, porcine heparin glycosaminoglycan (1,200–20,000 Da), dextran sulfate (500,000 Da), BSA, and tosyl-L-Gly-Pro-Lys-p-nitroanilide (TGPL)3 were obtained from Sigma (St. Louis, MO). ß-Tryptase from human lung (tetramers with N-glycosylated subunits of 29 to 35 kDa by SDS-PAGE) was prepared and purified to homogeneity, as described previously, by sequential affinity chromatography on B2 mAb Affi-Gel, and heparin-agarose to about 25 TGPL U/mg protein (14). Tryptase molar concentrations are given with respect to its subunits (30,000 Da average molecular mass) throughout the text, unless stated otherwise. Purified preparations of active ß-tryptase (100–250 µg/ml) were stored at -70°C in 10 mM Mes buffer, pH 6.5, containing 0.8 M NaCl and 20% (v/v) glycerol.
Measurement of ß-tryptase
ß-Tryptase enzymatic activity was assessed in 1-ml plastic cuvettes or 96-well microtiter plates. The standard assay to measure initial rates of hydrolysis of TGPL was performed by adding 10 µl of ß-tryptase to 1 ml of substrate buffer (0.1 mM TGPL in 40 mM HEPES, pH 7.4, containing 0.12 M NaCl). The amounts of p-nitroanilide liberated were monitored continuously at 405 nm in a Cary 3 UV-VIS spectrophotometer with a Peltier temperature controller (Varian Associates, Palo Alto, CA) for 5 min. Initial velocities were measured over a time period, during which less than 10% of the total substrate had been cleaved, and calculated using a mM extinction coefficient for product of 8.8. Enzyme activity is expressed in U, in which 1 U of enzyme cleaves 1 µmol of substrate/min. Microtiter plate assays were performed by adding 10 µl of sample into a well containing 200 µl of 40 mM HEPES buffer, pH 7.4, containing 0.12 M NaCl and 0.4 mM TGPL. Changes in the absorbance at 405 nm were measured in a Bio-Tek EL312, Bio-Kinetic Reader (Bio-Tec Instruments, Winooski, VT). ß-Tryptase protein was measured by a sandwich ELISA using the B12 mAb for capture and biotin-G4 mAb for detection, as described (19). The lower limit of detection was 0.05 ng of ß-tryptase.
Inactivation and reactivation of ß-tryptase
ß-Tryptase (5–18 µg, 0.167–6 nmol) was inactivated by incubation in 1 ml of 10 mM HEPES buffer, pH 7.4, containing 0.12 M NaCl at 37°C for 90 min in presence or absence of 0.5 mg/ml BSA. Portions tested for activity in the standard TGPL assay after mixing each with a fourfold weight excess of heparin were completely inactive. To study reactivation, the pH of inactivated tryptase solutions was adjusted by adding 0.1 vol of 0.5 M Mes, pH 5 to 6.5, or 0.5 M HEPES, pH 7 and 7.4, typically containing a fourfold weight excess of heparin over tryptase and appropriate amounts of NaCl, and incubated at the appropriate temperature. Portions were removed at each time point and assessed for enzyme activity by TGPL assay and for the quaternary state of tryptase by gel filtration. Recovered enzyme activity was normalized to the original enzyme activity before inactivation. Conversions between monomer and tetramer were stopped when the NaCl concentration was adjusted to 1 M by addition of ice-cold 4 M NaCl, following which the sample was injected immediately onto the gel filtration column.
Gel filtration
Gel filtration studies were performed on a Superose 12 HR 10/30 size exclusion column (1 x 30 cm; Pharmacia, Uppsala, Sweden) using a series 410 BIO LC solvent delivery system (Perkin-Elmer, Foster City, CA) at a flow rate of 1 ml/min. The column was equilibrated and run in 10 mM Mes buffer, pH 6, containing 1 M NaCl. The column was calibrated, as described previously (8, 3), with dextran blue (2 x 106 Da), catalase (232,000 Da), aldolase (158,000 Da), BSA (67,000 Da), chymotrypsinogen (25,000 Da), and acetone (58 Da).
Kinetic studies
Enzyme activity data were converted to activity ratios of
measured (v) to the total enzymatic activity of the original tryptase
sample (vT), or to the
inactivity ratio (1 -
v/vT). Initial
reaction rates were used for kinetic analyses. Plots of reactivation
and inactivation with apparent first order rate-determining steps were
plotted as the natural log of the inactivity and activity ratios,
respectively, vs time, and the first order rate constants
(k1) calculated from the slope determined
by linear regression using the following formulas:
 |
 |
 |
 |
 |
 |
 |
The charge product for reactivation was calculated using the
Debye-Hückel equation for a reaction in an aqueous solution:
 |
Activation energies
(Ea) were estimated
from the linear form of the Arrhenius equation.
 |
Regression analyses were performed with SigmaPlot and SigmaStat (Jandel, San Rafael, CA).
|
Results |
|---|
|
TopAbstractIntroductionExperimental ProceduresResultsDiscussionReferences |
|---|
In the absence of a stabilizing macromolecular anion, ß-tryptase
rapidly lost activity at neutral pH, and showed first order kinetics
with respect to active tetramer (Fig. 1
,
A and C), as described previously (13, 15, 16, 20). At 5 µg/ml (0.167 µM with respect to the 30,000 Da
subunit), pH 7.4, 37°C, and an ionic strength equal to 0.16 M NaCl,
the first order t1/2 for inactivation was
1.8 ± 0.1 min (mean ± SD). After at least 1 h of
incubation under these inactivating conditions, ß-tryptase had
completely converted to monomers, as analyzed by gel filtration (see
below). Complete inactivation of ß-tryptase was achieved at
ß-tryptase concentrations up to 0.5 µM. At higher concentrations of
ß-tryptase, complete inactivation was not achieved. Addition of
either dextran sulfate or heparin (4:1 weight ratios to ß-tryptase)
to completely inactivated ß-tryptase and incubation of this mixture
for up to 24 h at either room temperature or 37°C failed to
result in any detectable TGPL-cleaving activity. Thus, complete
inactivation appeared to be irreversible under these conditions.
|
(A and B),
reactivation of ß-tryptase (0.167 µM) to its original enzymatic
activity occurred at 20°C in a pH 6 buffer containing 50 mM Mes, 9 mM
HEPES, 0.12 M NaCl, 0.5 mg/ml BSA, and 20 µg/ml heparin. The ionic
strength was equivalent to 0.16 M NaCl. Reactivation followed first
order kinetics when plotted against the natural log of the inactivity
ratio of ß-tryptase vs time. The calculated
t1/2 of reactivation was 2.7 ± 0.3 min
(mean ± SD). The concentration dependence of reactivation under
these conditions was examined at ß-tryptase concentrations from 0.033
to 0.5 µM. As shown in Figure 2,
the first order rate constant was invariant at ß-tryptase
concentrations of 0.25 to 0.50 µM, but appeared to decrease slightly
at lower concentrations. This suggests that the overall process of
reactivation is not a simple first order reaction, even though the
rate-limiting step at ß-tryptase concentrations of 0.25 to 0.50 µM
best followed first order kinetics.
|
Reactivation of inactivated ß-tryptase (0.167 µM) was tested
at pH values from 5 to 7.4, as shown in Figure 3. The optimal pH was 6, with a sharp
fall in the initial rate of reactivation between pH 6 and 7. At pH 7,
20% of ß-tryptase activity could be recovered after an extended
incubation time of 1 h, but no ß-tryptase activity was recovered
at pH 7.4, suggesting a pH-dependent equilibrium between active and
inactive can be achieved. However, we chose to evaluate initial rate
data rather than equilibria in the current study. Initial rates of
reactivation appeared to decrease by 50% just below pH values of 6.5
and 5.
|
. Reactivation followed first order
kinetics in each case. As shown in the Arrhenius plot (Fig. 4B) from 4°C (0.0036°K-1)
to 22°C (0.0034°K-1), the reactivation rate
increased. However, as temperatures higher than 22°C were used, the
increase in reaction rate declined, indicating a potential temperature
transition near 22°C. From the linear portion of the Arrhenius plot
between 4°C and 22°C, an Ea of 27
kcal/mol was calculated. In contrast to reactivation, inactivation
rates in an isotonic buffer at neutral pH increased linearly when
plotted as an Eyring plot
(log(k1/T) against
1/T) from 4°C to 37°C (15), suggesting that the
rate-limiting steps of reactivation are more complex than for
inactivation.
|
A. NaCl equivalents from 120
to 1000 mM were examined. First order kinetics was observed from 0.12
to 0.36 M NaCl. The optimal ionic strengths for initial rates of
reactivation were observed to range from 120 to 200 mM NaCl
equivalents. At NaCl concentrations of 500 mM or greater, no
reactivation was observed. This is of interest because active
ß-tryptase at neutral pH is stabilized by high NaCl concentrations
(6, 15, 16). As shown in Figure 5B, the charge product
calculated according to the Debye-Hückel equation was -9.4 for
reactivation. The charge product calculated for tetramer formation
alone was -7.2 (data not shown). These values compare to a charge
product of -2.5 calculated previously for inactivation at neutral
pH (15).
|
A. Weight ratios of
heparin to ß-tryptase from 0 to 1000 were examined. First order
kinetics was found in the absence of heparin and at all concentrations
of heparin tested. The log of the first order rate constants vs the
heparin:tryptase weight ratios is plotted in Figure 6B. An
optimal weight ratio of 4:1 was found, the rate of reactivation being
slightly higher than in the absence of heparin and at a 2:1 weight
ratio of heparin to ß-tryptase. As the heparin:ß-tryptase weight
ratio increased above 24:1, the rate of reaction substantially slowed.
Thus, reactivation of ß-tryptase at pH 6 can occur without a
stabilizing macromolecular anion such as heparin. However, because
heparin resides with tryptase in vivo, binds inactive tryptase monomer
as well as active tetramer at neutral and acidic pH at physiologic
ionic strength, and is needed to stabilize tryptase that migrates into
a neutral pH milieu, kinetic experiments were conducted in the presence
of the optimal ratio of heparin to tryptase, as described above.
|
Gel filtration was performed to assess the quaternary state of
ß-tryptase during the reactivation process. This method has been used
previously to assess the apparent m.w. of different forms of tryptase
(8, 13), and has been verified by analytic ultracentrifugation (20).
Reactivation reactions were stopped by adjusting the NaCl concentration
to 1 M, and the resultant mixtures immediately applied to a Superose 12
column equilibrated in 10 mM Mes buffer, pH 6, containing 1 M NaCl at
room temperature. Under these conditions, previously prepared inactive
monomer and active tetramer each remained as such during
chromatography. Neither heparin nor dextran sulfate binds to
ß-tryptase under these conditions, and therefore do not affect the
retention times. As shown in Figure 7,
A and B, inactivated ß-tryptase, measured by a
sandwich ELISA and by OD280 nm, respectively, eluted only
at retention times consistent with a monomeric configuration at 0 min.
As shown in the lower panel of Figure 7A, no
enzymatic activity was detected in these monomeric fractions.
Inactivated ß-tryptase (0.167 µM, 20°C, 0.16 M NaCl-eq, pH 6,
±0.5 mg/ml BSA, and 20 µg/ml heparin) was then reactivated, and
samples from the reactivation mixture were analyzed at various time
points. The portion of immunoreactive ß-tryptase eluting with a
retention time consistent with a tetrameric configuration increased
over a 60-min time span. Neither dimer nor trimer peaks were detected
under these experimental conditions. Enzyme activity also increased
over time, but only in fractions corresponding to tetramer. At no time
point was enzyme activity detected in fractions corresponding to
monomers. The kinetics of tetrameric and active ß-tryptase formation
is analyzed in Figure 7, C and D. Tetramer
formation appeared to precede reactivation during the early portion of
the time course (120 s), while at later time points these parameters
appeared to converge (Fig. 7C). As above, the rate of
reactivation analyzed during the initial 5 min followed first order
kinetics (Fig. 7D, closed circles), in this case with a
t1/2 of 2.7 min. In contrast, tetramer formation
followed second order kinetics (Fig. 7D, open circles), with
a t1/2 of 2.3 min. Thus, the rate-determining
steps for formation of tetramer and active enzyme appeared to differ
qualitatively under these experimental conditions.
|
A. In
each case, the disappearance of monomer followed second order kinetics
during the initial 5 min of the reaction. The Arrhenius plot in Figure 8B shows that the initial reaction rate for tetramer
formation increased from 4°C (0.0036°K-1)
to 22°C (0.0034°K-1)
(t1/2 of 20 to 1.6 min) but did not
appreciably change from 22°C to 37°C
(0.0032°K-1), analogous to the effect of
temperature on reactivation (Fig. 4B). The
Ea was calculated to be 20 kcal/mol
between 4°C and 22°C.
|
). No
tetramer formed at pH 7.4 (Fig. 9A). At pH 7, a
modest amount of tetramer formation occurred, but the kinetics followed
a fourth order reaction with a t1/2 of 152 min
(Fig. 9B). This compares to a first order
t1/2 for reactivation of 230 min at pH 7,
consistent with tetramer formation preceding the return of enzyme
activity. At pH values of 5 to 6.5, second order reaction kinetics for
initial rates of tetramer formation was observed (Fig. 9C). The calculated t1/2 for
tetramer formation were nearly identical from pH 5 to 6, and then
increased more than fourfold at pH 6.5. Variations in second order
t1/2 between Figures 7, 8, and 9 (2.3, 1.7, and
0.9 min) most likely result from interexperimental variation, but also
may reflect the presence of BSA in the experiments shown in Figure 7, and the absence of BSA in experiments shown in Figures 8 and 9. BSA
(0.5 mg/ml) appears to slightly retard the initial rate of formation of
tetramers from inactive monomers at acidic pH.
|
|
Discussion |
|---|
|
TopAbstractIntroductionExperimental ProceduresResultsDiscussionReferences |
|---|
). Although a weight ratio of heparin
to ß-tryptase of 4:1 is optimal for reactivation, heparin is not
required for reactivation to occur at pH 6. The lack of a substantial
requirement for heparin is at variance with the requirement for heparin
during the processing of tryptase from ß-pro’tryptase monomers to
active tetramer (8). Omission of heparin during this processing step
results in inactive ß-tryptase monomers, even though processing is
performed at acidic pH. Thus, reactivation is distinct from activation
associated with processing of ß-tryptase precursors.
|
. The first rate-determining step of the reactivation
process at a pH of 6 is dimerization of ß-tryptase monomers, a second
order process at pH values from 5 to 6.5. However, because dimers were
never observed in gel filtration elution patterns, such intermediates
remain speculative. At pH 7, tetramer formation proceeds much more
slowly, the initial rate of formation appearing to follow fourth order
kinetics. It is conceivable that dimerization may not occur at this pH.
Consequently, tetramers may form directly from monomers, a less
efficient process. The second step at pH 6, formation of inactive
tetramer from putative dimers, is predicted to be more rapid than the
first step, making it difficult to detect such intermediates by a slow
process such as gel filtration. The rate-determining step leading to formation of active enzyme at pH 6 appears to be conversion of inactive ß-tryptase tetramer to active ß-tryptase tetramer, a first order process at pH values from 5 to 7. The pH optimum of 6 for reactivation as well as for tetramer formation suggests that certain histidine residues may play a critical role at multiple steps. This mechanism for reactivation in the current study is compatible with previous studies of ß-tryptase inactivation and of reactivation of partially inactivated enzyme at neutral pH, which provided evidence for a transient inactive tetrameric intermediate (15, 16, 20).
The concentration dependence of reactivation does not fit a simple
first order reaction, because the apparent first order rate constant
decreases when the initial tryptase concentration is below 0.25
µM. This may in part be explained because the overall rate constant
includes rate constants from at least two steps, one of which is second
order. As the starting concentration of inactive ß-tryptase monomer
is diminished, the t1/2 will increase for the
second order, rate-determining step leading to tetramer formation
(monomer to dimer), whereas the t1/2 will remain
constant for the first order, rate-determining step leading to active
enzyme formation (inactive to active tetramer). Consequently, as
dimerization becomes rate limiting with respect to formation of active
enzyme, the apparent overall rate constant is likely to diminish. This
may explain the results shown in Figure 2B in which
k1 values for conversion of inactive monomers to
active enzyme begin to diminish below a starting monomer concentration
of 0.25 µM, even though the overall reaction continues to fit a first
order time course better than a second order time course.
The optimal ionic strength for reactivation was near physiologic, equivalent to NaCl concentrations ranging from 0.12 to 0.2 M. Higher ionic strengths inhibited reactivation. However, the rate-determining step remained first order. Complete inhibition of reactivation and of tetramer formation occurred at NaCl concentrations of 0.5 M and above. Because high ionic strength also stabilizes the active tetramer, the inhibitory effect on reactivation presumably results from destabilizing one or more transition states, thereby preventing the reaction from going in either direction. A practical experimental benefit of the inhibitory effect of high ionic strength was that gel filtration could be performed at 1 M NaCl to effectively freeze the protein in its prechromatography monomeric and tetrameric states. At pH 6, application of the Debye-Hückel equation yielded a charge product of -9.4 for reactivation. This suggests the number of charged residues, most certainly including histidine residues, involved in the reactivation process at acidic pH is greater than the number involved in inactivation at neutral pH, in which the charge product has been reported to be -2.5 (15).
Both the first order rate constant for reactivation and the second order rate constant for tetramer formation were essentially unchanged from 22°C to 37°C, but declined at lower temperatures. This nonlinear relationship between °K-1 and log(k) over the entire temperature range is consistent with a complex mechanism involving two or more separate steps, rate constants, and activation energies. In spite of this complexity, Ea values between 4°C and 22°C were calculated to be 27 kcal/mol for reactivation and 20 kcal/mol for tetramer formation, consistent with reactivation requiring a higher activation energy than formation of tetramer alone. These values are similar to those of 19 kcal/mol reported for inactivation (15) or of 11 kcal/mol for TGPL cleavage (13). An explanation for the apparent transition occurring at 22°C for reactivation and tetramer formation is not apparent from current studies, but presumably must reflect the complexity of the reactivation process.
Reversal of inactivation has been reported to occur at neutral pH by addition of heparin (15). Whether this results from conversion of an inactive tetrameric intermediate or an inactive monomeric intermediate to active enzyme is uncertain. One of the difficulties in deciding whether heparin-free ß-tryptase has been completely inactivated at neutral pH occurs when the enzyme is further diluted by adding it to substrate. In the absence of stabilizer, this will tend to inactivate any residual active enzyme. Mixing another portion of residual active enzyme with a stabilizer before adding it to substrate will preserve this activity, creating the impression that ß-tryptase has been reactivated. We avoid this by adding heparin or dextran sulfate to inactivated enzyme before addition to substrate, and find no evidence for reactivation of inactive ß-tryptase monomers at neutral pH with these anionic macromolecules. Another confounding factor is that at ß-tryptase concentrations above 0.5 µM (subunit concentration) without stabilizer, residual tetramer remains even after an overnight incubation at neutral pH in an isotonic salt solution. This is similar to the concentration calculated based on sedimentation equilibrium data for 99% of unstabilized ß-tryptase to reside in a monomeric form near neutral pH at 0.2 M NaCl (20). At higher ß-tryptase concentrations, residual tetramer in equilibrium with monomer would be rapidly inactivated if diluted into substrate without stabilizer, but would be preserved as active tetramer or converted from inactive to active tetramer if a stabilizer was added before dilution. Thus, we conclude that reactivation of ß-tryptase at neutral pH occurs primarily through conversion of inactive tetramer to active tetramer.
Whether ß-tryptase monomers can exhibit enzyme activity is somewhat controversial. Because both inactivation and partial reactivation follow first order kinetics, inactivation was predicted to occur by active tetramer being converted to inactive tetramer, a potentially reversible rate-determining step, followed by inactive tetramer being converted to inactive monomers (15, 20). This model was not consistent with active monomer formation. On the other hand, evidence for active monomers was reported based on gel filtration experiments (16). ß-Tryptase, partially inactivated at pH 7.4, was subjected to gel filtration in a pH 6.1 buffer containing 0.3 M NaCl, and 10% glycerol with or without heparin. ß-Tryptase activity was then detected in fractions having retention times of monomers as well as tetramers. Based on the findings of the current study, reactivation of inactive monomers to active tetramers may have occurred after elution at the acidic pH used and needs to be considered as an alternative explanation for those results.
The ß-tryptase concentration calculated to be present in the acidic, heparin-rich interior of mast cell secretory granules, about 3.3 mM (10–35 pg/mast cell (21); secretory granules account for about 40% of the cell volume; mean mast cell diameter is about 10 µm), clearly favors maintenance of the active tetrameric configuration. Once released into the extracellular space, tryptase will be considerably diluted. Because mast cell concentrations in dermis, lung, and bowel range from 1 to 20 x 106 cells/cm3, release of 100% of the tryptase from 100% of the mast cells at such sites would result in a maximal tryptase concentration of about 3.3 µM. A more reasonable estimate of release of perhaps 10% of the total tryptase burden (because activated mast cells often do not release all of their granule contents, and particularly with an allergen challenge, not all mast cells may be activated) would result in a maximal, overall tryptase concentration in the extracellular tissue space of about 0.3 µM, within the range of concentrations of tryptase examined in the current study. An acidic pH environment, as might be anticipated at sites of inflammation, of wound healing, or of poor vascularity, would favor stabilization of active tetrameric tryptase as well as reactivation of inactive monomers, even if heparin were removed or destroyed. Consistent with these favorable effects of pH on tryptase reactivation are the observations that tryptase-mediated fibrinogenolysis (18) and kinin formation (17) also occur best at an acidic pH. Whether this property of tryptase can be extended to other activities of the enzyme, such as its ability to stimulate endothelial cells (22), fibroblasts (23, 24, 25), smooth muscle (26, 27), and epithelial cells (28), remains to be seen. In blood, ß-tryptase concentrations are considerably lower than in tissues, being <0.03 nM in normal blood and peaking between 0.17 and 6 nM in most cases of severe, mast cell-dependent systemic anaphylaxis (29, 30, 31). Thus, once ß-tryptase converts to inactive monomers and diffuses from its tissue site of release, reactivation would seem to require recruitment to an acidic site. For example, uptake of tryptase into the acidic compartment of a cell, or binding to available heparin proteoglycan in an extracellular space, in theory, could result in reactivation. Reactivation of tryptase in tissues at sites of allergic reactions might increase bronchial hyperreactivity to histamine in the airway, stimulate fibroblasts to produce more collagen, stimulate epithelial cells to produce inflammatory cytokines, or initiate angiogenesis by stimulating endothelial cells to begin to form tubules. Reactivation of tryptase in the endosomal compartment of cells might facilitate Ag processing if engulfed by cells having this capability. Such possibilities now need to be considered.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Lawrence B. Schwartz, Department of Internal Medicine, Virginia Commonwealth University, P.O. Box 980263, Richmond, VA 23298. E-mail address:
3 Abbreviation used in this paper: TGPL, tosyl-L-Gly-Pro-Lys-p-nitroanilide.
Received for publication November 10, 1997. Accepted for publication January 5, 1998.
|
References |
|---|
|
TopAbstractIntroductionExperimental ProceduresResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  Y. Fukuoka and L. B. Schwartz The B12 Anti-Tryptase Monoclonal Antibody Disrupts the Tetrameric Structure of Heparin-Stabilized beta-Tryptase to Form Monomers That Are Inactive at Neutral pH and Active at Acidic pH. J. Immunol., March 1, 2006; 176(5): 3165 - 3172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hallgren, S. Backstrom, S. Estrada, M. Thuveson, and G. Pejler Histidines Are Critical for Heparin-Dependent Activation of Mast Cell Tryptase J. Immunol., August 1, 2004; 173(3): 1868 - 1875. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. M. Strik, A. Wolbink, D. Wouters, B. A. Bladergroen, A. R. Verlaan, I. S. van Houdt, S. Hijlkema, C. E. Hack, and J. A. Kummer Intracellular serpin SERPINB6 (PI6) is abundantly expressed by human mast cells and forms complexes with {beta}-tryptase monomers Blood, April 1, 2004; 103(7): 2710 - 2717. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Fajardo and G. Pejler Human Mast Cell {beta}-Tryptase Is a Gelatinase J. Immunol., August 1, 2003; 171(3): 1493 - 1499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. SOMMERHOFF Mast Cell Tryptases and Airway Remodeling Am. J. Respir. Crit. Care Med., November 15, 2001; 164(10): S52 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Kambe, M. Kambe, J. P. Kochan, and L. B. Schwartz Human skin-derived mast cells can proliferate while retaining their characteristic functional and protease phenotypes Blood, April 1, 2001; 97(7): 2045 - 2052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.M. Braganza Mast cell: pivotal player in lethal acute pancreatitis QJM, July 1, 2000; 93(7): 469 - 476. [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Sommerhoff, W. Bode, P. J. B. Pereira, M. T. Stubbs, J. Sturzebecher, G. P. Piechottka, G. Matschiner, and A. Bergner The structure of the human beta II-tryptase tetramer: Fo(u)r better or worse PNAS, September 28, 1999; 96(20): 10984 - 10991. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
