Báo cáo Y học: Inhibition of the SERCA Ca21 pumps by curcumin Curcumin putatively stabilizes the interaction between the nucleotide-binding and phosphorylation domains in the absence of ATP pot

Inhibition of the SERCA Ca 21 pumps by curcumin Curcumin putatively stabilizes the interaction between the nucleotide-binding and phosphorylation domains in the absence of ATP Jonathan G. Bilmen 1 , Shahla Zafar Khan 1 , Masood-ul-Hassan Javed 2 and Francesco Michelangeli 1 1 School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK; 2 Shifa College of Medicine, Islamabad, Pakistan. Curcumin is a compound derived from the spice, tumeric. It is a potent inhibitor of the SERCA Ca 2þ pumps (all isoforms), inhibiting Ca 2þ -dependent ATPase activity with IC 50 values of between 7 and 15 mM. It also inhibits ATP- dependent Ca 2þ -uptake in a variety of microsomal membranes, although for cerebellar and platelet micro- somes, a stimulation in Ca 2þ uptake is observed at low curcumin concentrations (, 10 m M). For the skeletal muscle isoform of the Ca 2þ pump (SERCA1), the inhibition of curcumin is noncompetitive with respect to Ca 2þ , and competitive with respect to ATP at high curcumin concentrations (< 10–25 m M). This was confirmed by ATP binding studies that showed inhibition in the presence of curcumin: ATP-dependent phosphorylation was also reduced. Experiments with fluorescein 5 0 -isothiocyanate (FITC)-labelled ATPase also suggest that curcumin stabilizes the E1 conformational state. The fact that FITC labels the nucleotide binding site of the ATPase (precluding ATP from binding), and the fact that curcumin affects FITC fluorescence indicate that curcumin must be binding to another site within the ATPase that induces a conformational change to prevent ATP from binding. This observation is interpreted, with the aid of recent structural information, as curcumin stabilizing the interaction between the nucleotide- binding and phosphorylation domains, precluding ATP binding. Keywords: SERCA; ATP binding; curcumin; phosphoryl- ation; fluorescence. Tumeric is extensively used as a spice in Asian cooking and as a colouring agent in both the food and cosmetic industries [1]. Curcumin (diferuoylmethane or 1,7-bis(4-hydroxy- 3-methoxyphenol)-1,6-heptadiene-3,5-dione) is a compound found in tumeric that gives it its distinctive yellow colour [2]. Recently it has been shown that curcumin has anti- carcinogenic effects [3] that may be linked to its antioxidant properties [4]. Studies have shown that curcumin can affect a number of cellular processes including: activation of apoptosis in Jurkat T-cells [5], inhibition of platelet aggregation [6,7] and inhibition of inflammatory cytokine production in macrophages [8]. Curcumin has also been shown to affect the activity of a number of key enzymes such as cyclooxygenase [9], protein kinase C [10], protein tyrosine kinases [11] and a Ca 2þ -dependent endonuclease [12]. Many of these processes/enzymes are also known to be regulated by Ca 2þ . Cytosolic free Ca 2þ concentration ([Ca 2þ ] cyt ) is tightly controlled, due its importance in the regulation of many cellular processes. The sarco/endoplasmic reticulum Ca 2þ ATPase (SERCA) is one of the major mechanisms by which the low levels of [Ca 2þ ] cyt are maintained within cells. Three isoforms of the SERCA family of Ca 2þ pumps have so far been identified [13,14] and these are expressed in a tissue- specific manner [14]. SERCA1 is found predominantly in fast-twitch skeletal muscle while SERCA2a is found within cardiac and slow-twitch muscle. The splice variant form of SERCA2 (SERCA2b), which has an extended C-terminus is found in most nonmuscle cells and is particularly abundant in neuronal tissues. SERCA3 is less widely distributed in nonmuscle tissues but is relatively abundant in macro- phages, platelets and large intestines. The crystal structure of the Ca 2þ bound form of the SR Ca 2þ -ATPase (SERCA1) was recently resolved and shown to contain three domains within the cytoplasmic head region [15]. These are: the nucleotide binding domain, which binds ATP; the phosphorylation domain, which can be phos- phorylated on Asp351; and the actuator, which may be involved in anchoring the other two domains together during phosphoryl transfer [15]. These domains are attached to the membrane by 10 transmembrane helices, containing the two Ca 2þ binding sites that sit side-by-side [15,16]. Using inhibitors to study the ATPase has proved invaluable in helping to elucidate mechanistic steps within the Ca 2þ transport process [16 –18]. These steps and their associated conformational changes now need to be placed in context with changes within the tertiary structure of the Ca 2þ -ATPase. In this study, we show that curcumin is a potent inhibitor of SERCA Ca 2þ pumps that affects a number of steps within its mechanism. We try to rationalize these effects in terms of domain interactions of the known structure. Correspondence to F. Michelangeli, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK. Fax: þ 44 121 414 5925, Tel.: þ 44 121 414 5398, E-mail: F.Michelangeli@bham.ac.uk (Received 11 July 2001, revised 5 October 2001, accepted 11 October 2001) Abbreviations: FITC, fluorescein 5 0 -isothiocyanate; SR, sarcoplasmic reticulum; SERCA, sarco/endoplasmic reticulum Ca 2þ ATPase. Eur. J. Biochem. 268, 6318–6327 (2001) q FEBS 2001 MATERIALS AND METHODS Curcumin was purchased from Sigma. [g 32 P]ATP was obtained from Amersham. Magnesium green was purchased from Molecular Probes. All other reagents were of analytical grade. Membrane and protein preparation Sarcoplasmic reticulum (SR) and the purified Ca 2þ ATPase were prepared from rabbit skeletal muscle as described by Michelangeli & Munkonge [19]. Porcine cerebellar and cardiac microsomes were prepared as described by Sayers et al. [20]. Human platelet microsomes were prepared based on the method as described by Le Peuch et al. [21] and resuspended in a buffer containing 5 m M Hepes, 0.32 M sucrose, 0.1 mM benzamidine, 0.1 mM phenylmethanesul- fonyl fluoride and 10 m M leupeptin. Curcumin was dissolved in ethanol to give a stock solution of 10 m M. Ca 21 -ATPase activity Ca 2þ -ATPase activity determination in microsomes was performed using the phosphate liberation assay as described by Longland et al. [22]. Briefly, microsomal extracts were resuspended in 1 mL of buffer containing 45 m M Hepes/ KOH (pH 7.0), 6 m M MgCl 2 ,2mM NaN 3 , 0.25 M sucrose, 12.5 mg·mL 21 A23187 ionophore, and EGTA with CaCl 2 added to give a free [Ca 2þ ]of1mM. Assays were pre- incubated at 37 8C for 10 min prior to activation with ATP (final concentration 6 m M). The reaction was stopped by addition of 0.25 mL 6.5% (w/v) trichloroacetic acid. The assays were put on ice for 10 min prior to centrifugation for 10 min at 20 000 g: 0.5 mL of the supernatent was added to 1.5 mL buffer containing 11.25% (v/v) acetic acid, 0.25% (w/v) copper sulfate, and 0.2 M sodium acetate. Two- hundred and fifty microliters of 5% (w/v) ammonium molybdate was then added and mixed thoroughly and 0.25 mL of ELAN solution was added [2% (w/v) p-methylaminophenol sulfate and 5% (w/v) sodium sulfite]. The colour intensity was measured after 10 min at 870 nm absorbance and related to a calibration curve of colour intensity vs. known amounts of phosphate, previously treated as above. Controls were performed in the presence of ethanol, which at maximal curcumin concentrations was equal to 0.5% (v/v) and had no effect on the ATPase activity. In experiments where the effects of curcumin were investigated on Ca 2þ ATPase activity as a function of [Ca 2þ ] and [ATP], these were carried out on purified SR Ca 2þ -ATPase using a coupled enzyme assay as previously described in [16,19] in a buffer containing 40 m M Hepes/ KOH, 5 m M MgSO 4 ,0.42mM phosphoenolpyruvate, 0.15 m M NADH, 7.5 U pyruvate kinase, 18 U lactate dehydrogenase, 1.01 m M EGTA, pH 7.2. Free Ca 2þ con- centrations were calculated based on the method and binding affinities described by Gould et al. [23]. Ca 21 -uptake measurements The effects of curcumin on Ca 2þ uptake into microsomes or SR was measured as described by Michelangeli [24]. Briefly, microsomes were added to a stirred cuvette containing 2 mL of 40 m M Tris/phosphate, 100 mM KCl at pH 7.2 in the presence of 1 m M Fluo-3 (except for platelet microsomes where, which due to their high Ca 2þ content, the lower affinity magnesium green indicator was used, 0.625 m M), 10 mg·mL 21 creatine kinase and 10 mM phosphocreatine. Ca 2þ uptake was initiated by the addition of 1.5 m M MgATP. Fluorescence intensity was measured at 506 nm excitation/526 nm emission for Fluo-3 and 506 nm excitation/531 nm emission for Magnesium green. The results were calculated from fluorescence intensities using the following equation: ½Ca 2þ ¼K d £ ðF 2 F min Þ/ðF max 2 FÞ Where F is the fluorescence level and the addition of 1.25 m M EGTA and 1.5 mM CaCl 2 (Fluo-3) or 2 mM CaCl 2 (Magnesium green) determines F min and F max , respectively. The K d values used for Fluo-3 and Magnesium green are 900 n M [24] and 6 mM, respectively. Effects of curcumin on fluorescein 5 0 -isothiocyanate FITC-labelled Ca 21 -ATPase ATPase from SR was labelled with FITC according to the method described by Michelangeli et al. [17], with minor modifications to monitor the E2 to E1 transition. The SR ATPase was added in equal volume to the starting buffer (1 m M KCl, 0.25 M sucrose and 50 mM potassium phosphate pH 8.0). FITC in dimethylformamide was then added at a molar ratio of FITC/ATPase, 0.9 : 1. The reaction was incubated for 1 h at 25 8C and stopped by 0.25 mL of stopping buffer (0.2 M sucrose, 50 mM Tris/HCl pH 7.0), incubated for 30 min at 30 8C prior to being placed on ice until required. Measurements were undertaken in a buffer containing 50 m M Tris, 50 mM maleate, 5 mM MgSO 4 and 100 m M KCl at pH 6.0. Fluorescence was measured on a PerkinElmer LS50B spectrofluorimeter at 25 8C (excitation 495 nm, emission 525 nm). EGTA (100 m M) and then Ca 2þ (400 mM) or vanadate (100 mM) were added to measure changes in fluorescence. ATP binding to Ca 21 -ATPase ATP binding to purified Ca 2þ ATPase was also measured using radiolabelled ATP as described by Champeil et al. [26]. Briefly, 0.3 mg·mL 21 of purified ATPase was added to a buffer containing 150 m M Tes/Tris, 2 mM Mg 2þ and 2 mM EGTA (pH 7.0), to which was added ATP doped with [g 32 P]ATP to give a final concentration of 20 mM (specific activity 10 Ci·mol 21 ). One milliliter of solution was then rapidly filtered through a 0.45-mm Millipore HA filter, and placed in scintillant for counting. This was carried out in the absence and presence of curcumin, and controls to estimate nonspecific binding of ATP to the filter were performed as described previously [16]. Phosphorylation studies Phosphorylation of the ATPase by [g- 32 P]ATP was performed at 25 8C as described by Michelangeli et al. [17]. Briefly, SR Ca 2þ -ATPase was diluted to 75 mg·mL 21 in 20 mM Hepes/Tris (pH 7.2) containing 100 mM KCl, q FEBS 2001 Inhibition of the Ca 2þ -ATPase by curcumin (Eur. J. Biochem. 268) 6319 5mM MgSO 4, 1mM CaCl 2 in a total volume of 1 mL. The reaction was initiated by addition of ATP doped with [g- 32 P]ATP (specific activity, either 10 or 100 Ci·mol 21 ) and stopped after 15 s by addition of ice-cold 40% (w/v) trichloroacetic acid. The assay was placed on ice for 30 min subsequent to the addition of BSA (final concentration 1 mg·mL 21 ). The protein was separated from solution by filtration through Whatman GF/C filters. The filters were washed with 12% (w/v) trichloroacetic acid/0.2 M H 3 PO 4 , and left to dry, then placed in scintillant and counted. Membrane permeability studies To measure the effects of curcumin on membrane permeability, an assay was carried out as described by Longland et al. [22]. Ten milligrams (12.5 mmol) of egg phosphatidylcholine was dissolved in 0.25 mL of chloro- form and evaporated to dryness under a stream of N 2 . The phospholipid film was dispersed in 400 mLof40m M Hepes/KOH (pH 7.2), 100 mM KCl buffer containing 100 m M calcein. To this was then added 35 mL of 10.5% (w/v) potassium cholate in 40 m M Hepes/KOH (pH 7.2) Fig. 1. Inhibition of Ca 21 -ATPase activity by curcumin. Graphs representing both Ca 2þ -dependent ATPase activities (X) and Ca 2þ uptake (B)in a variety of microsomes are presented: (A) and (B), skeletal muscle SR; (C) and (D), cardiac SR; (E) and (F), platelet microsomes; and (G) and (H), cerebellar microsomes. All experiments were performed at 37 8C, pH 7.2. Each data point represents the mean ^ SD of three determinations. IC 50 for inhibition compared to controls (i.e. in the absence of curcumin) are as follows: (A) 15.0 ^ 0.8 m M (B) 5.0 ^ 0.3 mM (C) 7.4 ^ 0.4 mM (D) 20.3 ^ 2.2 m M (E) 8.8 ^ 1.3 mM (F) 34.3 ^ 1.5 mM (G) 13.7 ^ 4.2 mM (H) 50 ^ 2 mM curcumin. 6320 J. G. Bilmen et al. (Eur. J. Biochem. 268) q FEBS 2001 buffer). The suspension was sonicated to clarity at room temperature. Excess detergent was then removed by passing the suspension through a pre-equilibrated Sephadex G-25 column (with 40 m M Hepes/KOH (pH 7.2), 100 mM KCl buffer at room temperature), followed by 200 mL of Hepes buffer prior to centrifugation at 200 g for 20 s into a clean conical centrifuge tube. The resulting column eluate was passed through a second column as before, providing a suspension of reconstituted lipid vesicles. The dye filled vesicles were diluted in 1.8 mL of Hepes buffer and fluorescence intensity was measured at excitation and emission wavelengths of 490 nm and 520 nm, respectively. Ten microliters of 3 m M CoCl 2 was then added to the vesicle suspension and the rate of fluorescence quenching was monitored in the absence or presence of various concentrations of curcumin. Fluorescence studies Experiments to investigate the fluorescence of curcumin bound to the ATPase were performed in a buffer containing 20 m M Mes, 20 mM Mops, 80 mM KCl, 1 mM EGTA (pH 6.0 or 7.0). In Ca 2þ binding experiments to cerebellar microsomes, 12.5 mg·mL 21 Ca 2þ ionophore (A23187) was also added. All experiments were performed with 1 m M curcumin unless otherwise stated. Fluorescence was measured at 25 8C (excitation 411 nm, emission 500 nm). Ca 2þ and EGTA were added to measure changes in fluorescence at appropriate free Ca 2þ concentrations, using the constants given previously [23]. Ca 2þ binding to the Ca 2þ -ATPase was measured by monitoring the change in tryptophan fluorescence [17,27]. Purified ATPase was used at 2 m M in a buffer containing 20 m M Mes, 20 mM Mops, 80 mM KCl, 1 mM EGTA (pH 6.0 or 7.0). Ca 2þ binding was measured as percent increase in initial fluorescence, over a range of free Ca 2þ concentrations as described in [27]. Fluorescence was monitored at 25 8C (excitation 295 nm, emission 340 nm). RESULTS Figure 1 shows the Ca 2þ -dependent ATPase activity and Ca 2þ uptake in microsomes from various tissue extracts. The tissues were selected for their differential expression of SERCA subtypes: Skeletal SR membranes (Fig. 1A,B) express predominantly SERCA 1; cardiac SR (Figs 1C,D) express predominantly SERCA 2a; cerebellar microsomes (Fig. 1E,F) express mostly SERCA 2b and platelet microsomes (Fig. 1G,H) express a mixture of SERCA 2b and SERCA 3. The activities were measured at various curcumin concentrations, using the phosphate liberation assay in the presence of A23187 ionophore, and so were fully uncoupled. The Ca 2þ ATPase activity in all of the microsomes showed a high degree of inhibition, with half-maximal inhibition (IC 50 ) values ranging from 7.4 ^ 0.4 m M (platelets) to 15.0 ^ 0.8 mM (SR), with almost complete inhibition occurring at about 50 m M in all membranes. It was found that the IC 50 values for curcumin inhibition of SR and the purified Ca 2þ -ATPase varied from 7 to 17 m M dependent upon the preparation and conditions used. In addition, curcumin was tested to see if it was reversible with respects to inhibition of the ATPase. This was performed by initially preincubating the ATPase with 60 m M curcumin for 10 min followed by dilution in assay buffer to 0.06 m M, where the activity was found to be similar to controls. From Fig. 1B,D,F,H where the effects of curcumin on Ca 2þ uptake were monitored, the IC 50 values were calculated (compared to control). Skeletal muscle SR appears to be most sensitive to curcumin inhibition (IC 50 ¼ 5 ^ 0.3 mM), whilst cerebellar microsomes was least affected (IC 50 ¼ 50 ^ 1.7 mM) Cardiac and platelet microsomes were inhibited at intermediate concentrations (IC 50 ¼ 20 ^ 2.2 mM and 34 ^ 1.5 mM, respectively). ATP-dependent Ca 2þ uptake was stimulated in cerebellar microsomes, and to a lesser extent platelets, upon addition of low concentrations of curcumin. Maximal stimulation in platelets occurred at approximately 5 m M curcumin, with an increase in uptake of 12% (P , 0.01, students t-test, when compared with control). Maximal stimulation in cerebellar microsomes occurred at approximately 10 m M with a 76% increase in stimulation (P , 0.001). To measure the effects of curcumin on membrane permeability to cations, reconstituted liposomes were loaded with calcein, a fluorescent dye, and exposed to Co 2þ . The rate of quenching was then monitored (Fig. 2). After addition of curcumin, the rate of quenching was increased, showing that curcumin permeabilizes the membrane to metal ions. The increase in membrane permeability was seen to be dependent on the amount of curcumin that is added. This result indicates that the stimulation of Ca 2þ uptake in some of the microsome preparations is unlikely to be due to a decrease in ion leakage through the phospholipid membrane. Figure 3 illustrates the dependence of purified Ca 2þ - ATPase activity on both Ca 2þ (Fig. 3A) and ATP (Fig. 3B) in the absence and presence of curcumin, using the coupled enzyme assay. In Fig. 3A, the half-maximal activation of the ATPase by Ca 2þ was measured in the absence of and in the presence of 10 and 25 m M curcumin. The EC 50 was found to change insignificantly, from 0.52 ^ 0.12 m M to 0.63 ^ 0.30 m M Ca 2þ , although the maximal activity decreased from 18.95 IU·mg 21 (control) to 4.33 IU·mg 21 Fig. 2. Curcumin increases membrane permeability. The traces represent experiments of Co 2þ quenching calcein trapped within liposomes. The drop in fluorescence intensity represents quenching of the fluorescent dye by Co 2þ ions. Upon addition of curcumin, the rate of quenching is substantially increased and dependent upon the concentration of curcumin. The traces are representative of three or more experiments. q FEBS 2001 Inhibition of the Ca 2þ -ATPase by curcumin (Eur. J. Biochem. 268) 6321 (25 mM drug). The inset on Fig. 3A shows a double reciprocal (Lineweaver–Burk) plot for activity against [Ca 2þ ] free . As can be seen from the plots, the lines converge at a single point on the 1/[Ca 2þ ] axis, indicating noncompetitive inhibition with respect to Ca 2þ . Figure 3B shows a complex stimulation of the Ca 2þ - ATPase with increasing concentrations of ATP [28]. This data could be fitted to a bi-Michaelis–Menton equation assuming two sites, designated the high affinity catalytic site and the lower affinity regulatory site [28,29]. The kinetic parameters that describe the data are given in Table 1. A range of values for the kinetic parameters could be used to define the data profiles. These fits suggest that the V max values for both the catalytic and regulatory sites are reduced by curcumin, while at higher concentrations of curcumin (25 m M) the K m for the catalytic site is also possibly increased. In order to further assess the possibility of curcumin affecting the interaction of ATP binding to the ATPase, this was directly measured using [ 32 P]ATP in the absence of Ca 2þ (Fig. 4A). The data showed that the amount of ATP bound to the ATPase was reduced by curcumin. The binding inhibition had a apparent K i (IC 50 ) of about 9 mM. Reversing the order of addition to the ATPase (i.e. curcumin then ATP) had a similar effect on the extent of ATP binding, i.e. in both cases, the amount of ATP binding to the ATPase was significantly decreased. Experiments to assess the effects of curcumin on the ATP- dependent phosphorylation of the ATPase, were also undertaken. Figure 4B shows that ATP-dependent phos- phorylation was inhibited by the presence of 50 m M curcumin. In the absence of curcumin maximal phosphoryl- ation occurred at around 10 m M ATP where 1.7 ^ 0.3 nmol E-P per mg ATPase was phosphorylated. At 50 m M curcumin, the maximum level of phosphorylation was reduced by about 80% to 0.40 ^ 0.1 nmol E-P per mg ATPase. Figure 5A shows the traces of experiments obtained with FITC-labelled Ca 2þ ATPase in SR at pH 6, upon addition of Ca 2þ and vanadate. These changes have been used to monitor the transition between the E2 and the E1 step [28,30]. In the absence of curcumin, a 9.5% change in fluorescence is observed upon addition of Ca 2þ . This is believed to occur as at pH 6 the ATPase is essentially all in an E2 conformation (high fluorescence state) while the Fig. 3. The effects of Ca 21 and ATP dependence of Ca 21 ATPase activity by curcumin. (A) Ca 2þ -ATPase activity was measured as a function of [Ca 2þ ] free in the absence (B) and presence of 10 mM (W) and 25 m M (X) curcumin. (B) shows ATPase activity as a function of [ATP] in the absence (B) and presence of 10 m M (W)or25mM (X) curcumin. The kinetic parameters are given in the text or Table 1. The experiments performed at 37 8C pH 7.2. Each data point is the mean ^ SD of three to five determinations. Table 1. Kinetic parameters of Purified Ca 21 ATPase activity as a function of ATP concentration in the presence of curcumin. Note: these values are calculated from the best fits to the data in Fig. 3B. The numbers in brackets correspond to the range of values for each parameter which could also give adequate fits to the experimental data (i.e. where chi 2 for the fits are # 0.7). Curcumin concentration (m M) Catalytic K m (mM) Catalytic V max (IU·mg 21 ) Regulatory K m (mM) Regulatory V max (IU·mg 21 ) 0 3.0 6.3 0.40 13.6 (2.7–6.6) (6.3–9.3) (3.8–1.0) (13.4–14.2) 10 3.0 2.3 0.40 12.1 (1.9–3.6) (1.3–2.5) (0.24–0.42) (11.9–12.2) 25 7.0 2.2 0.40 5.1 (4.9–8.0) (1.8–2.4) (0.23–0.41) (4.7–5.2) 6322 J. G. Bilmen et al. (Eur. J. Biochem. 268) q FEBS 2001 addition of Ca 2þ shifts it to an E1 conformation (low fluorescence state) [28,30]. Vanadate, on the other hand, would shift the ATPase towards E2 and therefore increase the FITC-ATPase fluorescence if it were in an E1 conformational state [30]. The fluorescence change due to addition of Ca 2þ was shown to decrease upon preincubation with curcumin (5 m M), as well as slowing down the rate of this transition. In order to establish that the decrease in Ca 2þ -induced fluorescence change was due to a shift in the E1 to E2 step towards E1, the experiments were repeated with vanadate. At pH 6, vanadate induces little change in the FITC-ATPase fluorescence, indicative of it already being in an E2 state. However, in the presence of curcumin (5 m M), vanadate caused a rise in fluorescence suggesting that curcumin had shifted the equilibrium towards E1. Figure 5B shows the concentration effects of curcumin on both the Ca 2þ -induced fluorescence decrease and vanadate-induced fluorescence increase of FITC-labelled SR. The data show that the concentration of curcumin inducing half-maximal fluorescence changes in both cases were similar (< 5– 6 m M). It was found that curcumin strongly fluoresces in the presence of ATPase (excitation 411 nm, emission 500 nm), but little in its absence. This observation was used to assess curcumin binding to the Ca 2þ -ATPase. Titrations were performed by addition of either curcumin or ATPase and interpreted using Langmuir isotherms. Binding can be described by the following equation: ½Eþ½L$½EL Where [E] and [L] are the concentrations of free sites and ligands, respectively, and [EL] is the concentration of bound ligand. The total concentration of sites can be expressed as N[E] 0 , the product of total protein concentration ([E] 0 ) and the number of binding sites per protein molecule (N ). The concentration of bound ligand [EL] can be derived in terms of the dissociation constant K d , defined as; K d ¼½E·½L/½EL¼½N·E 0 2 EL·½L 0 2 EL/½EL where [L 0 ] and [E 0 ] are the total ligand and protein concentrations. This equation can then be rearranged to give Fig. 4. Effects of curcumin on ATP binding and ATP-dependent phosphorylation. (A) Displacement of [ 32 P]ATP (20 mM) bound to the Ca 2þ -ATPase by curcumin, measured at pH 7.2, 25 8C. Values represent the mean ^ SD of eight determinations. (B) Phosphorylation of SR Ca 2þ -ATPase by [ 32 gP]ATP (0–100 mM) in the absence (B) and presence of 50 m M curcumin (W), measured at pH 7.2, 25 8C. Values represent the mean ^ SD of three to five determinations. Fig. 5. The measurement of E2–E1 conformational change using FITC-labelled Ca 21 ATPase in SR. (A) Effects of curcumin on the fluorescence decrease in FITC-Ca 2þ ATPase induced by either 400 mM Ca 2þ or 100 mM vanadate, initially preincubated in the presence or absence of 5 m M curcumin at pH 6. (B) The effects of curcumin concentration on the fluorescence changes induced by either Ca 2þ (B) or vanadate (W). The experiments were performed at 25 8C and each data point is the mean ^ SD of three determinations. q FEBS 2001 Inhibition of the Ca 2þ -ATPase by curcumin (Eur. J. Biochem. 268) 6323 the following quadratic equation: ½EL 2 2 ½EL·ðK d þ½NE 0 þ½L 0 ÞþN½E 0 ½L 0 ¼ 0 Using the formula for the solution of a quadratic equation, the concentration of bound ligand [EL] is then given by: ½El¼ðA 2 ½A 2 2 4N½E 0 ½L 0  0:5 Þ/2 where A ¼ K d þ N[E] 0 þ [L] 0 , N is the number of binding sites per protein molecule, [E] 0 is the protein concentration, [L] 0 is the total concentration of ligand, and K d is the dissociation constant for binding Using this equation, curves can be fitted to the fluorescence data assuming a K d ¼ 0.8 mM and a stoichi- ometry of 1 (i.e. 1 curcumin per ATPase), either by varying curcumin concentration and keeping ATPase constant (Fig. 6A) or varying ATPase concentration, keeping the curcumin constant (Fig. 6B). The data in Fig. 6A could also be fitted assuming two binding sites for curcumin on the Ca 2þ -ATPase with differing affinities (a high affinity site with a K d of 0.55 mM and lower affinity site with a K d of 10 m M). In addition to enhancement of fluorescence in the presence of ATPase, curcumin bound ATPase also decreased its fluorescence intensity by up to 25% when Ca 2þ was added (Fig. 7A). To characterize this, the [Ca 2þ ] free was varied in the presence of ATPase and 1 m M curcumin at pH 6 and 7 and the fluorescence decrease measured (Fig. 7B). In order to assess whether this change is directly monitoring the Ca 2þ binding steps, additional experiments were performed to monitor tryptophan fluorescence of the ATPase as a function of [Ca 2þ ] free as this fluorescence change has also Fig. 6. Fluorescence of curcumin bound to the Ca 21 ATPase. (A) Fluorescence change upon addition of curcumin (0–8 m M) to the Ca 2þ -ATPase (2 mM) ATPase, pH 7.0, 25 8C. (B) Addition Ca 2þ ATPase into 1 mM curcumin at 25 8C, pH 7.0. Fluoresence intensity was measured at 500 nm, and excited at 411 nm. All points represent mean ^ SD of three determinations. The curves were fitted assuming a single binding site for curcumin on the ATPase, with a K d of 0.8 mM. Equally good fits to the data could also be achieved assuming two curcumin binding sites with K d values of 0.55 mM and 10 mM. Fig. 7. Fluorescence of curcumin bound to purified Ca 21 -ATPase in the presence Ca 21 . (A) Spectra of 1 mM curcumin in: (i) buffer alone at 25 8C, pH 7.0; (ii) in the presence of 2 m M purified Ca 2þ ATPase and (iii) after addition of 2.5 mM Ca 2þ . Results show approximately 25% decrease in fluorescence upon addition of Ca 2þ . (B) Fluorescence changes of either curcumin bound to the ATPase or tryptophan residues within the ATPase, upon addition of a range of free Ca 2þ concentrations (3 nM to 100 mM). Experiments were performed at 25 8C either at pH 6.0 (V, tryptophan, P, curcumin) or at pH 7.0 (B, tryptophan, O, curcumin). (C) Curcumin fluorescence change induced by Ca 2þ , monitored when bound to cerebellar microsomes (200 mg·mL 21 ). Experiments were performed at 25 8C pH 7.0. All data points represent means ^ SD of three determinations. Curcumin fluorescence was monitored using the following wavelengths: Excitation l ¼ 411 nm, emission l ¼ 500 nm. Tryptophan fluor- escence was monitored by exciting at 295 nm and detecting the emission at 340 nm. 6324 J. G. Bilmen et al. (Eur. J. Biochem. 268) q FEBS 2001 been associated with Ca 2þ binding to the ATPase [16]. At pH 7, the curcumin decrease and tryptophan increase in fluorescence can be superimposed and give a single curve with the EC 50 for Ca 2þ occurring at 0.22 ^ 0.11 mM (^ SEM, n ¼ 9). At pH 6, the curves could again be superimposed but were shifted to the left (EC 50 ¼ 5.0 ^ 1.0 mM,SEM,n ¼ 7) as Ca 2þ binds more weakly at lower pH. Experiments were performed with cerebellar microsomes (Fig. 7C) to see if the change in curcumin fluorescence with respect to Ca 2þ could be used with crude membrane preparations, where the presence of Ca 2þ ATPase is low and where tryptophan measurements are impracticable due to abundance of other proteins. It was found that a Ca 2þ binding curve could be derived from the curcumin fluorescence data that gave a EC 50 of 1.6 ^ 0.5 mM (^ SEM n ¼ 7) and a maximal decrease of 20%. DISCUSSION From the Ca 2þ -ATPase activities, it can be seen that all subtypes of SERCA are inhibited to a similar degree by curcumin suggesting it is not a subtype specific inhibitor of the Ca 2þ -ATPase. Interestingly, the corresponding Ca 2þ uptake shows marked differences. For platelet and cerebellar microsomes, an increase in Ca 2þ uptake at low concen- trations of curcumin was observed followed by inhibition at higher concentrations. This biphasic response has been observed in microsomes upon exposure to ethanol [31,32]. Mitidieri & de Meis [31] and Mezna et al. [32,33] showed that at concentrations where ethanol had no effect or inhibitory effects on Ca 2þ -ATPase activity, there was a significant increase in uptake. It is unlikely that the enhancement of Ca 2þ uptake is due to curcumin reducing the permeability of ions through the phospholipid bilayer, as our data shows that curcumin makes phospholipid membranes more, not less, leaky (Fig. 2). Therefore at present the simplest explanation would be that curcumin inhibits Ca 2þ release from these microsomes through a Ca 2þ channel. Studies on the inositol-trisphosphate-sensitive Ca 2þ channel, which is abundant in cerebellum and platelets, have shown it to be inhibited by curcumin and therefore it seems the most likely target [25]. If the biphasic response of curcumin on Ca 2þ uptake in cerebellar and platelet microsomes were to occur in intact nonmuscle cells, this may well explain many of the reported cellular effects observed with curcumin. For instance high doses of curcumin can induce apoptosis [34]. It is also known that prolonged elevation of [Ca 2þ ] cyt induces apoptosis [35] and agents such as thapsigargin [36] and alkylphenols [37], which inhibit ER Ca 2þ pumps can trigger this process. Therefore if high curcumin concentrations act in a manner similar to thapsigargin and elevates [Ca 2þ ] cyt (i.e. inhibit Ca 2þ uptake), this would be the most obvious mode of action. Platelet aggregation, inflammation, and arachadonic acid production are all processes that have been shown to be inhibited by curcumin [6,7,38] as well as requiring Ca 2þ [39–41]. If cells undergoing these processes were exposed to curcumin concentrations that were able to stimulate Ca 2þ uptake, this would have the effect of reducing [Ca 2þ ] cyt , leading to a reduction in stimulation. Therefore the effects of curcumin on these activities could be explained, at least in part, in terms of its effects on intracellular Ca 2þ levels. The mechanism by which the ATPase transports Ca 2þ is usually discussed in terms of the model proposed by DeMeis & Vianna [42], involving two major conformational states defined as E1 and E2. In the E1 form the ATPase is able to bind two Ca 2þ with high affinity on the cytoplasmic side of the membrane. While in the E2 form the Ca 2þ binding sites have translocated across the membrane to the luminal side and are of low affinity. Furthermore, in the E1 confor- mational state, the ATPase can be phosphorylated by ATP, which drives the translocation process. From the data presented here it appears that curcumin preferentially stabilizes the E1 form of the ATPase as well as acting as a noncompetitive inhibitor with respect to Ca 2þ . However, the situation is more complex with respect to ATP, with low concentrations of curcumin (up to 5–10 m M) acting as a noncompetitive inhibitor with respect to ATP, but higher concentrations appearing to be competitive. This is also confirmed from the ATP binding data, which showed that curcumin inhibited ATP binding (IC 50 ¼ 9 mM). The hydrophobic nature of curcumin would make it unlikely to compete for the nucleotide binding site by mimicking ATP, and therefore it is more likely to act as a competitive inhibitor by inducing a conformational change, which precludes ATP from binding. This suggestion is further supported by the fact that the Ca 2þ -induced conformational change monitored by the fluorescence of the FITC-labelled ATPase is affected by curcumin. FITC is known to label the ATPase on Lys515 within the ATP binding pocket of the nucleotide binding domain, also precluding ATP binding [15,43]. Therefore if curcumin affects this conformationally induced fluorescence change, it must be binding elsewhere as the ATP binding site is already occupied with FITC. In comparing the 2.6-A ˚ resolution crystal structure of the Ca 2þ bound (E1) form of the ATPase with the 8 A ˚ low resolution structure of the decavanadate-bound (E2) form of the ATPase, Toyoshima et al. [15], have shown by modeling that several major re-arrangements within the three cytoplasmic domains need to occur. They predicted that in going from E1 to E2, the nucleotide-binding domain has to move more than 25 A ˚ to come into close contact with Asp351 within the phosphorylation domain, for phosphoryl transfer to occur. These two domains are linked via a hinge or bridging region that encompasses amino-acid sequences 355–365 and 595–605. The actuator domain also under- goes a large motion, moving approximately 30 A ˚ and rotating almost 908 to come into closer contact with both the nucleotide-binding and phosphorylation domains. In addition, it was suggested that the nucleotide-binding domain is highly mobile in the presence of Ca 2þ and can come into close contact with the phosphorylation site by simple thermal fluctuation [15]. From this type of analysis of the probable conformational changes that need to occur in the tertiary structure of the Ca 2þ -ATPase in going from E1 to E2, a model can be proposed to explain the competitive nature of curcumin with respect to ATP, without it binding directly to the ATP- binding site (Scheme 1). In this model, we also postulate that the nucleotide-binding and phosphorylation domains are sufficiently mobile to allow them to come into contact with each other in the presence or absence of ATP. If both ATP and Ca 2þ are bound to the ATPase during this contact then phosphorylation can occur and Ca 2þ can be transported across them membrane. However, if the two domains come q FEBS 2001 Inhibition of the Ca 2þ -ATPase by curcumin (Eur. J. Biochem. 268) 6325 into contact in the absence of ATP, curcumin is then able to bind to the ATPase (possibly at the hinge region) locking the two domains together and therefore precluding ATP binding (i.e. inhibiting the ATPase in a ‘competitive manner’). It would appear unlikely that curcumin can ‘occlude’ ATP binding, in the same way as chromium-ATP, by trapping the ATP in the binding site when the two domains come together [44], as our ATP binding data shows that little ATP is bound to the ATPase when it is added prior to curcumin. The fluorescence data of curcumin bound to the ATPase indicates that it may bind with either a high affinity (< 1 m M) to a single site or to two sites of differing affinities (K d values of 0.55 mM and 10 mM, respectively). However, to inhibit the ATPase activity by 50% would require between 7 and 15 m M curcumin. Therefore this would be more consistent with the presence of two distinct binding sites for this molecule of differing affinities. Our data would suggest that the lower affinity binding site (K d ¼ 10 mM) might contribute more towards the inhibition on the ATPase, than the higher affinity one (K d ¼ 0.55 mM), as this correlates to the IC 50 value gained from the activity data (IC 50 values of between 7 and 17 m M). In addition, this would also be consistent with the [ 32 P]ATP binding data (where the IC 50 value was 9 mM) and FITC-ATPase data (where the IC 50 and EC 50 values were 5–6 mM). As the high affinity binding site for curcumin can sense Ca 2þ binding events, it could also be speculated that this site is either close to the Ca 2þ binding sites, or at a site which undergoes major changes upon Ca 2þ binding (i.e. the actuator domain or transmembrane helices M1 and M3 which lead into this domain [15]). In conclusion, curcumin inhibits the Ca 2þ -ATPase, by inducing a conformational change, which blocks the ATP from binding. ACKNOWLEDGEMENTS We would like to thank the BBSRC for a PhD studentship to J. G. 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