The Mechanism of Inhibition of the Ca2+-ATPase by Mastoparan
MASTOPARAN ABOLISHES COOPERATIVE Ca2+ BINDING*

Clare L. Longland, Mokdad MeznaDagger , and Francesco Michelangeli§

From the School of Biochemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT United Kingdom

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ABSTRACT
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MATERIALS AND METHODS
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The amphiphilic peptide mastoparan, isolated from wasp venom, is a potent inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. At pH 7.2, ATPase activity is inhibited with an inhibitory constant (Ki) of 1 ± 0.13 µM. Mastoparan shifts the E2-E1 equilibrium toward E1 and may affect the regulatory ATP binding site. The peptide also decreases the affinity of the ATPase for Ca2+ and abolishes the cooperativity of Ca2+ binding. In the presence of mastoparan, the two Ca2+ ions bind independently of one another. Our results appear to support the model that describes the relationship between the two Ca2+ binding sites as "side-by-side," because this model allows the possibility of independent Ca2+ entry to the two sites. Mastoparan shifts the steady-state equilibrium between E1'Ca2 and E1'Ca2·P toward E1'Ca2·P, by possibly affecting the conformational change that follows ATP binding. The peptide also causes a reduction in the levels of phosphoenzyme formed from [32P]Pi.

Some analogues of mastoparan were also tested and were found to cause inhibition of the Ca2+-ATPase in the range of 2-4 µM. The inhibitory action of mastoparan and its analogues appears dependent on their ability to form alpha -helices in membranes.

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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 right-arrow 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 alpha -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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Mastoparan (MP), mastoparan X (MPX), mastoparan 7 (MP7), and mastoparan 17 (MP17) were all obtained from Bachem. 45CaCl2, [3H]glucose, [gamma -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 [gamma -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 [gamma -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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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.

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 (black-square); 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.

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 approx 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.

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 (black-square) of 20 µM mastoparan, measured at pH 7.2 and 25 °C. Each data point is the mean ± S.D. of three determinations.

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 (black-square) 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.

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 chi 2 values, see Table II)
&Dgr;F=A<SUB>1</SUB>(1−<UP>exp</UP><SUP><UP>−</UP>k<SUB>1</SUB>t</SUP>)+A<SUB>2</SUB>(1−<UP>exp</UP><SUP><UP>−</UP>k<SUB>2</SUB>t</SUP>) (Eq. 1)
where Delta 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.

In the presence of mastoparan, the data for both Ca2+ binding and dissociation can be fitted equally well to the following monoexponential equation.
&Dgr;F=A(1−<UP>exp</UP><SUP><UP>−</UP>kt</SUP>) (Eq. 2)

Phosphorylation of the ATPase by [gamma -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 (black-square) of 20 µM mastoparan. Each data point is the mean ± S.D. of three determinations.

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 approx 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.

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 alpha -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 alpha -helical contents and hydrophobic moments are derived from other studies (see Refs. 15, 29, and 30).

MP17 is a relatively inactive synthetic mastoparan analogue, in which two amino acid substitutions reduce alpha -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 alpha -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 approx 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 approx 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.

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 [gamma -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 left-right-arrow  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 alpha -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.

    FOOTNOTES

* This work was financially supported by BBSRC and the Wellcome Trust.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.

Dagger Present address: Fluorescience Ltd, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9ST, UK.

§ To whom correspondence should be addressed. Tel.: 44-121-414-5398; Fax: 44-121-414-3982; E-mail: F.Michelangeli{at}bham.ac.uk.

    ABBREVIATIONS

The abbreviations used are: SR, sarcoplasmic reticulum; ER, endoplasmic reticulum; FITC, fluorescein 5'-isothiocyanate; NBD, nitrobenzo-2-oxa-1,3-diazole; TNP-ADP, trinitrophenyl adenosine diphosphate; MP, mastoparan; Mops, 4-morpholinepropanesulfonic acid; Mes, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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