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-

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

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

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 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

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.

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

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

(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

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

(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

(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

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

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

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

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

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

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

]
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

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-

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

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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