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:
(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. B.
and the government of Pakistan for a PhD scholarship to S. Z. K.
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