From the
Thapsigargin is a high affinity inhibitor of sarco- and
endoplasmic reticulum (SERCA) type ATPases. We have used kinetics to
determine the dissociation constant of thapsigargin-sarcoplasmic
reticulum Ca
Thapsigargin (TG)
TG appears to inhibit
the Ca
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy The apparent K
On-line formulae not verified for accuracy
The present study employs a transfer technique for TG
migration from inactive enzyme to native enzyme. By continuous
monitoring of activity of the native ATPase, its rate of inhibition
could be established and an off rate constant for TG obtained
(Fig. 1).
Of critical importance is that the
cross-link has no effect on the dissociation kinetics of TG. This was
tested in three ways; TG did not inhibit the rate of glutaraldehyde
cross-link formation as investigated by formation of E(125) on
PAGE analysis
(12) using ratios of 4:1 TG:ATPase (data not
shown), the E
ATPase activity using the coupled
enzyme assay is shown in Fig. 1. Addition of Ca
ATPase activity
of cross-linked SRV (E
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Fig. 2shows the difference between the initial and final
rates of activity of added E
Detergent has been shown previously to denature
the Ca
The observed rate constant
(k
The final TG concentration in stopped flow
experiments was varied from 0.75 µM; the lower limit of
detection of fluorescence changed to 4 µM for 1
µM active sites. Fluorescence signals decreased in
amplitude below 1 µM TG, while observed rate constants of
25 ± 2 s
An Arrhenius plot (Fig. 4)
of the observed binding rate constants (k
Observed rate constants for simulated binding are shown in
Fig. 4
, (inset). The simplest kinetic model to account
for experimental findings is given by Reaction 3, where
E
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy The model incorporated a second step with a rate-limiting
conformational change for three reasons. (i) The observed on rate
k
Determination of remaining rate constants was restricted by the
above findings. Values greater than 0.5 s
The best fit to the data therefore
yielded rate constants of k
The upper limit for the diffusion of a hydrophobic and
relatively large molecule, such as TG, was derived from simulation of a
two-step model as above (5
Finally, the present study provides information on the use of TG in
kinetic studies of the Ca
We thank Dr. P. Adams for assistance in kinetic
analysis and Professor M. C. Berman for conceptualization of the
transfer methodology.
-ATPase interaction in the absence and
presence of non-ionic detergent. The observed ``off'' rate
constant was measured as 0.0052 s
at 26 °C by
the kinetics of inhibition of ATPase activity following transfer from
an inactivated thapsigargin-ATPase complex to native ATPase. Inactive
ATPase was produced by cross-linking the active site with
glutaraldehyde. The observed dissociation rate constant was increased
7-fold by 0.1% Triton X-100, indicating that perturbation of the
transmembrane and stalk region by detergent altered the binding
parameters of the inhibitor. In addition, thapsigargin stabilized the
ATPase against inactivation caused by detergent in the absence of
Ca
. The observed ``on'' rate constant of
thapsigargin was measured at 26 °C as 25 s
irrespective of thapsigargin concentration, by the kinetics of
thapsigargin- induced change in intrinsic fluorescence. An Arrhenius
plot showed a temperature dependence of this rate constant, indicative
of a conformational change in the protein with an activation energy of
9.5 kcal/mol for thapsigargin binding. The affinity of the
Ca
-ATPase for thapsigargin was calculated to be
greater than 2 pM at pH 7.0 and 26 °C.
(
)
is a naturally
occurring sesquiterpene lactone isolated from the umbelliferous plant
Thapsia garganica (Apiaciae)
(1) , and it inhibits
Ca
-dependent responses in a number of cellular
systems
(2) . It has been found to be a high affinity inhibitor
specifically of sarco- and endoplasmic reticulum (SERCA) pumps,
providing a useful probe of intracellular Ca
functions (3-6). However, functional studies are performed
without knowledge of diffusion and transfer rates of the inhibitor
between target ATPases. Elucidation of the TG binding constants and
kinetics are therefore of crucial importance.
-ATPase in the subnanomolar range by
stabilizing the E
conformation
(4) . Neither
``on'' nor ``off'' rate constants have previously
been measured. Studies using dilution or extensive washing have not
demonstrated dissociation of the inhibitor
(7) . In the present
study, TG dissociation kinetics were monitored by a novel transfer
assay of TG from inactivated ATPase to active ATPase. This technique
also permitted the measurement of off rate constants in the presence of
detergent. Inhibition rates by TG can be monitored by a decrease in
intrinsic tryptophan fluorescence signal
(8) . The affinity was
obtained from analysis of these kinetics.
Materials
TG was purchased from L.C. Services Corp. (Woburn, MA) and
made up by weight in MeSO. A23187 was purchased from
Calbiochem. SR vesicles (SRV) were prepared from the back and hind leg
muscles of rabbits according to the method described in Ref. 9. This
gave 4.8 nmol/mg phosphorylable sites (data not shown) when the enzyme
was phosphorylated from Ca
ATP in the absence of Mg
and presence of 1 M KCl and 1 mM ATP, pH 7.0,
at 26 °C
(10) . The standard buffer used throughout the study
was 50 mM MOPS, pH 6.8, 5 mM MgCl
, 100
mM KCl, and 0.5 mM EGTA.
Methods
ATPase Activity
ATPase activity was measured at
26 °C with an Aminco DW-2 spectrophotometer by coupled NADH
oxidation monitored at 340 nm under continuous stirring as described
previously (5). Both cross-linked ATPase and native ATPase were
pretreated with 4% (w/w) A23187 and diluted to concentrations of 30
µg/ml in the assay medium. For detergent studies, 0.1% Triton X-100
was added to the assay, followed by sequential addition of SRV. Control
studies for the assay included measurement of the rate of oxidation of
NADH after addition of 10 µM ADP in the presence of Triton
X-100, and/or of TG in the absence of Ca-ATPase, to
determine the maximal rate of the coupled enzyme system itself. Analog
absorbance readings were digitized on a 12-bit A/D convertor at 1
reading/s, with the time base derived from the computer clock.
Inactivation of the Ca
SR vesicles (0.5 mg/ml), were incubated at 25
°C in 50 mM MOPS/tetramethylammonium chloride, pH 8.1, 50
µM CaCl-ATPase by
Cross-linking
, 0.2 M sucrose, and 150
µM glutaraldehyde for 70 min
(11) . Cross-link
formation was detected by the abnormal mobility of derivatized ATPase
on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The
native 110-kDa band converts to a 125-kDa band
(E(125))
(12) . Cross-link formation was tested to
levels of up to 4:1 TG:SRV (data not shown), showing no interference
with E(125) formation by thapsigargin.
Fluorescence Measurements
Intrinsic tryptophan
fluorescence was performed on an Aminco-SPF 500 spectrofluorimeter with
excitation and emission wavelengths at 295 and 330 nm at 4- and 10-nm
band passes, respectively. Conditions were 0.2 mg/ml SRV in standard
buffer with addition of 0.5 mM EGTA and varying concentrations
of TG at 26 °C.
Stopped Flow Fluorescence
Kinetic fluorescence
studies were performed on an Applied Photophysics DX.17MV stopped flow
fluorimeter using an excitation wavelength of 290 nm with 2-nm slit.
Emission wavelengths were selected using a cut-on filter that had
half-maximum transmittance at 321 nm and less than 0.006% T below 290
nm. Buffers were filtered (0.45 µm) and degassed prior to addition
of SRV (0.4 mg/ml) and TG. Changes in light scattering for mixing SRV
with TG were checked by excitation at 330 nm and found to be absent.
The accumulated average of 10-20 experiments was tested for the
best fit of the simulated reaction.
Simulation of Binding Kinetics
The TG binding and
dissociation reactions were simulated using a 4th order Runge-Kutta
algorithm. The sum of enzyme species was checked for equality at all
time stages of the simulation. Derived rate constants were determined
by least squares fits of the simulated data. The reaction model
employed is shown in Reaction 1, where E*TG and
E
TG are active and inactive enzyme species
with bound TG, respectively.
was calculated as
shown in Equation 1.
Figure 1:
Transfer of TG from glutaraldehyde
cross-linked Ca-ATPase to control
Ca
-ATPase. Ca
-ATPase activity was
measured by the coupled enzyme assay as described under
``Methods.'' Experiments (A and C) were
initiated with addition of 50 µg/ml cross-linked
Ca
-ATPase (E
) to the assay
medium containing 1 mM ATP and followed by 0.6 mM
CaCl
as indicated. TG was added to a stoichiometry of 0.9:1
for TG:enzyme. Exchange experiments were performed in the presence
(traceA) or absence (traceB) of
0.1% (w/w) Triton X-100. Both E and E
were treated with A23187 (4%, w/w) prior to addition. The
dottedlines represent slopes of the traces during
the final 1 min of activity. Control studies of ATPase activity in the
presence (B) and absence (D) of Triton X-100 for 50
µg/ml native Ca
-ATPase E
was followed by TG to 0.9:1, and 2 mM EGTA. The
inset shows the TG concentration dependence for inhibition of
50 µg/ml E
as performed in traceD.
The Ca pump of sarcoplasmic
reticulum was inactivated by reaction with glutaraldehyde, which has
been shown previously to inhibit the E
P to
E
P isomerization step
(11) , by forming an
intramolecular cross-link between Lys-492 and Arg-678
(13) .
Cross-linking results in 95% inactivation of
Ca
-ATPase activity
(11) and provided the ideal
TG donor when inhibited by TG.
E
isomerization step in cross-linked ATPase was inhibited by TG
(Fig. 3), and the activation energy for TG binding was not
altered by cross-linking (Fig. 4).
Figure 3:
Intrinsic tryptophan fluorescence changes
upon addition of TG. The intrinsic fluorescence of 0.2 mg/ml SRV in the
presence of 0.5 mM EGTA was monitored for the response to 0.6
mM CaCl (traceA) or to 1
µM TG followed by 0.6 mM CaCl
(traceB). Cross-linked SRV in the presence of
0.5 mM EGTA was treated with 0.1% Triton X-100 and followed by
1 µM TG (traceC). Inset,
kinetics of TG binding as measured by intrinsic tryptophan fluorescence
by stopped flow fluorimetry. Syringe A contained 0.4 mg/ml SRV and
syringe B 1.5 to 8 µM TG in standard buffer at 26 °C.
The trace represents fluorescence signals averaged from 20
successive runs. Data were fitted using a simulation of a model (see
``Methods'') that gave monoexponentials with observed rate
constants of 25 s
± 1 s
for all traces (solidline). Simulations using
a diffusion limit below 1
10
M
s
are
represented by the dottedline.
Figure 4:
Arrhenius plot of observed on rate
constants. Observed rate constants from stopped flow data derived from
data described in Fig. 3 (inset) for native (closedcircles) and cross-linked ATPase (opencircles) for the temperature range 6-30 °C were
plotted in the form of an Arrhenius plot. The fitted line has a slope
of -4.8. Inset, the observed binding rate constants at
26 °C (circles) for the given final TG concentrations from
Fig. 3 (inset). Simulations using diffusion rates of 5
10
(dottedline), 1
10
(solidline), and 5
10
(dashedline) M
s
over the TG concentration range of
0.1-4 µM, were analyzed with a least squares fit to
provide observed binding constants
(k
).
TG has been shown to bind
in the stalk region and not at the catalytic
site
(14, 15) , and has been reported to inhibit ATP
binding in a non-competitive manner at the catalytic site of SRV (0.1
mg/ml) (TG = 20 µM) at ratios of 65:1 TG to active
sites (3 nmol/mg)
(16) .
to native SRV stimulated ATPase activity from basal (105
nmol/mg/min) to maximal activity (1020 nmol/mg/min). Addition of just
substoichiometric TG (0.9:1) gave a rapid onset of inhibition,
indicative of the absence of possible binding reservoir that could
sequester TG and release it slowly (traces B and D).
Subsequent addition of EGTA inhibited turnover by 2-5%,
indicating near complete inhibition by TG. Titration of sarcoplasmic
reticulum with TG using this method gave a 1:1 inhibition curve for our
sarcoplasmic reticulum preparation with 4.8 nmol/mg active site
(Fig. 1, inset), indicative of the concentration of TG
and enzyme stoichiometry used in the present study.
) had a residual 5%
activity plus basal ATPase (Fig. 1, traceC).
Addition of TG inhibited the 5% residual activity, but not basal
activity, as shown previously
(5) . Subsequent addition of native
SRV (E
) (Fig. 1, traceC) gave an initial high rate of activity that became
progressively inhibited over a period of 5 min. Residual activity after
10 min represents basal activity of native SR plus basal activity of
cross-linked inactivated SR (Fig. 1, dottedlines). This indicates that TG, previously bound to one
form of Ca
-ATPase is able to migrate to and inhibit
normal SR. Under these experimental conditions, in the presence of
Ca
, E
is predominantly in
E
(2Ca) and occluded conformation,
E
P
2Ca
, after liberation of
TG
(11, 17) . The E
(2Ca)
conformation has been shown not to accept TG
rapidly
(4, 5) , while E
(1Ca) and
E
are inhibited
(6, 18) . Native
enzyme (E
) is cycling through
E
-E
conformations and can
rapidly accept TG during the E
phase, ultimately
resulting in 100% inhibition. Thus the reaction scheme employed was as
shown by Reaction 2, with the advantage that free TG remained at low
concentrations at all times.
. This was fitted
with a monoexponential giving a rate constant of 0.0052 ± 0.0004
s
for n = 10 and was taken as the
overall dissociation rate constant at pH 7.0 and 26 °C.
Figure 2:
Off rate constants of TG. Difference
between initial and final rates of data from Fig. 1 in the presence
(traceB) or absence (traceA) of
0.1% Triton X-100. Monoexponential fits (solidlines)
gave rate constants of 0.0052 ± 0.0004 s and
0.035 ± 0.001 s
for n = 10,
respectively.
Effect of Detergent on the Off Rate Constant
Previous
data have shown profound effects on catalytic function from
intercalation of non-ionic detergents into the lipid
bilayer
(19, 20, 21, 22) . In view of
suggested binding of TG in the stalk region, it was decided to test the
effects of the non-ionic detergent Triton X-100 on the off rate of TG
from its inhibitory site on the cross-linked ATPase (Fig. 2,
uppertrace). The critical micelle concentration for
Triton X-100 under ATPase conditions was 0.02% when measured by 90°
light scattering at 600 nm, as shown previously
(21) . Overall
ATPase activity was diminished up to 60% by 1.0% Triton X-100
(Fig. 1, traceB), as shown
previously
(21) . The apparent off rate constant was increased
7-fold from 0.005 to 0.035 s by detergent (traceA).
-ATPase in the presence of EGTA
(20, 24) and was monitored by intrinsic fluorescence (Fig. 3,
traceC) as described previously
(20) . TG (1
µM) (1:1) stabilized denaturation without reversing the
process. Addition of Ca
did not reverse fluorescence,
indicating full inhibition by TG in the presence of detergent.
Measurement of On Rate Constants
Intrinsic
tryptophan fluorescence was increased by 4% upon the addition of
Ca to the E
species
(Fig. 3, uppertrace), as described
previously
(25) . Intrinsic fluorescence was employed to measure
the on rate constant of TG under the same conditions as used for the
ATPase assay except for the absence of ATP. Sagara et al.(8) have shown that the fluorescence decrease upon the addition
of TG in EGTA is closely correlated with E
TG
formation and that fluorescence signals asymptote above levels of 1:1
TG:Ca
-ATPase. Addition of TG to the Ca
free enzyme (E
) resulted in a 3% decrease in
intrinsic fluorescence (Fig. 3, traceB), which
did not respond to the readdition of Ca
, indicative
of stabilization of the ATPase in the E
TG
conformation. These data were repeated for cross-linking the active
site with glutaraldehyde, which had no effect on the fluorescence
change induced by TG.
) for TG binding was a monoexponential with a
rate of 25 s
at 26 °C (Fig. 3,
inset).
were monoexponential over the entire
concentration range tested (Fig. 4, inset). This
indicates that binding forms an initial precursor complex followed by a
rate-limiting step for inhibition.
) of TG
to native (closedcircles) and cross-linked (opencircles) ATPase, over the temperature range 6-30
°C, showed that the cross-link did not affect the kinetics, and
that the process occurs with an activation energy of 9.5 kcal/mol.
*TG and E
TG are
precursor and inhibited conformations, respectively.
was independent of TG concentration
(Fig. 4, inset), whereas low values for k
(<10
M
s
) in combination with a high values for
k
(1000 s
), to achieve a
simulated observed rate constant of 25 s
, gave a
linear dependence of observed rate constant to the TG concentration.
(ii) Diffusion limits of less than 5
10
M
s
produced a
pronounced dip in k
at 1:1 binding
(Fig. 4, inset, dottedline). (iii)
There was a marked lag phase at the initial stages of the formation of
E
TG (Fig. 3, inset).
for
k
in combination with 25 s
for k
led to subsaturation of
E
TG at equilibrium. The reverse rate
constant (k
= 0.005
s
; Fig. 2) was chosen as a fraction of the
forward rate (k
) to achieve maximal inhibition.
The dissociation rate constant (k
) was 55
s
by default.
= 5
10
M
s
,
k
= 55 s
,
k
= 25 s
, and
k
= 0.005 s
. The
apparent affinity of the enzyme for TG from derived rate constants gave
an apparent binding constant of 2.2 pM for TG, while diffusion
limits above 10
M
s
gave sub-picomolar values.
DISCUSSION
Transfer Methodology
Establishing binding
constants for very high affinity ligands presents difficulties in that
signals from the low concentrations of enzyme used for equilibrium
titration are below the limits of detection. One approach has been to
determine the kinetics of the binding reaction using high
concentrations of protein. Measurement of dissociation rate constants
by dilution or filtration may be too slow, since the rapid on rate
ensures that the enzyme cannot be cleared of TG. The method employed in
the present study is a transfer reaction from inactivated to normal
sarcoplasmic reticulum, keeping free concentrations of TG to a minimum.
Subsequent to liberation of TG, the donor changes to a conformation
that does not readily re-accept the inhibitor, resulting in near 100%
transfer and inhibition of the acceptor. We have shown that TG does not
bind at the catalytic site and does not affect the reactivity or
relative positions of Lys-492 and Arg-678 during cross-link formation.
Conversely, the cross-link did not change the kinetics of TG
inhibition. Our conclusion is that the cross-link does not influence
the TG-ATPase interaction, providing the ideal inhibitor for this type
of transfer reaction.
Binding and Dissociation Kinetics of TG
The
dissociation rate constant of 0.0052 s was similar
to previous values observed using the change in NBD-labeled
Ca
-ATPase fluorescence upon the addition of
Ca
to E
in the absence of
Mg
at pH 7.0 (6, 18). Their study
(26) concluded that the rate of approximately 0.009
s
is an apparent E
TG
E
Ca
TG isomerization, and not the
dissociation rate of the inhibitor. The Arrhenius plot of intrinsic
tryptophan fluorescence changes indicates a temperature-dependent
conformational change within the enzyme for TG binding. This implies a
rate-limiting step following initial TG binding, with an activation
energy for the protein conformational change of 9.5 kcal/mol for
binding. Guillain et al.(25) have extensively studied
the intrinsic tryptophan fluorescence signal changes occurring during
binding and dissociation of Ca
and concluded that the
fluorescence accurately reported binding kinetics. The present study
assumes that kinetics of TG binding are also monitored accurately by
this method.
10
M
s
), resulting in
an apparent K
= 2 pM.
More rapid binding rates, with delayed reporting of conformational
changes by tryptophan fluorescence at a secondary rate-limiting step,
are possible. This would then place the affinity of the ATPase for TG
in the femtomolar range.
Inferred TG Binding Site
Takeyasu and Ishii
(27) showed that the first 200 amino acid residues of the
Ca-ATPase were not important for TG sensitivity,
while Norregaard et al. (14) have shown sensitivity between
residue 200 and 348, suggesting that transmembrane segments M3 and M4
are involved in the TG binding site. Modification of TG aliphatic side
chain extensions to impart a more hydrophobic interaction have shown a
net loss of inhibitory potency in endoplasmic reticulum Ca
pumps
(27) , indicating that the hydrophobic component is
not critical for binding. Weaker perturbation was achieved in TG
diastereoisomers showing a 3000-fold decrease in inhibitor potency. The
detergent-treated SRV, in addition to their ability for coupled
vectorial Ca
transport
(23) with a
2-5-fold decrease in affinity for
Ca
(21) , showed a 7-fold acceleration of the
TG off rate constant (Fig. 2). Thus, intercalation of detergent
molecules does not impede TG diffusion from its binding site. The fact
that TG stabilized the partially solubilized ATPase in the absence of
Ca
implies that two or more transmembrane helices
must be involved in the TG binding site. TG promises to be a useful
stabilizer for studies involving lipid removal by detergent.
-ATPase of sarcoplasmic
reticulum. Christensen et al.(28) measured inhibition
at 0.17 nM in bovine cerebellar microsome Ca
pumps, and we have demonstrated an apparent
K
of 2.2 pM or less in
Ca
-ATPase of sarcoplasmic reticulum. TG provides a
useful probe for kinetic investigation of the partial reactions in the
ATPase of sarcoplasmic reticulum, notably in the analysis of
Ca
binding, as well as the
Ca
-ATPases in cellular systems where knowledge of
rates of diffusion and an estimate of the rates of inhibition are
required.
and
E
, conformations of the non-phosphorylated enzyme
in the absence and presence of EGTA, respectively;
E
P and E
P, phosphorylated
forms of E
and E
,
respectively; SERCA, sarco- and endoplasmic reticulum.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.