Testing the Versatility of the Sarcoplasmic Reticulum Ca2+-ATPase Reaction Cycle When p-Nitrophenyl Phosphate Is the Substrate*

Francisco Fernandez-BeldaDagger, Maria-Isabel Fortea, and Fernando Soler

From the Departamento de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia en Espinardo, 30071 Murcia, Spain

Received for publication, September 21, 2000, and in revised form, December 4, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A detailed characterization of p-nitrophenyl phosphate as energy-donor substrate for the sarcoplasmic reticulum Ca2+-ATPase was undertaken in this study. The fact that p-nitrophenyl phosphate can be hydrolyzed in the presence or absence of Ca2+ by the purified enzyme is consistent with the observed phenomenon of intramolecular uncoupling. Under the most favorable conditions, which include neutral pH, intact microsomal vesicles, and low free Ca2+ in the lumen, the Ca2+/Pi coupling ratio was 0.6. A rise or decrease in pH, high free Ca2+ in the lumenal space, or the addition of dimethyl sulfoxide increase the intramolecular uncoupling. Alkaline pH and/or high free Ca2+ in the lumen potentiate the accumulation of enzyme conformations with high Ca2+ affinity. Acidic pH and/or dimethyl sulfoxide favor the accumulation of enzyme conformations with low Ca2+ affinity. Under standard assay conditions, two uncoupled routes, together with a coupled route, are operative during the hydrolysis of p-nitrophenyl phosphate in the presence of Ca2+. The prevalence of any one of the uncoupled catalytic cycles is dependent on the working conditions. The proposed reaction scheme constitutes a general model for understanding the mechanism of intramolecular energy uncoupling.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It is well established that the free energy released from ATP hydrolysis can be used for the vectorial translocation of cations across cellular membranes (1). In this sense, clear structural and functional similarities among P-type ATPases have been observed (2-4). These similarities include the hydrophobic transmembrane segments containing the porelike region, the extramembranous globular head where the ATP binding site is located, two major conformational states of the enzyme, the acyl phosphate intermediate and the catalytic cycle. Although these common features suggest that ion-transporting ATPases share the same energy transduction mechanism, this idea was challenged when non-nucleotide compounds were used as phosphorylating substrate (5). In fact, it was observed that the transport activity supported by non-nucleotide substrates is dependent on the cation-ATPase involved. In other words, the same non-nucleotide substrate does not always perform the same task since it is dependent on the energy transduction system. For instance, it has been reported that Ca2+ is transported by sarcoplasmic reticulum (SR)1 Ca2+-ATPase during the hydrolysis of pNPP or acetyl phosphate (6-9). However, the hydrolysis of pNPP supports neither Ca2+ transport by the erythrocyte membrane Ca2+-ATPase (10, 11) nor H+ transport by the H+-ATPase from the yeast plasma membrane (12). Other studies have reported that reconstituted Na+,K+-ATPase displays slight Na+ uptake during the hydrolysis of acetyl phosphate (13), whereas the H+,K+-ATPase from gastric mucosa cannot actively transport H+ and K+ when hydrolyzing acetyl phosphate (5). Thus, it has been recognized that the hydrolysis of non-nucleotide substrates in the absence of transport activity is related to low energy conformations of the enzyme (5, 12, 14), whereas the simultaneous occurrence of both substrate hydrolysis and ion transport is associated with high energy conformations (7, 9, 15).

In an attempt to clarify the role of non-nucleotide substrates in the energy coupling process, we selected pNPP as energy-donor substrate and SR Ca2+-ATPase as a transduction system. Initially, the enzyme was exposed to conditions that allowed Ca2+ transport at the expenses of pNPP hydrolysis. Then, the assay conditions were modified to induce uncoupling. The use of a purified enzyme preparation (16) and the highly specific inhibitor TG (17, 18) shed light on the hydrolytic activities measured in the presence or absence of Ca2+. The experimental strategy for analyzing the energy uncoupling routes was based on the inhibitory effect of vanadate and TG, namely vanadate interacts selectively with the E2 conformation of the enzyme whereas TG inhibits the accumulation of E1 forms.

Our functional model provides a mechanistic description of the intramolecular uncoupling phenomenon when the SR Ca2+-ATPase hydrolyzes pNPP. This is relevant for understanding the uncoupling of different P-type ATPases in the presence of different phosphorylating substrates and in the absence of a cationic leak.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [45Ca]CaCl2 was obtained from Amersham Pharmacia Biotech. The Ca2+ standard solution (Titrisol) was purchased from Merck. Ionophore A23187 from Streptomyces chartreusensis was a product of Calbiochem. Ammonium metavanadate was from Acros Organics. 4-Nitrophenyl phosphate (disodium salt) was from Roche Molecular Biochemicals. TG was obtained from Molecular Probes Europe. The liquid scintillation mixture (S 4023), deoxycholic acid (sodium salt), and other reagents of analytical grade were from Sigma. HAWP filter units with pore diameter of 0.45 µm were from Millipore. 45Ca2+-loaded vesicles were filtered under vacuum with a Hoefer manifold filtration box from Amersham Pharmacia Biotech.

Sample Preparation-- Right side-oriented (intact) vesicles were obtained from the SR membrane of fast twitch rabbit leg muscle as described by Eletr and Inesi (19). Purified Ca2+-ATPase was prepared by deoxycholate treatment according to method 2 of Meissner et al. (16). Final pellets were resuspended and stored in frozen aliquots at -80 °C until use. Vesicles leaky to Ca2+ were obtained by including A23187 in the reaction medium.

Protein Quantitation-- The protein concentration was measured by the Lowry et al. (20) procedure using bovine serum albumin as standard.

Ca2+ Concentration-- Free Ca2+ in the external medium was calculated as described by Fabiato (21), taking into account the Ca2+-EGTA absolute stability constant (22), the pK values for the EGTA protonation (23), pH, and the presence of relevant ligands. Free Ca2+ in the lumenal medium was fixed by equilibrating the vesicles in the standard reaction medium but including potassium oxalate in the range of 0.5-5 mM.

Vanadate Solution and Enzyme Inhibition-- Stock solutions containing mostly monovanadate were prepared by dissolving ammonium metavanadate in ultrapure water (Milli-Q grade) adjusted at pH 10.0 with NaOH. The absence of a yellow/orange color indicated the absence of any decavanadate species (24). The sensitivity to vanadate was studied by measuring the dependence of the pNPP hydrolysis rate on the vanadate concentration.

Enzyme Hydrolytic Activity-- Linear rates of pNPP hydrolysis were measured at 25 °C by following the time-dependent accumulation of p-nitrophenol (25). The standard reaction medium consisted of 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2 (50 µM free Ca2+), 0.2 mg/ml SR protein, and 5 mM potassium oxalate. The reaction was started by adding a given pNPP concentration. Aliquots containing 0.5 ml of reaction mixture were withdrawn at different time intervals and mixed with 0.5 ml of 10% (w/v) trichloroacetic acid. For each sample, the membrane protein was sedimented at 10,000 rpm and 4 °C for 5 min in an Eppendorf microcentrifuge and the supernatant containing p-nitrophenol (0.9 ml) was supplemented with 45 µl of 10 N NaOH (final concentration, 0.5 N). A blank assay was performed by adding 0.5 ml of 10% trichloroacetic acid to 0.5 ml of reaction mixture containing no phosphorylating substrate. After mixing, pNPP was added and processed as described before. p-Nitrophenol was quantitated by colorimetric reading at 420 nm. The extinction coefficient of p-nitrophenol (1.62 × 104 M-1·cm-1) was determined previously under the experimental conditions used in this study. pNPP hydrolysis was also measured under nonstandard conditions. A detailed description of the reaction media composition is provided in the corresponding figure legends.

Transport Experiments-- Linear rates of active Ca2+ accumulation were measured at 25 °C with the aid of radioactive tracer and sample filtration (26). The standard reaction medium consisted of 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2 (free Ca2+ was 50 µM), ~7,000 cpm/nmol 45Ca2+, 0.2 mg/ml SR protein, and 5 mM potassium oxalate. After equilibration, the reaction was initiated by adding pNPP and stopped at various times by filtering aliquots of 0.5 ml. Filters retaining the microsomal vesicles were rapidly rinsed with 10 ml of ice-cold La3+ medium (10 mM Mops, pH 7.0, 2 mM LaCl3), solubilized, and subjected to liquid scintillation counting. Unspecific 45Ca2+ retained by the filters was subtracted by performing a blank assay. In this case, an aliquot of 0.5 ml of reaction mixture before the addition of pNPP was filtered and processed as described previously. Other assay conditions for Ca2+ transport are given in the figure captions and in the text where appropriate.

TG Effect on the Purified Ca2+-ATPase-- Hydrolysis of pNPP in the presence or absence of Ca2+ was measured at 25 °C, as described for SR vesicles. Measurements in the presence of Ca2+ were carried out in a medium containing 20 mM buffer (Mes, pH 6.0, Mops, pH 7.0, or Tris, pH 8.0), 80 mM KCl, 20 mM MgCl2, CaCl2 and EGTA to yield 50 µM free Ca2+, 0.2 mg/ml purified enzyme, and 10 mM pNPP. For measurements in a nominally Ca2+-free medium, EGTA was raised to 1 mM and Ca2+ was omitted. Some experiments at neutral pH were performed in the presence of 40% (v/v) Me2SO. The reaction medium was supplemented with 2 µM TG when indicated. The degree of activation by Ca2+ was calculated as the ratio between the enzyme activity observed in the presence or absence of Ca2+ (both measured in the absence of TG). The TG inhibition factor was calculated as a ratio between the enzyme activity in a Ca2+-containing medium measured in the absence or presence of TG.

Passive Permeability and Me2SO-- SR vesicles (0.4 mg/ml) were equilibrated at 25 °C in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, ~9,000 cpm/nmol 45Ca2+, and 5 mM potassium oxalate. The hydrolytic reaction was initiated by adding 10 mM pNPP. The final volume was 2.5 ml and the temperature 25 °C. A blank assay was carried out by processing a sample with no added pNPP. At t = 6 min, the reaction medium was supplemented with 5 µM TG and a volume of 2.5 ml containing 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, 5 mM potassium oxalate, and 80% (v/v) Me2SO was added immediately. The time course of Ca2+ accumulation was followed at different times by filtering aliquots containing 0.1 mg of membrane protein. Filters were rinsed with 10 ml of ice-cold La3+ medium, solubilized, and counted by liquid scintillation.

Data Presentation-- Experimental data are presented as the mean plus or minus the standard error and correspond to at least three independent assays, each performed in duplicate. Kinetic parameters were evaluated after curve fitting by nonlinear regression algorithm using the SigmaPlot software (Jandel Scientific).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

pNPP as a Substrate of the Enzyme-- The initial experimental conditions included a preparation of microsomal vesicles showing negligible passive Ca2+ leakage (27), and a reaction medium containing 20 mM Mg2+. Free Ca2+ in the external medium was maintained at 50 µM by including a Ca2+/EGTA-buffered system while 5 mM oxalate ensured a low free Ca2+ in the lumen. The dependence of the hydrolysis rate on the pNPP concentration measured at pH 7.0 and in the Ca2+-containing medium displayed a hyperbolic profile, as shown in Fig. 1A. A reaction medium at pH 6.0 or 8.0 did not significantly modify the hydrolysis rate measured at each pNPP concentration.



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Fig. 1.   Effect of pH when pNPP was hydrolyzed in the presence of Ca2+ at different substrate concentrations. A, the hydrolytic reaction at 25 °C was started by adding a given pNPP concentration and stopped by quenching aliquots of 0.5 ml of reaction mixture with 0.5 ml of 10% trichloroacetic acid at different times. Hydrolysis rates were measured by following the accumulation of the product, p-nitrophenol. The initial reaction medium containing SR vesicles in the presence of 50 µM free Ca2+ consisted of: 20 mM Mes, pH 6.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.16 mM CaCl2, 0.2 mg/ml SR protein, and 5 mM K+-oxalate (); 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, 0.2 mg/ml SR protein, and 5 mM K+-oxalate (black-square); or 20 mM Tris-HCl, pH 8.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.25 mM CaCl2, 0.2 mg/ml SR protein, and 5 mM K+-oxalate (black-triangle). B, Ca2+ transport associated with pNPP hydrolysis was measured in parallel experiments by including ~7,000 cpm/nmol 45Ca2+ in the corresponding reaction medium. The reaction was started by the addition of pNPP and stopped at different times by filtering 0.5-ml aliquots of reaction mixture. Filters were processed as described under "Experimental Procedures." Rates of transport at pH 6.0 (open circle ), pH 7.0 (), or pH 8.0 (triangle ) are shown.

The dependence of the Ca2+ transport rate on pNPP concentration was also measured. The rate of active Ca2+ accumulation was highly dependent on the reaction medium pH (Fig. 1B). A hyperbolic dependence was observed at pH 6.0 or 7.0, even though higher rates were obtained at neutral pH. Negligible rates were observed after a fast and small component of Ca2+ transport when the experiments were carried out at pH 8.0.

An analysis of the experimental curves (Table I) revealed that Km for pNPP hydrolysis in the presence of Ca2+ and that for Ca2+ transport were in the 2-3 mM range when measured from pH 6.0 to 8.0. Moreover, Vmax for pNPP hydrolysis in the presence of Ca2+ was around 55 nmol/min/mg of protein, with the pH having only a slight effect whereas Vmax for Ca2+ transport was clearly affected by pH. The Ca2+/Pi coupling ratio at neutral pH was around 0.6. The coupling ratio decreased to 0.4 when measured at pH 6.0, and complete uncoupling was observed at pH 8.0. 


                              
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Table I
Effect of pH on the SR Ca2+-ATPase performance in the presence of pNPP and Ca2+
Kinetic parameters and coupling values were obtained from the data shown in Fig. 1. Km and Vmax units are mM and nmol/min/mg protein, respectively.

Free Ca2+ in the lumen affects to the enzyme coupling ratio when ATP is the substrate (28, 29); therefore, this parameter was also checked. We selected a reaction medium at pH 7.0 containing 20 mM Mg2+, 50 µM free Ca2+, and 10 mM pNPP that was equilibrated with different concentrations of oxalate. The hydrolysis rate in the presence of Ca2+ displayed a small increase from 42 to 50 nmol/min/mg of protein when the oxalate concentration was raised from 0 to 5 mM (Fig. 2A). The rate of Ca2+ transport increased as oxalate concentration was raised in the millimolar range, displaying a sigmoidal dependence (Fig. 2B). The rate was negligible in the absence of precipitating anion, whereas maximal values of 31 nmol of Ca2+/min/mg of protein were obtained in the presence of 5 mM oxalate.



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Fig. 2.   Effect of lumenal Ca2+ on pNPP hydrolysis when 50 µM free Ca2+ was present in the external medium. A, linear rates of pNPP hydrolysis (black-square) were measured at 25 °C in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, 0.2 mg/ml SR protein, and 10 mM pNPP. K+-oxalate in the range of 0.5-5 mM was also included when indicated. B, linear rates of Ca2+ transport in the absence or presence of K+-oxalate were measured by including 45Ca2+ in the reaction medium described for the hydrolysis of pNPP ().

The catalytic properties of the enzyme are sensitive to the presence of Me2SO (30, 31); therefore, we evaluated the organic solvent effect in the standard reaction medium at pH 7.0 containing 50 µM free Ca2+, 10 mM pNPP, and native SR vesicles. The pNPP hydrolysis rate in the presence of Ca2+ increased when the organic solvent concentration was raised. Maximal activity was observed at 20% Me2SO, whereas a further increase in the solvent concentration up to 40% induced a progressive decrease in the hydrolysis rate (Fig. 3A). In contrast, the hydrolysis rate measured in the absence of Ca2+ increased progressively as the Me2SO concentration was raised. Furthermore, maximal Ca2+ transport rates were observed in the presence of 10% Me2SO and higher solvent concentrations decreased the transport activity, tending to the zero value (Fig. 3B). The dependence of pNPP hydrolysis in the presence of Ca2+ and that of Ca2+ transport on the Me2SO concentration did not show the same profile. Possible effects of Me2SO on SR vesicle permeability (i.e. in Ca2+ transport measurements) were ruled out experimentally using 45Ca2+-loaded vesicles. An active loading was started by adding 10 mM pNPP to the standard reaction medium and stopped after 6 min by adding 5 µM TG. The 45Ca2+ load was ~170 nmol/mg of protein, and a subsequent addition of 40% Me2SO had no effect on the Ca2+ content retained by the vesicles (data not shown).



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Fig. 3.   Effect of Me2SO when SR vesicles are hydrolyzing pNPP in the presence or absence of Ca2+. A, linear rates of hydrolysis in the presence of Ca2+ were measured at 25 °C in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM mgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, 0.2 mg/ml SR protein, 5 mM K+-oxalate, and 10 mM pNPP. Me2SO was also included when indicated (black-square). Enzyme activity data in the absence of Ca2+ were obtained by omitting the addition of CaCl2 and raising the EGTA concentration up to 1 mM. Me2SO was included when indicated (black-triangle). B, linear rates of Ca2+ transport were measured in the reaction medium described for pNPP hydrolysis in the presence of Ca2+ but supplemented with 45Ca2+ ().

Effect of Vanadate on Enzyme Activity-- Vanadate is known to interact with the enzyme conformation in the absence of Ca2+ by acting as a Pi analog (32, 33). When the reaction medium was at pH 7.0 and contained 20 mM Mg2+, 1 mM EGTA (i.e. absence of Ca2+), 10 mM pNPP, and 5 mM oxalate, the hydrolysis of pNPP by native SR vesicles was completely inhibited by ~20 µM vanadate (Fig. 4). The inhibition profile in the absence of Ca2+ was exactly the same when a preparation of purified Ca2+-ATPase was used. Nevertheless, the pNPP hydrolysis rate in a medium containing 50 µM free Ca2+ and leaky vesicles required ~100 µM vanadate to induce complete inhibition. A partial inhibition of 45% was observed in the presence of 100 µM vanadate when A23187 was removed (i.e. when intact vesicles were used) and oxalate was included.



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Fig. 4.   The effect of vanadate on pNPP hydrolysis when measured at neutral pH. The enzyme activity was measured at 25 °C in the following reaction media: 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 1 mM EGTA, 0.2 mg/ml SR vesicles, and 10 mM pNPP (); 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 1 mM EGTA, 0.2 mg/ml purified Ca2+-ATPase, and 10 mM pNPP (open circle ); 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, 0.2 mg/ml SR vesicles, 5 mM K+-oxalate, and 10 mM pNPP (black-square); or 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, 0.2 mg/ml SR vesicles, 15 µM A23187, and 10 mM pNPP (). The vanadate effect was studied by including different concentrations in the reaction media.

Similar experiments using SR vesicles were performed at pH 6.0 (Fig. 5A). In the absence of Ca2+, complete inhibition of the pNPP hydrolysis was now observed in the presence of ~50 µM vanadate. Interestingly, the hydrolysis rate in a Ca2+-containing medium and A23187 (leaky vesicles) displayed the same dependence on vanadate concentration. However, when the enzyme activity was measured in the presence of extravesicular Ca2+ and oxalate (intact vesicles), the sensitivity to vanadate was lower. A vanadate concentration of 100 µM induced a 60% inhibition of the pNPP hydrolysis rate.



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Fig. 5.   Vanadate effect on pNPP hydrolysis measured at pH 6.0 (panel A) or pH 8.0 (panel B). Experiments in panel A were performed in a medium containing: 20 mM Mes, pH 6.0, 80 mM KCl, 20 mM MgCl2, 1 mM EGTA, 0.2 mg/ml SR vesicles, and 10 mM pNPP (); 20 mM Mes, pH 6.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.16 mM CaCl2, 0.2 mg/ml SR vesicles, 5 mM K+-oxalate, and 10 mM pNPP (black-square); or 20 mM Mes, pH 6.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.16 mM CaCl2, 0.2 mg/ml SR vesicles, 15 µM A23187, and 10 mM pNPP (). Experiments in panel B were performed in a medium containing: 20 mM Tris-HCl, pH 8.0, 80 mM KCl, 20 mM MgCl2, 1 mM EGTA, 0.2 mg/ml SR vesicles, and 10 mM pNPP (black-triangle); 20 mM Tris-HCl, pH 8.0, 80 mM KCl, 20 mM MgCl2, 0. 2 mM EGTA, 0.25 mM CaCl2, 0.2 mg/ml SR vesicles, 5 mM K+-oxalate, and 10 mM pNPP (open circle ); or 20 mM Tris-HCl, pH 8.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.25 mM CaCl2, 15 µM A23187, and 10 mM pNPP (triangle ). Vanadate concentrations up to 100 µM were included when indicated.

Fig. 5B shows the sensitivity to vanadate when measured at pH 8.0. The enzymatic activity of SR vesicles in the absence of Ca2+ was completely inhibited by 10 µM vanadate. Likewise, the pNPP hydrolysis rate measured in a Ca2+-containing medium and A23187 (leaky vesicles) was inhibited by 45% in the presence of 100 µM vanadate. The percentage of inhibition induced by 100 µM vanadate was only 32% when the Ca2+ ionophore was substituted for oxalate (intact vesicles).

Fig. 6 shows the effect of lumenal Ca2+ on vanadate sensitivity. The lumenal free Ca2+ was manipulated by modifying the initial concentration of oxalate added (34). Thus, the standard reaction medium at pH 7.0 containing 20 mM Mg2+ and 50 µM free Ca2+ in the external medium was equilibrated with either 0.5 or 5 mM oxalate before the addition of 10 mM pNPP. Intact vesicles and an oxalate concentration of 5 mM, i.e. a low free Ca2+ in the lumen, displayed low sensitivity to vanadate. As a reference, 100 µM vanadate produced a 45% inhibition of the enzyme activity. When the oxalate concentration was cut to 0.5 mM to increase the free Ca2+ in the lumen, there was a further decrease in vanadate sensitivity. In this case, 100 µM vanadate induced a 35% inhibition of the pNPP hydrolysis rate.



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Fig. 6.   Sensitivity to vanadate of SR vesicles hydrolyzing pNPP in the presence of high or low lumenal Ca2+. Linear rates of pNPP hydrolysis were measured at 25 °C in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, 0.2 mg/ml SR vesicles, 10 mM pNPP, and either 0.5 mM () or 5 mM K+-oxalate (black-square). Vanadate was also included when indicated.

The Me2SO effect on the enzyme activity was also analyzed by studying vanadate sensitivity (Fig. 7). In one case, the reaction medium at pH 7.0 and in the absence of Ca2+ was supplemented with 40% Me2SO and the reaction was started by adding 10 mM pNPP. The inhibition of pNPP hydrolysis in the absence of Ca2+ showed a hyperbolic dependence with respect to vanadate reaching an asymptotic level at ~50 µM vanadate. In the other case, the reaction medium contained 50 µM free Ca2+, 5 mM oxalate and was also supplemented with 40% Me2SO. The inhibition of the enzyme activity in the presence of Ca2+ showed the same vanadate dependence observed in the absence of Ca2+.



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Fig. 7.   Sensitivity to vanadate of SR vesicles hydrolyzing pNPP in the presence of 40% Me2SO. The hydrolysis of pNPP was measured at 25 °C in a 50 µM free Ca2+-containing medium, i.e. 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 0.2 mM EGTA, 0.247 mM CaCl2, 0.2 mg/ml SR vesicles, 5 mM K+-oxalate, 40% (v/v) Me2SO, and 10 mM pNPP (black-square), or in a Ca2+-free medium, i.e. 20 mM Mops, pH 7.0, 80 mM KCl, 20 mM MgCl2, 1 mM EGTA. 0.2 mg/ml SR vesicles, 40% (v/v) Me2SO, and 10 mM pNPP (black-triangle). Vanadate was included in the reaction medium when indicated.

Inhibition of Purified Enzyme by TG-- A key question is to know whether or not Ca2+-ATPase is able to hydrolyze pNPP in the absence of Ca2+. The hydrolysis of pNPP in the presence or absence of Ca2+ and the effect of TG on these activities were measured using a preparation of purified enzyme. Under these conditions, any hydrolytic activity should be attributed to the Ca2+-ATPase protein. Fig. 8 (panels A-C) show data obtained at pH 6.0, 7.0, and 8.0, respectively. The rate of pNPP hydrolysis at pH 6.0, in the presence of 10 mM pNPP and in the absence of Ca2+ was 11.0 nmol/min/mg protein. This value decreased to 7.7 nmol/min/mg of protein when measured at pH 7.0 and was 2.7 nmol/min/mg of protein at pH 8.0. The corresponding hydrolytic activities measured in the presence of 50 µM free Ca2+ and 10 mM pNPP were 45.7, 62.0, and 42.0 nmol/min/mg of protein, respectively. Therefore, the activating effect of Ca2+ on the pNPP hydrolysis rate increased from 4.2-fold at pH 6.0, to 8.0-fold at pH 7.0 and 15.5-fold at pH 8.0. In any case, the addition of TG to the enzyme in the presence of Ca2+ gave the corresponding activity values measured in the absence of Ca2+. Moreover, TG had no effect on the pNPP hydrolysis rate measured in the absence of Ca2+. Data obtained at neutral pH and in the presence of 40% Me2SO (Fig. 8, panel D) show that the enzyme activity in the presence or absence of Ca2+ had the same value and also that TG had the same effect on the rate of pNPP hydrolysis when Ca2+ is present or absent. The activation by Ca2+ or the inhibition by TG calculated as described (see Fig. 8 legend) are dependent on the experimental conditions, increasing from ~4 to ~15 as the pH increased from 6.0 to 8.0. Neither activation nor inhibition occurred (the factor is 1) in the presence of 40% Me2SO.



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Fig. 8.   Ca2+ and TG effects on pNPP hydrolysis measured in purified Ca2+-ATPase. Experiments at pH 6.0 (A) in a Ca2+-containing medium included 0.2 mM EGTA and 0.16 mM CaCl2. Experiments at pH 7.0 (B) in the presence of Ca2+ contained 0.2 mM EGTA and 0.247 mM CaCl2. Experiments at pH 8.0 (C) in the presence of Ca2+ contained 0.2 mM EGTA, 0.25 mM CaCl2. The final free Ca2+ was always 50 µM. Experiments in a Ca2+-free media contained 1 mM EGTA and no added Ca2+. Experiments in the presence of 40% Me2SO (D) were performed in the reaction media described for panel B but included the organic solvent. The effect of TG was studied by including a concentration of 2 µM. The Ca2+ activation factor was calculated by dividing pNPP hydrolysis obtained in the presence of Ca2+ by that in the absence of Ca2+ when TG was absent. The TG inhibition factor was calculated by dividing the activity in a Ca2+-containing medium when TG was absent by that when TG was present. The inhibition factor in panel D was corrected taking into consideration the effect of TG on pNPP hydrolysis in the absence of Ca2+.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The coupling efficiency of SR Ca2+-ATPase during the hydrolysis of pNPP is clearly dependent on pH (Fig. 1 and Table I). It is known that Ca2+ binding at equilibrium is a pH-dependent process involving different enzyme conformations and a sequential mechanism (35), as follows.
E<SUB>2</SUB><UP>H</UP><SUP>+</SUP><SUB>3</SUB> ↔ E<SUB>2</SUB>H<SUP>+</SUP> ↔ E<SUB>1</SUB> ↔ E<SUB>1</SUB><UP>Ca</UP> ↔ E<SUB>1</SUB><UP>Ca<SUB>2</SUB></UP>

<UP><SC>Reaction</SC> 1</UP>
Therefore, acidic pH stabilizes the enzyme in the absence of Ca2+ (protonated E2 conformation) and alkaline pH favors Ca2+ binding (E1Ca2 conformation). The activating effect of Ca2+ on the pNPP hydrolysis rate in the purified enzyme is also pH-dependent and increases when the H+ concentration decreases (Fig. 8, A-C). This indicates that, under turnover conditions and acidic pH, the protonated E2 conformations will be more abundant, whereas the E1 species will predominate at alkaline pH. Qualitatively similar results were obtained when a preparation of intact vesicles and an oxalate-containing medium were used (data not shown).

The highest sensitivity to vanadate was observed during pNPP hydrolysis in the absence of Ca2+ (Figs. 4 and 5). Higher vanadate concentrations were necessary to produce complete inhibition when the H+ concentration was raised. This is to be expected since the target species for vanadate, the protonated E2 forms, will be more abundant at acidic than at alkaline pH. Data obtained with a preparation of purified enzyme (Fig. 4) indicated that pNPP hydrolysis in the absence of Ca2+ is associated with the catalytic activity of the Ca2+-ATPase protein and not with any other protein.

The effect of vanadate in the presence of Ca2+ was also pH-dependent (Figs. 4 and 5). The hydrolysis of pNPP in leaky vesicles and in the presence of Ca2+ at pH 6.0 was highly sensitive to vanadate. The inhibition profile was the same for leaky vesicles in a Ca2+-containing medium as for intact vesicles in a Ca2+-free medium (Fig. 5A). High sensitivity to vanadate is indicative of the predominant accumulation of E2 forms, especially when pNPP is the substrate. The non-nucleotide substrate does not prevent vanadate binding and inhibition, as occurs with mM ATP (33, 36). Moreover, the degree of activation by Ca2+ and inhibition by TG in a preparation of purified enzyme was low (Fig. 8A). TG selectively inhibits the Ca2+-dependent activity (17), and so high inhibition by TG indicates the predominant accumulation of E1 forms, whereas low TG inhibition is indicative of a low accumulation of E1 or what is the same, the predominant presence of E2 forms.

Enzyme activity in leaky vesicles and in the presence of Ca2+ at pH 8.0 was clearly resistant to vanadate, the inhibition profile being very similar to that displayed by intact vesicles in the presence of Ca2+ and oxalate (Fig. 5B). The Ca2+ activation and TG inhibition factors at pH 8.0 were high (Fig. 8C). These data suggest that E1 forms will be the predominant species.

Lumenal free Ca2+ has profound effects on coupling when the phosphorylating substrate is ATP (37, 38), but not when the phosphorylating substrate is pNPP. The reaction cycle at neutral pH and in the presence of a low lumenal Ca2+ concentration was already highly uncoupled (coupling ratio = 0.6), and the sensitivity to vanadate was relatively low. An increase in internal free Ca2+ produced complete uncoupling and slightly decreased the sensitivity to vanadate. Uncoupling through E1 forms induced by lumenal Ca2+ has already been characterized when the phosphorylating substrate is UTP (39).

The rate of pNPP hydrolysis had the same value in the presence or absence of Ca2+ when Me2SO is 40% (Fig. 3A). In other words, Ca2+ does not activate pNPP hydrolysis in a medium containing 40% Me2SO. These are conditions of complete uncoupling (Fig. 3B), and we confirmed that Me2SO does not alter membrane permeability (data not shown). This lack of Ca2+ activation is associated with a high sensitivity to vanadate, and was the same whether pNPP hydrolysis was measured in the presence or absence of Ca2+ (Fig. 7). In addition, pNPP hydrolysis in the presence of Ca2+ and using purified enzyme was insensitive to TG inhibition (Fig. 8D). These observations can be explained if Me2SO favors the reaction cycle in the absence of Ca2+ even when Ca2+ is present. It suggests that one sole catalytic route involving E2 forms can be forced when the Me2SO concentration is high enough.

The activation by Ca2+ and inhibition by TG in a preparation of purified enzyme had different absolute values, but, for each experimental condition tested, the activation and inhibition factors had the same value (Fig. 8). We know that TG produces stabilization of the E2 conformation (18). Thus, TG inhibits the Ca2+-dependent activity and allows the hydrolysis of pNPP in the presence of Ca2+ through E2 forms. This points to the existence of a relationship between the Ca2+-dependent and Ca2+-independent activities measured in SR vesicles and suggests that both hydrolytic activities are related to the Ca2+-ATPase protein.

It has been reported that SR Ca2+-ATPase is phosphorylated by and/or hydrolyzes ATP (40), UTP (39), or even the non-nucleotide substrate furylacryloylphosphate (41) in a Ca2+-free medium. In the case of pNPP, the rate of phosphorylation, which is much lower than that by ATP (25), does not allow the accumulation of phosphorylated intermediate during the enzyme turnover. The rates of Ca2+ binding in the presence of pNPP and phosphorylation by pNPP in the absence of Ca2+ must be of similar magnitude in contrast to those observed when ATP is the substrate. This explains why pNPP hydrolysis in a Ca2+-containing medium can partly occur through E2 forms.

In conclusion, the low efficiency of Ca2+ transport sustained by pNPP may be attributed to the coexistence of one coupled (E1-E2 cycle) and two uncoupled routes (E1 cycle and E2 cycle), as depicted in Scheme I. This means that: (i) alkaline pH favors the operation of the E1 cycle, whereas acidic pH potentiates the E2 cycle; (ii) uncoupling through the E1 cycle is favored by the integrity of the vesicles and occurs even when the free Ca2+ in the lumen is low; and (iii) pNPP can be hydrolyzed through the E2 cycle even in a Ca2+-containing medium. This study confirms that the catalytic cycle of P-type ATPases does not follow a rigid sequence of reactions and is consistent with the existence of one sole energy transduction mechanism.



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Scheme I.   Catalytic routes of SR Ca2+-ATPase in the presence of pNPP and Ca2+. The addition of pNPP to the enzyme in the presence of Ca2+ (E1Ca2) leads to the formation of the phosphorylated intermediate E1P·Ca2. When Ca2+ dissociation inside the vesicles takes place before Pi release into the external medium, there is an E1-E2 cycle (solid line). This route yields vectorial translocation of Ca2+ from the cytoplasmic to the lumenal compartment. When Pi release occurs first, Ca2+ dissociates to the external medium and there is an E1 cycle. E2 generated in a coupled cycle can interact with pNPP producing a phosphorylated intermediate without bound Ca2+ (E2P) and giving rise to the E2 cycle. Futile cycles are marked with a dashed line. E1Ca2 and E1P·Ca2 are unphosphorylated and phosphorylated conformations, respectively, of the enzyme with high affinity Ca2+ bound. E2 and E2P are unphosphorylated and phosphorylated conformations, respectively, with low Ca2+ affinity.



    FOOTNOTES

* This work was supported by Spanish Ministerio de Ciencia y Tecnologia Grant PB97-1039.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 To whom correspondence should be addressed: Dept. de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain. Fax: 34-968-364-147; E-mail: fbelda@um.es.

Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M008648200


    ABBREVIATIONS

The abbreviations used are: SR, sarcoplasmic reticulum; pNPP, p-nitrophenyl phosphate; TG, thapsigargin; Mes, 4-morpholineethanesulfonic acid; Mops, 4-morpholinepropanesulfonic acid; A23187, calcimycin; E1 and E2, enzyme conformations with high or low Ca2+ affinity, respectively.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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