©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetics of Thapsigargin- Ca-ATPase (Sarcoplasmic Reticulum) Interaction Reveals a Two-step Binding Mechanism and Picomolar Inhibition (*)

George A. Davidson (§) , Richard J. Varhol

From the (1) Medical Research Council Biomembrane Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, Cape Town, South Africa

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

Thapsigargin (TG)() 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.

TG appears to inhibit the Ca-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.


EXPERIMENTAL PROCEDURES

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 CaATP 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-ATPase by Cross-linking

SR vesicles (0.5 mg/ml), were incubated at 25 °C in 50 mM MOPS/tetramethylammonium chloride, pH 8.1, 50 µM CaCl, 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 ETG are active and inactive enzyme species with bound TG, respectively.

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 Kwas calculated as shown in Equation 1.

On-line formulae not verified for accuracy


RESULTS

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).


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 EP to EP 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.

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 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 10M 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) .

ATPase activity using the coupled enzyme assay is shown in Fig. 1. Addition of Ca 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.

ATPase activity of cross-linked SRV (E) 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, EP2Ca, 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.

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. 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).

Detergent has been shown previously to denature the Ca-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 ETG 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 ETG conformation. These data were repeated for cross-linking the active site with glutaraldehyde, which had no effect on the fluorescence change induced by TG.

The observed rate constant (k) for TG binding was a monoexponential with a rate of 25 s at 26 °C (Fig. 3, inset).

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 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.

An Arrhenius plot (Fig. 4) of the observed binding rate constants (k) 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.

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*TG and ETG are precursor and inhibited conformations, respectively.

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 was independent of TG concentration (Fig. 4, inset), whereas low values for k (<10M 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 10M 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 ETG (Fig. 3, inset).

Determination of remaining rate constants was restricted by the above findings. Values greater than 0.5 s for k in combination with 25 s for k led to subsaturation of ETG 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.

The best fit to the data therefore yielded rate constants of k = 5 10M 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 10M 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 ETG ECaTG 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.

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 10M 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.

Finally, the present study provides information on the use of TG in kinetic studies of the Ca-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 Kof 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: TG, thapsigargin; SR, sarcoplasmic reticulum; SRV, SR vesicles; MOPS, 4-morpholinepropanesulfonic acid; E and E, conformations of the non-phosphorylated enzyme in the absence and presence of EGTA, respectively; EP and EP, phosphorylated forms of E and E, respectively; SERCA, sarco- and endoplasmic reticulum.


ACKNOWLEDGEMENTS

We thank Dr. P. Adams for assistance in kinetic analysis and Professor M. C. Berman for conceptualization of the transfer methodology.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.