On the Inhibition Mechanism of Sarcoplasmic or Endoplasmic Reticulum Ca2+-ATPases by Cyclopiazonic Acid*

(Received for publication, January 3, 1996, and in revised form, October 15, 1996)

Fernando Plenge-Tellechea Dagger , Fernando Soler and Francisco Fernandez-Belda §

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Ca2+-ATPase inhibition by stoichiometric and substoichiometric concentrations of cyclopiazonic acid was studied in sarcoplasmic reticulum preparations from rabbit fast-twitch muscle. The apparent affinity of the nonphosphorylated enzyme for ATP showed a Kd of ~3 µM in the absence of cyclopiazonic acid and ~28 µM in the presence of the drug. Fractional saturation of the enzyme by cyclopiazonic acid was accompanied by the appearance of two ATP-binding populations (enzyme with and without drug) and a progressive increase in the half-maximal concentration for saturating the ATP-binding sites. Enzyme turnover in the presence of stoichiometric concentrations of cyclopiazonic acid displayed lower apparent affinity for ATP and lower maximal hydrolytic activity than in the absence of the drug. When cyclopiazonic acid is in the substoichiometric range, the observed kinetic parameters will correspond to the simultaneous contribution of two different reaction cycles sustained by the enzyme with and without drug. The inhibition could be elicited by adding ATP to allow the enzyme turnover when cyclopiazonic acid was preincubated with the enzyme in the presence of Ca2+. The onset of inhibition during enzyme cycling was observed over a period of seconds, revealing the existence of a low inhibition rate constant. It is concluded that cyclopiazonic acid decreases enzyme affinity for ATP in non-turnover conditions by approximately one order of magnitude. This allows enzyme cycling after drug binding, provided that a high ATP concentration is used. Cyclopiazonic acid and ATP do not compete for the same binding site.


INTRODUCTION

Cyclopiazonic acid (CPA)1 is a mycotoxin produced by certain strains of Penicillium cyclopium and Aspergillus flavus. This indole tetramic acid with the molecular formula C20H20N2O3 was first isolated in 1968 (1). It may be found as a natural contaminant in some cereal products and mold-fermented cheese and meat (2), representing a potential risk to human and animal health. The clinical signs of CPA toxicity are usually related to muscle functionality, suggesting a direct effect of CPA on this tissue (3). By using isolated SR vesicles from skeletal muscle, it was proved that CPA is a potent inhibitor of both Ca2+-ATPase activity and ATP-dependent Ca2+ transport (4). The specificity of CPA for SR Ca2+-ATPase and not for other cation ATPases was also established (5). CPA, along with thapsigargin (TG) and 2,5-di(tert-butyl)-1,4-benzohydroquinone, are three structurally unrelated compounds that constitute a group of highly specific inhibitors of SERCA proteins (6).

Selective inhibition by CPA has been exploited in countless reports (for examples, see Refs. 7-12) to analyze Ca2+ signaling mechanisms. Indeed, a detailed characterization of the CPA action is a critical aspect in the manipulation of intracellular Ca2+ stores and may be helpful in providing information on the Ca2+-ATPase energy transduction mechanism. The literature available does not provide a satisfactory description of the CPA effect. For instance, an initial report indicated that the Ca2+-ATPase inhibition was essentially complete at stoichiometric amounts of CPA; however, the type of inhibition was studied in the range of 10-100 nmol CPA/mg of protein (5).

The present study was devoted to clarify the effect of CPA when the drug was used in the stoichiometric range with respect to the enzyme concentration. To this end, a series of measurements on enzyme turnover were performed to elucidate the functional characteristics of the CPA interaction. Ligand binding at equilibrium by using radioactive ATP and TNP-ATP fluorescence allowed us to decipher the relationship between the ATP- and the CPA-binding sites. Measurements of radioactive EP, including different conformational states of the protein, preincubation conditions, and reaction schedules shed light on the enzyme behavior with respect to protection, sensitivity, and onset of inhibition by CPA. All of these data are compiled in a functional model and are discussed with respect to the well characterized inhibitor TG.


EXPERIMENTAL PROCEDURES

Materials

[gamma -32P]ATP was obtained from Amersham Corp. and used at a specific activity of approximately 20,000 cpm/nmol. [3H]Glucose and 45CaCl2 were products of DuPont NEN. They were used at ~3,000 and ~15,000 cpm/nmol, respectively. The Ca2+ ionophore A23187 was purchased from Boehringer Mannheim. TNP-ATP was from Molecular Probes Europe, The Netherlands. The liquid scintillation mixture was Sigma-Fluor (S-4023) from Sigma. A stock solution of cyclopiazonic acid from Penicillium cyclopium (Sigma) was prepared in ethanol. The volume of ethanol added did not exceed 1% of the total volume. CPA was always added to the incubation/reaction media after the SR vesicles, which were then preincubated for at least 5 min before the reactions were started.

Membrane Preparation and Quantitation

Sarcoplasmic reticulum vesicles from fast-twitch muscle of rabbit hind leg were prepared according to Eletr and Inesi (13) and stored in frozen aliquots until use. The membrane protein concentration was determined by the colorimetric procedure of Lowry et al. (14) using bovine serum albumin as the standard.

Free Ca2+ Concentration

The concentration of free Ca2+ in the EGTA-containing solutions was calculated by the computer program of Fabiato (15) using the binding constant of the Ca2+-EGTA complex (16) and binding constants for EGTA protonation (17).

Ca2+-dependent ATPase Activity

The steady-state rate of enzyme activity in leaky vesicles was measured at 25 °C. The assay medium was buffered at pH 7.0 and contained Mg2+, K+, Ca2+ ionophore, an ATP-regenerating system, and 10 µM free Ca2+. CPA was also present when indicated. Complete descriptions of the experimental media are given in the corresponding figure captions. The appearance of inorganic phosphate during the first minutes of the reaction was evaluated with molybdovanadate reagent (18). Ca2+-ATPase activity was corrected for Mg2+-dependent ATPase activity measured in the presence of 1 mM EGTA with no added Ca2+.

ATP Binding to the Enzyme

Nucleotide binding in the absence of Ca2+ was measured by the double labeling radioactive technique (19). SR vesicles (0.2 mg protein/ml) were suspended at 22 °C in the incubation medium consisting of 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, and specified CPA concentrations. Aliquots of 1 ml (0.2 mg protein) were layered onto 0.45 µm Millipore filters (HAWP) under vacuum and then rinsed with 1 ml of incubation medium supplemented with 1-50 µM [gamma -32P]ATP and 1 mM [3H]glucose. The ATP bound to the protein was estimated from the radioactive counting of the filters after correction for unspecific nucleotide absorption (3H labeling).

Titration of the TNP-ATP Binding Site

The fluorescence signal of TNP-ATP was measured with an optical system from Bio-Logic Co. (Claix, France) by using a 150-watt mercury-xenon lamp. The experimental protocol was essentially as described previously (20). The excitation wavelength was 410 nm, and the emission light was selected with a 515-nm cutoff filter (Ealing Electro-Optics, Holliston, MA). Samples were maintained at 22 °C under continuous stirring. The incubation medium contained 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 0.2 mM EGTA, 0.1 mg/ml SR protein, and 2 µM TNP-ATP in the absence or presence of CPA. The decrease in fluorescence was elicited by successive additions of ATP.

Enzyme Phosphorylation by ATP

The phosphorylated intermediate of the enzyme was studied by using [gamma -32P]ATP as a substrate. Unless otherwise stated, all the reactants were preincubated in an ice-water bath before the reactions were initiated. The phosphorylation reaction was started by manual mixing under vortexing of the enzyme suspension with the radioactive ATP and stopped by the addition of 1 volume of ice-cold perchloric acid (0.25 M) plus sodium phosphate (2 mM). The quenched samples were filtered under vacuum using 0.45-µm pore size Millipore filters (type HAWP), and the filters were then washed five times with 5 ml of 0.125 M perchloric acid and 1 mM sodium phosphate. The filters were subjected to radioactive counting after solubilization in 3 ml of liquid scintillation mixture. Different enzymatic forms, reaction media, and experimental schedules were applied to study the effect of CPA as follows:

(i) Maximal levels of EP initiated from E1Ca2. The microsomal vesicles (0.2 mg protein/ml) were suspended in the presence of 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2 (10 µM free Ca2+), and 15 µM A23187. This reaction mixture was preincubated with a defined CPA concentration. The phosphorylation reaction was started by mixing 0.5 ml of this medium with 20 µl of medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, and 1.25 mM [gamma -32P]ATP (50 µM final concentration). The reaction was quenched after 5-s by an equal volume (0.5 ml) of quenching solution.

(ii) Maximal levels of EP initiated from E2. SR vesicles (0.2 mg/ml) were incubated with 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, and 15 µM A23187. Following an additional incubation with a defined CPA concentration, 0.5-ml aliquots were mixed with the phosphorylation medium (20 µl) containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 24.2 mM CaCl2, and 1.25 mM [gamma -32P]ATP. The final ATP and Ca2+ concentrations were 50 µM and 0.967 mM, respectively. The reaction was stopped by the addition of acid after 5 s.

(iii) EP evolution initiated from E1Ca2. The enzyme suspension containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, 0.2 mg protein/ml, and 15 µM A23187 was preincubated with 0.8 µM CPA. CPA was omitted from the control assay. The phosphorylation medium contained 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, and 10 mM [gamma -32P]ATP. The reaction was started by mixing 0.5 ml of enzyme suspension with 20 µl of radioactive ATP medium (400 µM [gamma -32P]ATP after mixing) at 22 °C and arrested in the second time scale by adding 0.5 ml of acid.

(iv) Chase of maximal EP initiated from E1Ca2. Pretreatment of SR vesicles (0.2 mg/ml) in a medium of 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, 15 µM A23187, 2 mM phosphoenolpyruvate, and 10 units/ml pyruvate kinase with a certain CPA concentration was carried out at 22 °C. The reaction was studied at the same temperature by an initial addition of 50 µM nonradioactive ATP. After 3 min, the reaction mixture was supplemented with ~500,000 cpm of [gamma -32P]ATP to give approximately 20,000 cpm/nmol after mixing. Samples were denatured 2 s later by the addition of the perchloric acid/phosphate solution (0.5 ml).

(v) Maximal levels of EP initiated from E2ATP. The initial incubation medium consisted of 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 20 or 200 µM [gamma -32P]ATP, 0.2 mg/ml SR protein, and 15 µM A23187. CPA was then added to the incubation medium. The reaction was started by mixing 0.5 ml of enzyme suspension with 20 µl of medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, and 24.2 mM CaCl2. The phosphorylation time was 2 s, and the reaction was terminated by acid quenching.

Data Presentation

The experimental values represent the average of at least three independent experiments performed in duplicate. The standard deviations of the mean values (plus or minus) are given when indicated.


RESULTS

CPA, Binding Experiments, and Enzyme Turnover

The inhibition of SERCA activity is critically dependent on enzyme, ATP, and CPA concentrations (5); therefore, we initially studied the CPA/enzyme stoichiometry under our experimental conditions. The steady-state rate of enzyme activity as a function of SR protein concentration was measured at 25 °C in leaky vesicles in the presence of an ATP-regenerating system and a fixed ATP concentration of 50 µM. As can be seen in Fig. 1, the rate of ATP hydrolysis was linearly dependent on the membrane protein concentration (open circle ). When the experiments were repeated in the presence of CPA, the linear dependence of the enzyme activity was delayed. This effect was more evident for a CPA concentration of 0.25 µM (bullet ) than for a concentration of 0.1 µM (black-triangle), which is clear proof of high affinity inhibition. An abscissa intersection value of 0.025 mg/ml was obtained in the experiments with 0.1 µM CPA and 0.055 mg/ml in the presence of 0.25 µM CPA. The asymptote intersection with the abscissa axis provides information on the drug/enzyme stoichiometry. Thus, a drug:enzyme molar ratio of approximately 1:1 can be deduced, assuming from the maximal EP level that 1 mg of SR protein contains ~4 nmol of Ca2+-ATPase active sites.


Fig. 1. Effect of CPA on Ca2+-ATPase activity as a function of membrane protein concentration. The assay medium at 25 °C contained 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, 2 mM phosphoenolpyruvate, 6 units/ml pyruvate kinase, 4% (w/w, with respect to the membrane protein) A23187, and different SR protein concentrations in the absence (open circle ) or presence of 0.1 µM (black-triangle) or 0.25 µM CPA (bullet ). The reaction was started by adding 50 µM ATP. The initial rate of ATP hydrolysis at each membrane protein concentration was evaluated in 1-ml aliquots of reaction mixture. Bars, S.D.
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It was published previously that ATP counteracts the inhibitory effect of CPA (5). Therefore, we studied whether CPA perturbed the interaction of ATP with the enzyme by measuring the equilibrium binding of ATP to the nonphosphorylated enzyme (Fig. 2A). The assay was carried out in the presence of EGTA to prevent any ATP hydrolysis. Protein concentration was 0.2 mg/ml, and radioactive ATP was used as a tracer. The ATP-binding isotherm in the absence of CPA (open circle ) indicates a single population of ATP-binding sites with a maximal binding capacity of ~4 nmol/mg protein and an apparent Kd of ~3 µM, which is in agreement with previous reports (20, 21). When the experiments were performed in the presence of 0.3 or 0.8 µM CPA, there was a progressive increase in the half-maximal ATP concentration necessary for saturating the high affinity binding sites. It was not possible to measure the saturation level in the presence of CPA due to unspecific low affinity ATP binding (21). Nevertheless, the high affinity ATP sites can be characterized by a Scatchard plot (Fig. 2B). The titration curve in the presence of 0.3 µM CPA displayed a biphasic pattern (Fig. 2B, inset), confirming the existence of two different ATP-binding populations (enzyme with and without CPA) at substoichiometric drug concentrations. The apparent Kd for ATP in the presence of CPA (the lower affinity component) can be more easily evaluated when the enzyme is saturated by the drug (i.e. with 0.8 µM CPA, equivalent to 1 mol of CPA/mol of enzyme). Under these conditions, we found a single population of ATP sites with maximal binding of 4 nmol/mg of protein and an apparent Kd of ~ 28 µM (Fig. 2B, main panel).


Fig. 2. Binding at equilibrium of ATP to the Ca2+-deprived enzyme in the presence of CPA. The incubation medium at 22 °C consisted of 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, and 0.2 mg/ml SR protein either in the absence (open circle ) or presence of 0.3 (black-triangle) or 0.8 µM (bullet ) CPA. Aliquots of 0.2 mg of protein loaded onto filters were perfused with 1 ml of incubation medium containing 1 mM [3H]glucose and various [gamma -32P]ATP concentrations. The specific high affinity binding of ATP was evaluated from double radioactivity counting. A, the binding data in a direct plot. Bars, S.D. B, Scatchard plots corresponding to experiments in the presence of 0.3 µM CPA (black-triangle), a substoichiometric drug concentration or 0.8 µM CPA (bullet ), a stoichiometric concentration.
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To complement the preceding data and also to test CPA concentrations above the stoichiometric level, we took advantage of the fluorescent probe TNP-ATP (22, 23). The binding of TNP-ATP to the enzyme in the absence of Ca2+ is accompanied by a specific fluorescence increase. An indirect titration of the catalytic site can be obtained by the subsequent sequential addition of ATP because the change in relative fluorescence can be correlated with nanomoles of ATP bound to the enzyme (20, 24). The results (Fig. 3) confirmed that CPA increases the half-maximal concentration necessary for saturating the ATP-binding sites and also that maximal ATP binding can reach the level observed in the absence of drug. Moreover, CPA lowered the Hill coefficient of the ATP-binding curve, when the CPA:enzyme molar ratio was <1. This is illustrated by comparing the fluorescence isotherm in the absence of CPA (open circle ) and that obtained at 0.25 µM CPA (black-square). The selected CPA concentration represents approximately 60% enzyme saturation by the drug because the SR protein concentration was 0.1 mg/ml. We also extended our measurements to 0.4 (bullet ) and 1.2 µM CPA (black-triangle), representing 1 or 3 mol of CPA/mol of enzyme, respectively. It is noteworthy that the half-maximal saturation in the fluorescence isotherms was independent of CPA when the CPA/enzyme was >= 1. Likewise, the ATP binding cooperativity reverted to a Hill coefficient of approximately 1 when the enzyme was saturated by CPA.


Fig. 3. Characterization of the catalytic site using the TNP-ATP fluorescence. SR vesicles (0.1 mg/ml) were suspended at 22 °C in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 0.2 mM EGTA, and 2 µM TNP-ATP. CPA was either absent (open circle ) or present at a concentration of 0.25 (black-square), 0.4 (bullet ), or 1.2 µM (black-triangle). The decrease of the TNP-ATP fluorescence was obtained by sequential additions of ATP. Samples under stirring were excited at 410 nm, and the emission was selected with a cutoff filter (OG 515). Bars, S.D.
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We also studied the Ca2+-ATPase activity as a function of the ATP concentration after incubation of the nonphosphorylated enzyme with CPA. The drug concentration was selected to give the same CPA:enzyme molar ratios used in the ATP-binding experiments of (Fig. 2). The reaction medium contained a protein concentration of 0.02 mg/ml and included Ca2+ ionophore and an ATP-regenerating system to permit steady-state conditions at ATP concentrations as low as 1 µM. The results of (Fig. 4A) indicate that samples incubated in the absence of CPA (open circle ) exhibit the expected increase of enzyme activity with increasing concentrations of ATP. The presence of 0.03 µM (black-triangle) or 0.08 µM CPA (bullet ) produced a progressive decrease in the apparent enzyme affinity for ATP and maximal hydrolytic activity. Note that in the presence of 0.08 µM CPA, a stoichiometric concentration of the drug, the enzyme may express hydrolytic activity, provided that the ATP concentration is high enough. It is relevant that CPA concentrations above stoichiometric levels led to further decreases in enzyme activity (data not shown). Enzyme activity data obtained at the higher ATP concentrations were also analyzed in a double reciprocal plot (Fig. 4B). The absence of a common intersection point on the ordinate axis confirms that the Vmax value is dependent on the CPA concentration.


Fig. 4. ATP dependence on enzyme activity rate measured in the presence of CPA. 0.02 mg/ml of SR protein was preincubated at 25 °C in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, 2 mM phosphoenolpyruvate, 6 units/ml pyruvate kinase, and 1.5 µM A23187. Different ATP concentrations were used to initiate the reaction. These experiments were performed in the absence (open circle ) or presence of 0.03 µM (black-triangle) or 0.08 µM CPA (bullet ). A, a direct plot. Bars, S.D. B, a double reciprocal plot of the data points collected at high ATP concentrations.
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CPA and Reaction Cycle

It was shown previously by using the extrinsic fluorescence of fluorescein-5'-isothiocyanate that CPA stabilizes the E2 form of the enzyme (5, 25). This observation on enzyme conformational states can be more deeply analyzed by considering the basic molecular transitions involved in the Ca2+-ATPase reaction cycle (see Scheme I). Thus, the reactivity of the E1 form toward ATP can be monitored by following the phosphorylation partial reaction (step 2). For this purpose, 0.2 mg/ml SR vesicles preequilibrated with saturating Ca2+ (E1Ca2) were supplemented with CPA up to 1 µM, and the accumulated EP was studied at 0 °C by measuring the 32P covalently bound to the protein after addition of [gamma -32P]ATP. The EP level reached the same maximal levels as those obtained in the absence of drug (Fig. 5). Alternatively, SR vesicles preincubated in the presence of excess EGTA (enzyme deprived of Ca2+ or E2) were incubated with CPA before the addition of [gamma -32P]ATP and Ca2+. The phosphorylation reaction was also performed at 0 °C and maintained for 5 s as before. Using this protocol, we can study the sequence of steps 1 and 2. In this case, the presence of increasing CPA concentrations produced a progressive inhibition of the radioactive EP level (Fig. 6).


Scheme I. Minimal species in the SERCA reaction cycle. Partial reactions are indicated by numbers in parentheses.
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Fig. 5. CPA effect on the phosphorylation of E1Ca2. The phosphorylation reaction was performed at 0 °C for 5 s in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, 15 µM A23187, 0.2 mg/ml SR protein, 50 µM [gamma -32P]ATP, and different CPA concentrations. See the schedule of additions in the figure.
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Fig. 6. Effect of CPA on the phosphorylation of E2. The final reaction medium consisted of 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, 15 µM A23187, 0.2 mg/ml SR protein, different CPA concentrations, and 50 µM [gamma -32P]ATP. The reaction was maintained at 0 °C for 5 s. See sequence of additions in the figure.
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The time course of the accumulated EP was also considered in the next assay. The experiment was performed at 22 °C and involved the addition of CPA to the enzyme in the presence of Ca2+, before mixing with radioactive ATP. The reaction was stopped at various time intervals by the addition of acid. The ATP concentration was relatively high (400 µM) to permit observation of the EP time course on the scale of minutes. This also permitted detection of enzyme turnover in the presence of 0.8 µM CPA (a stoichiometric concentration). As illustrated by (Fig. 7), CPA inhibition can be elicited by allowing enzyme turnover. The EP hydrolytic cleavage was monophasic until complete exhaustion of the substrate in the absence of CPA. However, when CPA was present, there was a clear biphasic response. Enzyme inhibition manifested as an initial transient decay of EP followed by a lasting steady-state level. As a result of the inhibition, the ATP was not completely consumed in the same time span as in the control experiment. The different temperature used may also explain why the EP level measured at the shorter phosphorylation times was lower than the control value obtained in Fig. 6. We then studied inhibition of the cycling enzyme as a function of different CPA concentrations. The reaction medium was modified to maintain enzyme turnover in the minute time scale without the necessity of adding high concentrations of radioactive ATP. CPA was added to the enzyme in the presence of Ca2+, as before, but the phosphorylating substrate was now 50 µM nonradioactive ATP. The reaction mixture also contained an ATP-regenerating system to ensure enzyme turnover during the assay. After a phosphorylation period of 3 min at 22 °C, EP labeling was detected by adding a pulse of [gamma -32P]ATP followed by acid quenching at 2 s. Fig. 8 shows that the accumulated EP under steady-state conditions decreased as a function of the CPA concentration. The degree of inhibition at 0.8 µM CPA was higher than in the experiment of Fig. 7 because of the presence of a lower ATP concentration.


Fig. 7. Time course of the EP level in the presence of CPA. The final reaction medium contained 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, 15 µM A23187, 0.2 mg/ml SR protein, 0.8 µM CPA, and 400 µM [gamma -32P]ATP. The reaction was performed at 22 °C and stopped by acid at different time intervals. The starting species was E1Ca2, and the sequence of additions was as indicated. Samples in the absence (open circle ) or presence (bullet ) of CPA are shown.
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Fig. 8. Accumulation of EP under turnover conditions as a function of the CPA concentration. The phosphorylation medium contained 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.967 mM CaCl2, 15 µM A23187, 2 mM phosphoenolpyruvate, 10 units/ml pyruvate kinase, 0.2 mg/ml SR protein, different CPA concentrations, and 50 µM ATP. After 3 min at 22 °C, a pulse of [gamma -32P]ATP (to give ~20,000 cpm/nmol after mixing) was added, and the reaction was prolonged for an additional 2 s.
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The last set of experiments was directed toward establishing whether ATP had any protective effect when CPA was added to the enzyme in the E2 form. In the experiment outlined in Fig. 9, we exposed SR vesicles in the absence of Ca2+ to 20 or 200 µM [gamma -32P]ATP before the addition of different CPA concentrations in the stoichiometric range. Then, Ca2+ was added to initiate enzyme phosphorylation. The reaction was carried out at the ice-water temperature for 2 s. The observed inhibition resembles that obtained when CPA was directly added to the enzyme in the absence of ATP and Ca2+ (compare Fig. 6). EP decreased progressively as the CPA concentration rose. However, for a given CPA concentration, the EP level was higher in the experiment performed in the presence of the higher ATP concentration.


Fig. 9. Effect of CPA on the phosphorylation of E2ATP. SR vesicles (0.2 mg/ml) were suspended at 0 °C in a medium containing 20 mM Mops, pH 7.0, 80 mM KCl, 5 mM MgCl2, 1 mM EGTA, 15 µM A23187, a certain CPA concentration, and 20 (open circle ) or 200 µM (bullet ) [gamma -32P]ATP. The phosphorylation reaction was started by adding a small volume of a Ca2+ medium (final concentration, 0.967 mM) and was stopped 2 s later according to the sequence of additions.
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DISCUSSION

The experiments reported above are helpful for understanding the functional properties of CPA as inhibitor of the SERCA family of proteins. It should be stressed that the present study has been restricted to stoichiometric and substoichiometric CPA concentrations. Because different CPA:enzyme molar ratios have been reported (5, 25) as producing complete inhibition of the Ca2+-ATPase activity, our first concern was to re-examine this parameter under well defined assay conditions. A plot of enzyme activity versus membrane protein concentration (Fig. 1), or the inhibitory effect of CPA on EP formation (Fig. 6), indicated a 1:1 stoichiometry with respect to active sites on the enzyme. Therefore, the number of CPA-binding sites is equivalent to that of the high affinity ATP-binding sites. The stoichiometric value is somewhat lower than that originally reported (6-8 nmol CPA/mg of protein) at low ATP concentrations (5). Small changes in the experimental conditions, including the purity of the membrane protein preparation or the methods used for the binding site estimation, may account for the observed difference.

When the enzyme is completely saturated by CPA, the Scatchard plot (Fig. 2B) indicates the existence of a single ATP-binding population. This is consistent with the binding cooperativity data (Fig. 3) showing a Hill coefficient of approximately 1. However, when the enzyme is partially saturated by CPA, there are two nucleotide binding populations (Fig. 2B, inset) and negative cooperativity (Fig. 3). The apparent Kd for ATP in the presence of CPA can be graphically estimated from a Scatchard plot, and the evaluation is easier when the enzyme is completely saturated by CPA (Fig. 2B, compare data points in main panel and low affinity component in the inset). Furthermore, the half-maximal ATP concentration necessary for saturating the high affinity sites will only give information on the apparent Kd when CPA:enzyme is >= 1. In any case, CPA reduced the apparent Kd for ATP by approximately one order of magnitude. This may explain why other phosphorylating substrates such as acetylphosphate, which has lower affinity for the enzyme, cannot be hydrolyzed after incubation with CPA (5). Interestingly, the effect of TG as a SERCA inhibitor is similar because TG also decreases the binding affinity of ATP (20). This effect may explain the observed ATP binding inhibition induced by TG (26). A decrease in the binding affinity at the phosphorylation site promoted by TG would also account for the loss in the phosphorylating capacity of Pi (27, 28).

Earlier studies have claimed a competitive relationship between CPA and ATP (5, 25). However, our ATP-binding experiments indicate that the observed decrease in the Kd for ATP is independent of CPA. Certainly, this observation cannot be reconciled with a direct competition between CPA and ATP. Furthermore, CPA does not modify the TNP-ATP fluorescence signal, suggesting that the ligands do not interact at the same binding site. This property is shared by TG. Studies on TNP-ATP fluorescence suggested that the interaction of TG with the protein was not at the ATP-binding site (20).

The reversal of CPA inhibition by increasing the ATP concentration has been interpreted as a protective effect of ATP and attributed to a competitive effect (5). From the present data (Figs. 2, 3, 4), it is apparent that CPA simply decreases the Kd for ATP so that the enzyme will express higher hydrolytic activity if ATP concentration is increased. A similar effect of ATP was also observed in the EP measurements of Fig. 9. Before it can be assured that CPA is a competitive inhibitor, it must be demonstrated that the Ca2+-ATPase activity in the presence of CPA can reach the maximal velocity measured in the absence of drug. A direct plot of enzyme activity versus ATP concentration (Fig. 4A) indicates that this is not the case. Double reciprocal plots of data conforming a high affinity inhibition mechanism give concave-down curves that are difficult to analyze (29). However, by plotting enzyme activity data obtained at the higher ATP concentrations, we were able to analyze the region near the 1/v axis in more detail. Fig. 4B confirms that CPA does not behave as a competitive inhibitor. The conclusion that CPA is a competitive inhibitor (5) was based on the analysis of nonlinear double reciprocal plots showing a poor resolution in the region near the ordinate axis. Furthermore, the experiments were performed at CPA concentrations (10-100 nmol/mg of protein), much higher than the stoichiometric level. In another study (25), a Dixon plot was the only experimental evidence offered to support the competitive character of CPA. This plot provides information on the Ki value but cannot be used to diagnose the type of inhibition.

The Ca2+-ATPase activity isotherms shown in Fig. 4A indicate that the enzyme may undergo turnover in the presence of stoichiometric CPA at high ATP concentrations. However, enzyme turnover displayed lower ATP affinity (K'ATP) and lower maximal velocity (V'max) than in the absence of CPA. Consequently, when the CPA concentration is in the substoichiometric level, the observed kinetic parameters will correspond to the simultaneous contribution of two different reaction cycles sustained by the enzyme without CPA (KATP and Vmax) and the enzyme with CPA (K'ATP and V'max). The inhibition of the SERCA activity by CPA can be illustrated by Scheme II. In this model, CPA does not interact with the E1 form, and the binding of CPA to E2 does not take place at the catalytic site. When CPA was in excess with respect to the enzyme protein concentration, there was a higher degree of inhibition. This suggests that the CPA effect is not restricted to the ATP-binding process, although this was not investigated further.


Scheme II. Inhibition model of SERCA proteins by CPA in the stoichiometric range.
[View Larger Version of this Image (8K GIF file)]


The phosphorylation of E1Ca2 by ATP was not altered by CPA (Fig. 5); hence, the catalytic site is probably not the drug target. This suggests that E1 is refractory to CPA. However, when the reaction cycle was started from E2, the addition of CPA inhibited EP formation (Fig. 6), suggesting a selective effect of CPA on the E2 form. This is in agreement with previous fluorescence measurements of the protein conformational states (5). The protective effect of Ca2+ on E1 can be eliminated by adding ATP. This is an indication that the enzyme turnover generates an intermediate species that is susceptible to CPA. No inhibition was observed in the presence of 50 µM ATP because EP decay took place over a short time span (data not shown). However, the inhibition becomes clear when the EP evolution was studied in the presence of a relatively high ATP concentration (400 µM) to allow enzyme turnover on a time scale of minutes (Fig. 7). The data of Figs. 7 and 8 reveal that the onset of inhibition during the enzyme turnover is a rather slow process. This implies that a high number of reaction cycles is required for enzyme inhibition to be observed. It can be postulated that after the initial binding of CPA to the enzyme, there is a slow isomerization step leading to an E2 state with a lower ATP binding affinity. The data for TG indicated that enzyme inhibition is also associated with the E2 conformation (28). TG inhibition by adding ATP also developed over seconds (30), although it was faster than in the case of CPA inhibition.

An important difference between CPA and TG is that the latter causes a drastic decrease in the apparent Kd for both ATP and Ca2+, producing for practical purposes a dead-end complex (26-28). The present data indicate that CPA diminishes ATP binding affinity but to a lesser extent than TG. Therefore, the enzyme with CPA bound can be forced to undergo turnover by increasing ATP concentration.

Location of the CPA-binding site will throw light on the role played by the region of the SERCA proteins that is recognized by the drug. This may be achieved from the construction of chimeric proteins as was done for the location of the TG target site (31).


FOOTNOTES

*   This work was supported by Grant PB94-1164 (to F. F. B.) from Direccion General de Investigacion Cientifica y Tecnica, Spain. 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    Recipient of a fellowship from Consejo Nacional de Ciencia y Tecnologia of Mexico. On leave of absence from Universidad Autonoma de Baja California, Ensenada, Baja California, Mexico.
§   To whom correspondence should be addressed. Tel.: 34 68 307100, ext. 2920; Fax: 34 68 364147.
1    The abbreviations used are: CPA, cyclopiazonic acid; SR, sarcoplasmic reticulum; TG, thapsigargin; SERCA, sarco- or endoplasmic reticulum Ca2+-ATPases; TNP-ATP, 2' (or 3')-O-(trinitrophenyl)adenosine-5'-triphosphate; EP, phosphorylated enzyme; A23187, calcimycin; Mops, 4-morpholinepropanesulfonic acid; E1 and E2, conformations of the nonphosphorylated enzyme either in the presence or absence of Ca2+, respectively.

Acknowledgments

We are grateful to Dr. Garcia-Carmona from our Department for helpful suggestions. We also thank the anonymous referee for very positive criticism.


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