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INTRODUCTION |
The sarco(endo)plasmic reticulum Ca2+-ATPase
transports Ca2+ from the cytosol to the lumen of the
sarcoplasmic reticulum
(SR)1/endoplasmic reticulum
(ER). The mechanism of this Ca2+-ATPase is usually
discussed in terms of the model proposed by de Meis and Vianna (1). The
model postulates two major conformational states of the enzyme, E1 and
E2. These two states differ in that the affinity for Ca2+
is high in the E1 conformation but low in the E2 conformation, and the
Ca2+-binding sites are exposed on the cytoplasmic side of
the SR in E1 but exposed to the lumen in E2.
It has been demonstrated that the binding of Ca2+ to the
ATPase is both sequential and co-operative (2). This suggests that binding of the first Ca2+ ion is followed by a slow
conformational change (E1Ca
E1'Ca), which allows binding of the
second Ca2+ ion. The second site is only formed on
transition to E1'Ca (2).
The Ca2+-ATPase belongs to a family of enzymes known as the
P2-type ATPases (3). Several peptide toxins have been shown
to inhibit the action of these enzymes. Both myotoxin a, from rattle snake venom, and melittin, isolated from bee venom, are basic peptides
that inhibit the SR Ca2+-ATPase (4-6). Melittin is also a
potent inhibitor of the H+/K+-ATPase and the
Na+/K+-ATPase (7-10).
Mastoparan (MP) is an amphiphilic tetradecapeptide isolated from wasp
venom (11). It is known to possess a variety of biological activities
including mast cell degranulation, mobilization of Ca2+
from cerebellar microsomes and sarcoplasmic reticulum, activation of
the ryanodine receptor and modulation of various enzymes, for example
the Na+/K+-ATPase of rat brain (12-14).
In aqueous solutions, mastoparan forms a random structure, however, in
a lipid environment, the peptide adopts an amphiphilic
-helical
structure, which is thought to be crucial for its interaction with
biological membranes (15). In a previous study (13), we showed that
mastoparan and a number of closely related analogues inhibit the SR
Ca2+-ATPase. Here we elucidate the mechanism of this inhibition.
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MATERIALS AND METHODS |
Mastoparan (MP), mastoparan X (MPX), mastoparan 7 (MP7), and
mastoparan 17 (MP17) were all obtained from Bachem.
45CaCl2, [3H]glucose,
[
-32P]ATP and [32P]Pi were
obtained from Amersham Pharmacia Biotech. All other reagents were
purchased from Sigma.
SR and the purified Ca2+-ATPase were prepared from rabbit
fast-twitch skeletal muscle as described by Michelangeli and Munkonge (16). Ca2+-ATPase activities were determined using the
coupled enzyme method described by Michelangeli and Munkonge (16) and
monitored in a buffer containing 40 mM Hepes/KOH (pH 7.2),
1 mM EGTA, 5 mM MgSO4, 2 mM ATP, 0.42 mM phosphoenolpyruvate, 0.15 mM NADH, 7.5 IU of pyruvate kinase, and 18 IU of lactate
dehydrogenase. Ca2+-ATPase (15 µg) was incubated for 10 min at 37 °C in 2.5 ml of assay buffer. ATPase activity was
initiated by the addition of 1 mM CaCl2 to give
a free Ca2+ concentration of 6.5 µM.
The Ca2+-ATPase/SR was labeled with
nitrobenzo-2-oxa-1,3-diazole (NBD) as described by Henderson et
al. (17). The Ca2+-ATPase was labeled with fluorescein
5'-isothiocyanate (FITC) at a ratio of FITC to ATPase of 0.5:1
according to the method of Michelangeli et al. (18).
Fluorescence measurements were performed at 25 °C using a
Perkin-Elmer LS-50B fluorimeter. Measurements of NBD fluorescence were
made at excitation and emission wavelengths of 430 and 510 nm,
respectively, in a buffer containing either 150 mM
Mops/Tris, 0.3 mM EGTA, 100 mM choline chloride
at pH 7.2, or 150 mM Mes/Tris, 0.3 mM EGTA, 100 mM choline chloride at pH 6.0. Tryptophan fluorescence was
monitored by exciting at 295 nm and measuring the emission at 330 nm.
These measurements were made in a buffer containing 20 mM
Hepes/Tris, 100 mM KCl, 5 mM MgSO4,
100 µM Ca2+ at pH 7.2.
Rapid kinetic fluorescence measurements were performed using a
stopped-flow spectrofluorimeter (Applied Photophysics, Model SX17 MV).
The sample handling unit possesses two syringes, A and B (drive ratio
10:1), which are driven by a pneumatic ram. Tryptophan fluorescence was
monitored (at 25 °C) by exciting the sample at 280 nm and measuring
the emission above 320 nm using a cut off filter. Ca2+
binding was measured at pH 7.2 in 20 mM Hepes/Tris, 100 mM KCl, 5 mM MgSO4, 50 µM EGTA, plus 1 mM Ca2+ from
syringe B, whereas Ca2+ dissociation was measured at pH 7.2 in 20 mM Hepes/Tris, 100 mM KCl, 5 mM MgSO4, 100 µM
Ca2+, plus 2 mM EGTA from syringe B, all values
being final concentration.
Ca2+ binding to the ATPase was measured using the dual
labeling technique of Michelangeli et al. (19). ATPase (0.1 mg) was incubated at 25 °C in 1 ml of buffer containing 20 mM Hepes/Tris, 100 mM KCl, 5 mM
MgSO4, 100 µM EGTA, 500 µM
[3H]glucose (0.2 Ci/mol) and
45CaCl2 (3 Ci/mol) to give the required free
Ca2+ concentration at pH 7.2. Samples were then rapidly
filtered through Millipore HAWP filters (0.45 µm). Filters were left
to dry, after which 8 ml of scintillant was added. The filters were
then counted for both 3H and
45Ca2+. The amount of [3H]glucose
trapped on each filter was used to calculate the wetting volume for the
filter, and the amount of Ca2+ trapped in this volume was
subtracted from the total Ca2+ bound to the filter to give
that bound to the ATPase. A correction was also applied for nonspecific
binding of Ca2+ to the filter.
Equilibrium levels of phosphorylation of the ATPase by
[32P]Pi were measured in 150 mM
Mes/Tris (pH 6.2), containing 5 mM EGTA, 10 mM
MgSO4, and 1 mM Pi (10 Ci/mol), at
25 °C and a protein concentration of 0.9 mg/ml. Samples were
incubated for 20 s and then quenched with 10% trichloroacetic
acid, 0.2 M H3PO4. The precipitate
was collected by rapid filtration through Whatman GF/C filters, washed with 30 ml of 12% trichloroacetic acid, 0.2 M
H3PO4, and then counted.
Steady-state levels of phosphorylation of the ATPase by
[
-32P]ATP were carried out in a similar manner as
above. Experiments were carried out at 25 °C in 20 mM
Hepes/Tris (pH 7.2) containing 100 mM KCl, 5 mM
MgSO4, 100 µM CaCl2, and 0.075 mg/ml ATPase. Two stocks of labeled ATP were made up to cover the range
of ATP concentrations up to 100 µM, with specific
activities of 10 and 100 Ci/mol. The reaction was initiated by addition
of [
-32P]ATP and quenched as described above after
10 s. The samples were then filtered, washed, and counted.
Dual wavelength spectrophotometry was performed on a Shimadzu UV-3000
dual wavelength-recording spectrophotometer. Experiments were carried
out at 25 °C in 20% (w/v) sucrose, 50 mM Mops/KOH (pH
7) containing 1 mM CaCl2 and 0.8 mg/ml ATPase.
Titration of the Ca2+-ATPase with trinitrophenyl adenosine
diphosphate (TNP-ADP) was then monitored by recording the absorbance
difference at 422 nm and 390 nm, as described by Coll and Murphy
(20).
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RESULTS |
Ca2+-ATPase Inhibition--
Fig.
1A shows the effect of
mastoparan on purified, fully uncoupled, Ca2+-ATPase
activity. The inhibitory constant is determined to be 1 ± 0.13 µM mastoparan. In sealed SR vesicles, ATPase activity is
low due to high Ca2+ concentrations in the vesicle lumen,
and the inhibition can be relieved by addition of the Ca2+
ionophore A23187. Effects of mastoparan on ATPase activity of SR
vesicles in the presence of A23187 are very similar to those determined
for the purified ATPase (data not shown).

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Fig. 1.
A, the effect of mastoparan on SR
Ca2+-ATPase activity. Activity of the purified ATPase (0.05 µM) was measured at pH 7.2 and 37 °C. Each data point
is the mean ± S.D. of three determinations. The effect of
mastoparan (1 µM) on SR Ca2+-ATPase activity
as a function of Ca2+ (B), ATP (C),
and Mg2+ (D) concentration. Activity of the
purified ATPase (0.05 µM) was measured at pH 7.2 and
37 °C. Each data point is the mean ± S.D. of three
determinations.
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However, in the absence of A23187, low concentrations of mastoparan
(<1 µM) actually increase the activity of the ATPase. Thus mastoparan increases the permeability of the SR membrane to
Ca2+, a phenomenon previously reported by Longland et
al. (13). At higher concentrations of mastoparan (
1
µM), ATPase activity in sealed SR vesicles decreases due
to inhibition of the pump.
Fig. 1B shows the effect of mastoparan on the
Ca2+ dependence of ATPase activity. A bell-shaped curve was
obtained both in the presence and absence of mastoparan. The
Km value for the high-affinity (activatory)
Ca2+ sites was increased from 0.16 µM in the
absence of peptide to 0.27 µM in the presence of
mastoparan. In contrast, the Km value for the lower
affinity (inhibitory) sites (0.2 mM) was not significantly
effected by the inhibitor. In addition, maximum ATPase activities were
observed at similar free Ca2+ concentrations in both the
presence and absence of mastoparan (i.e. 6.5 µM).
The effect of mastoparan on the dependence of ATPase activity on the
concentration of ATP is shown in Fig. 1C. The data are fitted to a modified form of the Michaelis-Menten equation, assuming that ATP interacts at 2 sites: a high-affinity (catalytic) site and a
low affinity (regulatory) site (21).
Addition of mastoparan had little effect on ATPase activity at low ATP
concentrations but considerably inhibited the pump at higher ATP
concentrations. In the absence of peptide, the data could be fitted
assuming Km and Vmax values
for the catalytic site of 0.85 µM and 1.68 IU/mg,
respectively, and Km and Vmax
values for the regulatory site of 0.15 mM and 4.34 IU/mg, respectively. In the presence of mastoparan, the data could be fitted
assuming the same values for the catalytic site and the same
Km value for the regulatory site. The
Vmax value for the regulatory site, however,
decreased to 1.24 IU/mg.
Fig. 1D shows the effect of mastoparan on the
Mg2+ dependence of ATPase activity. In the absence of
mastoparan, ATPase activity decreases with increasing concentrations of
Mg2+. At low concentrations of Mg2+, the
presence of mastoparan results in strong inhibition of the ATPase. As
the Mg2+ concentration is increased from 2-10
mM, stimulation of ATPase activity is then observed
followed by inhibition.
NBD and FITC Fluorescence--
It has been shown that the E2-E1
equilibrium for the ATPase can be monitored by changes in the
fluorescence intensity of the ATPase labeled with NBD (22). The
fluorescence intensity of the labeled ATPase is higher in the E1
conformation than in the E2 conformation (22).
Addition of mastoparan to NBD-labeled SR at pH 7.2 (Fig.
2) results in an increase in fluorescence
intensity with an apparent Kd value of 1.8 µM. The maximal fluorescence increase observed is
17-18%. Therefore mastoparan shifts the E2-E1 equilibrium toward
E1.

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Fig. 2.
The effect of mastoparan on the fluorescence
intensity of NBD-labeled SR (0.5 µM ATPase) measured at
pH 7.2 in 150 mM Mops/Tris, 0.3 mM EGTA, 100 mM choline chloride ( ); measured at pH 6.0 in 150 mM Mes/Tris, 0.3 mM EGTA, 100 mM
choline chloride ( ). Each data point is the mean ± S.D. of
three determinations.
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It has been suggested that the E2-E1 equilibrium is
pH-dependent with low pH favoring the E2 form (23).
Consequently pH can also be used to trigger the E2-E1 transition. The
addition of mastoparan to NBD-labeled SR at pH 6.0 (Fig. 2) also causes an increase in fluorescence intensity, however, with a higher apparent
Kd (14.1 µM) and a maximal
fluorescence increase of
31%.
The E2-E1 equilibrium can also be studied by monitoring changes in the
fluorescence intensity of FITC-labeled ATPase. In contrast to
NBD-labeled ATPase, addition of Ca2+ to FITC-labeled ATPase
results in a decrease in fluorescence, which has been attributed to the
E1 conformational state (23).
At pH 7.0, where the E1/E2 ratio has been determined to be 0.5 (23),
calcium induces a decrease in fluorescence of 6%, whereas in the
presence of 20 µM mastoparan, this decrease is reduced to
3%. This is consistent with mastoparan having shifted the E2-E1 equilibrium toward the E1 conformation. Furthermore, addition of 20 µM mastoparan to FITC-labeled ATPase in the absence of
calcium also caused a 3% decrease in fluorescence.
Calcium Binding and Dissociation--
Table
I shows the level of
45Ca2+ bound to the ATPase at a free
Ca2+ concentration of 50 µM, a concentration
at which both high-affinity Ca2+ binding sites should be
fully saturated. This level is unchanged in the presence of 30 µM mastoparan (added either before or after the labeled
Ca2+), demonstrating that mastoparan does not effect the
stoichiometry of Ca2+ binding. Levels of Ca2+
binding to the native ATPase are higher than expected as a result of
nonspecific binding of Ca2+ to the ATPase and associated
lipids (19).
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Table I
Effect of mastoparan on 45Ca2+ binding to purified
ATPase (1 µM) at a free Ca2+ concentration of 50 µM
Each value is the mean ± S.D. of three determinations.
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Fig. 3 shows Ca2+ binding to
the ATPase as a function of Ca2+ concentration. It clearly
shows that mastoparan reduces the affinity of Ca2+ binding
to the ATPase, increasing the Kd from 0.6 to 3.7 µM. In the absence of mastoparan, binding of
Ca2+ to the ATPase is cooperative, as expected, with a Hill
coefficient of 1.60. In the presence of mastoparan, this cooperativity
is no longer observed, with the Hill coefficient being reduced to 0.9.

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Fig. 3.
Binding of 45Ca2+ to
purified ATPase as a function of free Ca2+
(pCa), in the absence ( ) and presence ( ) of 20 µM mastoparan, measured at pH 7.2 and 25 °C. Each data
point is the mean ± S.D. of three determinations.
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Calcium binding and dissociation can also be studied through changes in
the tryptophan fluorescence of the ATPase. On addition of
Ca2+ to the ATPase, there is an increase in tryptophan
fluorescence that has been attributed to the E1Ca-E1'Ca transition,
with the E2, E1, and E1Ca forms having relatively low tryptophan
intensities and the E1'Ca and E1'Ca2 forms having higher
fluorescence intensities (24).
Fig. 4 shows that the addition of 20 µM mastoparan to the ATPase causes a shift in the
Ca2+ concentration dependence of this transition to higher
Ca2+ concentrations. The apparent Kd
value is increased from 1.4 to 25 µM, suggesting once
again that mastoparan may decrease the affinity of the ATPase for
Ca2+. The presence of the peptide also halves the maximum
change in the fluorescence intensity observed.

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Fig. 4.
Percent change in tryptophan fluorescence of the
purified ATPase (0.5 µM) as a function of free
Ca2+ (pCa), in the absence ( ) and presence
( ) of 20 µM mastoparan. Measured at pH 7.2 in 20 mM Hepes/Tris, 100 mM MgSO4, 100 µM Ca2+. The calcium dependence of
fluorescence was observed by addition of EGTA to give the required free
Ca2+ concentration. Each data point is the mean ± S.D. of three determinations.
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It has been shown in stopped-flow experiments with the
Ca2+-ATPase that both Ca2+ binding and
dissociation are biphasic in nature (25). Fig. 5 shows the binding (A) and
dissociation (B) of calcium to and from the ATPase in the
absence and presence of 30 µM mastoparan. The kinetic
parameters obtained from these experiments are given in Table
II. In the absence of peptide, the data
for both Ca2+ binding and dissociation can be fitted to the
following biexponential equation (fitting these data to a
monoexponential equation resulted in much larger
2
values, see Table II)
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(Eq. 1)
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where
F equals fluorescence change,
A1, A2,
k1, and k2 are the
amplitudes and rate constants for the fast and slow phases of
Ca2+ binding/dissociation, respectively, and t
is time (s).

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Fig. 5.
A, kinetics of the increase in
tryptophan fluorescence caused by Ca2+ binding to the
ATPase, measured in the absence (upper trace) and presence
(lower trace) of 30 µM mastoparan. In the
stopped-flow experiment, syringe A contained 0.5 µM
ATPase in buffer at pH 7.2 (20 mM Hepes/Tris, 100 mM KCl, 5 mM MgSO4, and 50 µM EGTA), and syringe B contained 10 mM
Ca2+ (final concentration = 1 mM). B,
kinetics of the decrease in tryptophan fluorescence caused by
Ca2+ dissociation from the ATPase, measured in the absence
(lower trace) and presence (upper trace) of 30 µM mastoparan. In the stopped-flow experiment, syringe A
contained 0.5 µM ATPase in buffer at pH 7.2 (20 mM Hepes/Tris, 100 mM KCl, 5 mM
MgSO4, and 100 µM Ca2+), and
syringe B contained 20 mM EGTA (final concentration = 2 mM).
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Table II
Kinetic data describing the binding and dissociation of Ca2+ to
and from the ATPase in the presence and absence of 30 µM
mastoparan
The data are fitted to either a monoexponential or biexponential
equation, as described in the text.
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In the presence of mastoparan, the data for both Ca2+
binding and dissociation can be fitted equally well to the following
monoexponential equation.
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(Eq. 2)
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Phosphorylation of the ATPase by [
-32P]ATP and
[32P]Pi--
Steady-state levels of
phosphorylation can be studied under conditions of low Ca2+
where the ATPase will be in a continuous state of turnover. At 25 °C, mastoparan increases the steady-state levels of phosphoenzyme formation over the range of ATP concentrations used (Fig.
6). In the absence of mastoparan,
apparent Kd and EPmax values of 46 µM and 9.1 nmol EP/mg ATPase, respectively, are obtained, whereas in the presence of mastoparan, these values are reduced to 9 µM and 9.1 nmol EP/mg. These results suggest that
mastoparan has either increased the affinity of the ATPase for ATP
and/or that the steady-state equilibrium between E1'Ca2 and
E1'Ca2·P has been shifted toward
E1'Ca2·P.

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Fig. 6.
Equilibrium phosphoenzyme levels of the
purified ATPase (0.7 µM) at pH 7.2, 25 °C, as a function of ATP concentration in the absence ( ) and
presence ( ) of 20 µM
mastoparan. Each data point is the mean ± S.D. of three
determinations.
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TNP-ADP is known to be a competitve inhibitor of the SR
Ca2+-ATPase (26, 27). It binds very tightly to the active
site of the enzyme. Consequently, if the nucleotide binding site of the ATPase is covalently blocked with FITC, TNP-ADP is unable to bind. As
described under "Materials and Methods," dual wavelength
spectrophotometry can be used to monitor the absorbance difference at
422 and 390 nm on titration of Ca2+-ATPase with TNP-ADP.
This method can be used to determine whether or not mastoparan alters
the affinity of the ATPase for TNP-ADP and hence ATP. Under conditions
where the ATPase is in the E1 conformation, we determined that the
apparent Kd for TNP-ADP was 3.76 ± 0.61 µM (data not shown), consistent with TNP-ADP binding to
the high affinity nucleotide binding site (28). In the presence of 70 µM mastoparan, this value was relatively unchanged, 3.45 ± 0.48 µM.
As shown in Fig. 7, mastoparan causes a
40% reduction in the levels of phosphoenzyme formed from 1 mM [32P]Pi at 25 °C. The
presence of mastoparan therefore shifts the equilibrium between E2 and
E2·Pi toward E2.

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Fig. 7.
The effect of mastoparan on phosphoenzyme
formation by [32P]Pi
at 25 °C, at a fixed Pi
concentration of 1 mM. Each data point is the
mean ± S.D. of three determinations.
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Mastoparan Analogues--
Since mastoparan was first discovered
several other analogues have been isolated from different species of
wasp. In addition several synthetic analogues have been made. The
effect of a selection of these analogues on the ATPase was undertaken,
and the results were compared with those obtained for mastoparan. Table
III summarizes the effect of these
peptides on the Ca2+-ATPase. The fractional
-helical
contents and hydrophobic moments of the peptides derived from other
studies are also shown in this table (15, 29, 30).
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Table III
Comparison of the effects of mastoparan analogues on the ATPase with
mastoparan
µ corresponds to the hydrophobic moment of the peptide (a measurement
of the asymmetry with which hydrophobicity is distributed around the
axis of a helix). Fractional -helical contents and hydrophobic
moments are derived from other studies (see Refs. 15, 29, and 30).
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MP17 is a relatively inactive synthetic mastoparan analogue, in which
two amino acid substitutions reduce
-helix formation as well as
decrease the hydrophobic moment of the peptide (15). It can be seen
that this peptide is a far weaker inhibitor of the ATPase. The
Ki value for MP17 is extrapolated, because at 30 µM MP17 only 10% inhibition was observed. This peptide
also has no effect on the fluorescence intensity of NBD-labeled ATPase.
MPX and MP7 retain the ability to form
-helices in lipids and have
similar hydrophobic moments to mastoparan. They also inhibit the
ATPase, with Ki values of 4.4 and 2.2 µM, respectively. Similar to mastoparan, these two
analogues increase the fluorescence intensity of NBD-labeled ATPase,
implying that they too shift the E2-E1 equilibrium toward E1.
Conclusion--
A number of P2-type ATPases, including
the Ca2+-ATPase, H+/K+-ATPase, and
Na+/K+-ATPase are inhibited by peptide toxins
such as mastoparan and melittin (4-10, 13). Because this family of
enzymes are believed to operate by similar mechanisms, a detailed
understanding of how mastoparan inhibits the Ca2+-ATPase
may also provide insight into the mechanism of inhibition of other
P2-type ATPases by this peptide.
Mastoparan acts as a potent inhibitor of the SR
Ca2+-ATPase, having a Ki value of 1 ± 0.13 µM. The two analogues, MPX and MP7, also inhibit
the ATPase with inhibitory constants of between 2.2 and 4.4 µM. Thus mastoparan acts as one of the most potent ATPase
inhibitors, apart from thapsigargin (31).
Using both NBD-labeled ATPase and FITC-labeled ATPase, mastoparan has
been shown to shift the E2-E1 equilibrium of the enzyme toward E1, in
contrast to thapsigargin which stabilizes the E2 form of the enzyme
(31). At pH 6.0, the maximum percent increase in NBD fluorescence is
approximately twice that at pH 7.2, because more of the ATPase can be
shifted from the E2 form to the E1 form. Froud and Lee (23) have shown
that the ratio E1/E2 varies from 0.1 to 0.5 with changing pH from 6 to
7. The apparent affinity of mastoparan for the ATPase has been reduced
at pH 6.0, demonstrating that ionic interactions are important in the
binding of the peptide to the ATPase.
Mastoparan may also affect the regulatory ATP binding site, because the
Vmax for the regulatory site is reduced from
4.34 IU/mg to 1.24 IU/mg in the presence of the inhibitor.
The presence of mastoparan halves the maximum change in tryptophan
fluorescence intensity observed, suggesting that mastoparan has altered
the normal sequence of conformational changes that occur either on
Ca2+ binding or Ca2+ dissociation.
The Ca2+ dependence of ATPase activity data suggested that
the affinity of Ca2+ binding to the E1 form of the ATPase
was reduced by
2-fold in the presence of mastoparan. As a result,
the Ca2+ binding step was isolated and investigated in more
detail. Both the tryptophan fluorescence data and the Ca2+
binding studies suggested that mastoparan decreased the affinity of the
ATPase for Ca2+ by between
10- and 20-fold. The
stoichiometry of Ca2+ binding, however, was shown to be unaffected.
In the absence of mastoparan, binding of Ca2+ to the ATPase
is cooperative, whereas this cooperativity is abolished in the presence of mastoparan. This suggests that the Ca2+ binding sites
have been effected in such a way that Ca2+ binding is no
longer a two-step process, comprising fast binding of a first
Ca2+ ion followed by a slow conformational change, which
allows binding of a second Ca2+ ion. In the presence of
mastoparan, the peptide appears to have altered the Ca2+
binding sites in such a way that the two Ca2+ ions now bind
independently of one another. Thus, binding of the second
Ca2+ ion is not dependent on a conformational change
produced by binding of the first Ca2+ ion.
Site-directed mutagenesis has localized the two calcium binding sites
between transmembrane helices M4, M5, and M6 (32, 33). The critical
residues have been identified as Glu309,
Glu771, Asp800, Thr799, and
Asn796. In addition Glu908, located within
transmembrane helix M8, may play a minimal role in Ca2+
binding. Two models have been proposed to describe the relationship between these two sites; the "stacked" model and the more recently proposed "side-by-side" model (34). Although both models can account for the cooperative nature of Ca2+ binding, the
later model also allows the possibility of independent Ca2+
entry to the two sites. Our results appear to support the side-by-side model, because the presence of mastoparan causes a conformational change that results in independent Ca2+ binding (see Scheme
1).

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Scheme 1.
A and B represent the
stacked and side-by-side models that have been proposed to describe the
relationship between the two Ca2+ binding sites (I and II).
Large arrows indicate the major route of Ca2+
translocation, whereas the smaller arrows indicate possible other sites
of Ca2+ entry and exit. C represents the two sites in the
presence of mastoparan. Ca2+ binding is now seen to be
independent.
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The data from the rapid kinetic measurements clearly show that in the
absence of mastoparan both Ca2+ binding and dissociation to
and from the ATPase are biphasic in nature. In the presence of
mastoparan these processes are now monophasic, lending further weight
to the argument that Ca2+ binding/dissociation is
independent in the presence of the peptide.
Mg2+ is an essential activator of the SR
Ca2+-ATPase. It is required for several of the steps, which
together make up the catalytic cycle of the enzyme. However,
Mg2+ is also in competition with Ca2+ for
binding at the two Ca2+ binding sites. Consequently, ATPase
activity decreases with increasing concentrations of Mg2+
in the absence of mastoparan. At low concentrations of
Mg2+, the presence of mastoparan results in strong
inhibition of the ATPase. As the Mg2+ concentration is
increased from 2 to 10 mM, stimulation of ATPase activity
is then observed, followed by inhibition. Perhaps the stimulatory
effects of Mg2+ (which are usually masked by the
competition between Ca2+ and Mg2+) are due to
the effect of mastoparan on the Ca2+ binding sites. We have
determined that the affinity of the Ca2+ binding sites for
Ca2+ is reduced in the presence of mastoparan, it could
therefore follow that the affinity of these sites for Mg2+
will also be reduced. For the stimulatory effects of Mg2+
to be observed, the affinity of the Ca2+ binding sites for
Mg2+ must have been more greatly reduced than they were for
Ca2+.
The effect of mastoparan on the steady-state levels of phosphorylation
of the ATPase by [
-32P]ATP was studied. The results
suggest that mastoparan increases the affinity of the ATPase for ATP
and that the steady-state equilibrium between E1'Ca2 and
E1'Ca2·P is pushed toward E1'Ca2·P.
However, mastoparan was shown not to effect the binding of ATP to the
catalytic site (see Fig. 1C) and was also shown to have no
effect on the affinity of the enzyme for TNP-ADP. Petithory and Jencks
(35) have suggested that phosphorylation of the ATPase by ATP is a two-step process in which ATP binding is followed by a conformational change. This active conformation of the enzyme is then able to undergo
rapid phosphorylation.
Because binding of ATP is unaffected by mastoparan, perhaps the peptide
effects the conformational change associated with ATP binding, thus
decreasing the apparent Kd for ATP in the
phosphorylation experiment.
Mastoparan causes a reduction in the levels of phosphoenzyme formed
from 1 mM [32P]Pi, thereby
pushing the E2
E2·Pi equilibrium toward E2. The reduction in levels of phosphoenzyme could be due to reduced levels of
E2 as a result of mastoparan shifting the E2-E1 transition toward E1.
The two mastoparan analogues, MPX and MP7, have similar effects on
ATPase activity as mastoparan itself. MP17, however does not. MP17 has
a reduced
-helical content in membranes and a much smaller
hydrophobic moment. Thus, the ability of mastoparan analogues to
interact with and inhibit the Ca2+-ATPase appears to
correlate with their ability to adopt ordered conformations in
membranes as well as their amphiphilicity.