Lakshmi D Devanur
email: lakshmi.devanur@kcl.ac.uk

Robert W Evans

Patricia J Evans

Robert C Hider

Chelator-facilitated removal of iron from transferrin: Relevance to combined chelation therapy

Keywords: Iron chelation, enhanced scavenging activity of iron chelators, transferrin, deferiprone, desferrioxamine, hydroxypyridin-4-ones desferrioxamine, DTPA, diethylenetriaminepentacetic acid, FO, ferrioxamine, HPO, hydroxypyridin-4-one, NTBI, non-transferrin-bound iron, Tf, transferrin

Current iron chelation therapy consists primarily of the use of desferrioxamine (DFO), which has to be administered via intravenous infusion, together with deferiprone and deferasirox, which are orally active chelators. These chelators, although effective at decreasing the iron load, are associated with a number of side effects. Grady suggested that the combined administration of a smaller bidentate chelator and a larger hexadentate chelator, such as DFO would result in greater iron removal than either chelator alone (Grady, Bardoukas & Giardina (1998) Blood 92, 16b). This, in turn, could lead to a decrease in chelator dose. To test this hypothesis the rate of iron transfer from a range of bidentate hydroxypyridin-4-one (HPO) chelators to DFO was monitored. Spectroscopic methods were utilised to monitor the decrease in the concentration of the Fe-HPO complex. Having established that the shuttling of iron from the bidentate chelator to DFO does occur under clinically relevant concentrations of chelator, studies were undertaken to evaluate whether this mechanism of transfer would apply to iron removal from transferrin. Again, the simultaneous presence of both a bidentate chelator and DFO was found to enhance the rate of iron chelation from transferrin at clinically relevant chelator levels. Deferiprone was found to be particularly effective at 'shuttling' iron from transferrin to DFO, probably due to its small size and relative low affinity for iron compared with other analogous HPO chelators.

3

INTRODUCTION.

Although current chelation therapy is effective at improving life expectancy of individuals with transfusion-dependent anaemias, its use causes numerous disadvantages. Desferrioxamine (DFO) is not orally active and must be administered by subcutaneous infusion over 8h periods up to 5 times per week. This property decreases compliance with the drug. Two orally active iron chelators have also been approved for clinical use; namely, deferasirox [START_REF] Cappellini | A phase 3 study of deferasirox (ICL670), a once-daily oral iron chelator, in patients with betathalassemia[END_REF] and deferiprone (1,2-dimethyl-3-hydroxypyridin-4-one) [START_REF] Hider | The design and properties of 3hydroxypyridin-4-one iron chelators with high pFe(3+) values[END_REF]. A comparative study of the safety and efficacy of deferiprone and DFO in -thalassaemia major patients has demonstrated a similar ability to remove iron from the liver, but a greater increase in cardiac function was observed with deferiprone treatment [START_REF] Peng | Safety monitoring of cardiac and hepatic systems in b-thalassemia patients with chelating treatment in Taiwan[END_REF]. Interest in the application of iron chelators in the treatment of non iron overload diseases has also increased over the last 5 years with considerable effort being concentrated in two important fields, the treatment of cancer [START_REF] Kalinowski | Future of toxicology-iron chelators and differing modes of action and toxicity: The changing face of iron chelation therapy[END_REF] and the treatment of neurodegeneration [START_REF] Gaeta | The crucial role of metal ions in neurodegeneration: the basis for a promising therapeutic strategy[END_REF][START_REF] Boddaert | Selective iron chelation in Friedreich ataxia: biological and clinical implications[END_REF].

It has previously been suggested that the combined administration of a bidentate and a hexadentate chelator can lead to enhanced scavenging activity of iron from certain ironcontaining proteins [START_REF] Grady | Iron chelation: combined therapy could be a better approach[END_REF]. In principle, the smaller, kinetically labile bidentate chelator can rapidly access intracellular iron pools. The scavenged iron is subsequently transferred to the kinetically inert hexadentate chelator, forming a more stable complex, which is excreted. In this way, the bidentate chelator acts as a 'shuttle' molecule and the hexadentate chelator as a 'sink'. The concept of combined chelation therapy has previously been utilised to remove excess metals from the body in the cases of both plutonium and cadmium poisoning [START_REF] Schubert | Mixed ligand therapy for plutonium and cadmium poisoning[END_REF]. A combination of diethylenetriaminepentacetic acid (DTPA) and salicylic acid, for instance, was utilised in the removal of plutonium. Combined administration of these two chelators had a greater effect than either DTPA or salicylic acid alone.

Enhanced scavenging, if operational, will be dependent on the transfer of iron from the bidentate to the hexadentate chelator (Figure 1). It is therefore of interest to investigate whether appreciable rates of transfer can occur at clinically relevant chelator concentrations.

It has been previously established that DFO cannot remove iron from transferrin at physiological concentrations, probably due to its relatively large size [9]. However, it is hypothesised that in S t a g e 2 ( a ) P O S T -P R I N T 4 the presence of a smaller bidentate chelator, iron could be 'shuttled' from transferrin to DFO.

Experiments investigating possible synergistic effects of combined administration of chelators in the removal of iron from transferrin have been carried out previously [START_REF] Pollack | Chelate mediated transfer of iron from transferrin to desferrioxamine[END_REF][START_REF] Pollack | Iron removal from transferrin. An experimental study[END_REF]. However, these studies were undertaken using high chelator concentrations. The observation that deferiprone is able to chelate transferrin-bound iron [9], indicates that it may also be able to access other iron pools that are inaccessible to DFO. Thus, the combination of deferiprone with DFO may permit the chelation of iron, which would not normally be removed by DFO therapy alone. As deferiprone has been shown to be able to access and remove iron from cardiac tissue [START_REF] Peng | Safety monitoring of cardiac and hepatic systems in b-thalassemia patients with chelating treatment in Taiwan[END_REF], the combination of deferiprone and DFO, for instance, might be expected to facilitate the removal of cardiac iron more efficiently.

This paper investigates the transfer of iron from bidentate chelators to DFO in an attempt to establish whether appreciable rates are achievable under clinical conditions and whether this phenomenon will lead to an increase in the rates of iron removal from transferrin, when compared to either chelator alone.

EXPERIMENTAL.

Materials.

1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone) was purchased from Sigma Chemical Co.

(Poole, U.K.). Nine hydroxypyridin-4-one (HPO) chelators were used in this study and each was prepared as previously described [START_REF] Dobbin | Synthesis, physicochemical properties, and biological evaluation of N-substituted 2-alkyl-3-hydroxy-4(1H)-pyridinones: orally active iron chelators with clinical potential[END_REF][START_REF] Liu | Design, synthesis and evaluation of novel 2-substituted 3-hydroxypyridin-4-ones: Structureactivity investigation of metalloenzymes inhibition by iron chelators[END_REF][START_REF] Liu | Design of clinically useful iron(III) selective chelators[END_REF][START_REF] Piyamongkol | The design and synthesis of 3-hydroxide-4-one chelators[END_REF][START_REF] Piyamongkol | Novel synthetic approach to 2-(1'hydroxyalkyl)-and 2-amido-3-hydroxypyridin-4-ones[END_REF]. These chelators fall into three main categories, the 'low' affinity chelators (deferiprone, CP95 and CP40, pFe 3+ < 20), chelators with a large ring substituent (CP357 and CP358) and the 'high' affinity chelators (CP363, CP375, CP502 and CP511, pFe 3+ > 20

Purification of transferrin

Commercially available transferrin was not utilised for the iron removal studies since these preparations are frequently contaminated with other proteins and low molecular weight molecules, which could interfere with the iron removal process. The presence of such low molecular weight contaminants, for instance citrate, could facilitate the transfer of iron from transferrin to DFO thereby resulting in artefacts. For these reasons, transferrin was purified from human serum. Solid ammonium sulphate was added to the serum sample (approximate volume 500ml) to give a 45% saturated solution. The amount of ammonium sulphate required was calculated as previously described [START_REF] Dawson | Data for biochemical research 3 rd Ed, Chapter 23[END_REF]. The solution was left stirring overnight at 4C and subsequently centrifuged for 30 min at 8,000g at 4C. A further amount of ammonium sulphate was added to the supernatant to increase the saturation to 70% and the solution was left to equilibrate for 3h at 4C with stirring. The solution was then centrifuged for 1h at 16,000g at 4C, after which the supernatant was discarded and the pellet was re-dissolved in 50mM sodium bicarbonate. The sample was then dialysed using 12kDa molecular weight cut-off tubing (Medicell International Ltd., London) overnight against de-ionised water at 4C to remove the ammonium sulphate. 0.1M Fe-NTA solution (calculated as in equation 1) was added to completely saturate the transferrin present in the sample. In all cases Fe-NTA was prepared by the addition of the free acid to ferric chloride in a 1:1 ratio. This solution was freshly prepared at the time of use. Sodium bicarbonate was then added to the solution to give a final concentration of 5mM, together with a solution of Tris/HCl (pH 8), which was added to a final concentration of 10mM.

…………….(1)

The partially purified transferrin was applied to a DEAE Sepharose column (30cm, radius 4cm) equilibrated with 500ml of 20mM Tris/HCl (pH 8). Both albumin and caeruloplasmin have a greater net negative charge than transferrin and therefore displace it from the DEAE Sepharose column. As the solution was passed through the column, albumin (brown) and caeruloplasmin (blue) were found to bind more tightly and transferrin (red) was collected in the eluent. Albumin and caeruloplasmin were displaced from the column by elution with 2M NaCl. The column was 6 re-equilibrated with 20mM Tris/HCl (pH 8) and the process repeated twice until transferrin bound to the column without being displaced, indicating that the other proteins had been removed. Once bound to the column, transferrin was eluted using a salt gradient of 20mM Tris/HCl and 0.2M NaCl. Fractions were pooled and samples run on an SDS gel to confirm the purity of the purified transferrin. Selected fractions were then dialysed against de-ionised water overnight at 4C and subsequently freeze-dried.

Preparation of diferric transferrin

Although an excess of Fe-NTA was added to the solution to completely saturate transferrin during the purification process, a mixture of all four forms of transferrin are usually present in the final product. In order to completely saturate the transferrin, the freeze-dried product was dissolved in 0.1M sodium bicarbonate and 0.1M Fe-NTA was added, the volume required being calculated using equation 1. A Sephadex G-25 column (30cm, radius 3cm) was equilibrated with 25mM ammonium bicarbonate and the transferrin solution was applied. This process removed excess Fe-NTA. The transferrin sample was again freeze-dried and stored at -20C.

Methods.

Transfer of iron from hydroxypyridinones to desferrioxamine.

A 1mM stock solution of hydroxypyridin-4-one (HPO) was prepared in water and treated with atomic absorption standard iron. The solution was made up with 50mM MOPS (pH 7.4). The final total concentrations of iron and HPO in the solution were 10M and 30M, respectively.

The HPOs are bidentate chelators, therefore a 3-fold molar excess of HPO over iron is required to fully complex the iron in solution. When an additional 300M citrate or 100M HPO was required, a calculated amount of either citric acid or HPO was subsequently added to the preformed iron(III) complex.

A solution of DFO was prepared in water and added to the Fe-HPO solution, to give a final concentration of 10M. The transfer of iron was monitored by spectroscopy at room temperature. The Fe-DFO complex has an absorbance maximum at 430nm and the Fe-HPO complexes have maxima in the region 450nm-460nm, therefore the transfer of iron from one Iron removal from transferrin -urea gels.

Diferric transferrin (32.5M) was prepared in 50mM MOPS buffer (pH 7.4). A combination of bidentate chelators followed by DFO were added and the sample was incubated for 2h at 37C.

The sample was then run on a urea gel. 6M urea gels were prepared as previously described [19].

Transferrin samples were dissolved in sample buffer prior to loading on the gel. The sample buffer contained 0.01% (w/v) bromophenol blue and 10% (w/v) glycerol in running buffer. The gel was run at 80V overnight. The bands were visualised by the addition of Coomassie Blue stain. 0.25% (w/v) Coomassie Blue was dissolved in 90ml acetic acid and 455ml methanol and made up to 1 litre with de-ionised water. After de-staining in a solution of 10% acetic acid and 10% methanol, the relative percentages of each of the forms of transferrin were calculated using densitometry scanning.

When iron removal from transferrin was investigated in the presence of serum, the sample buffer was replaced with Rivanol solution. Rivanol is a dye, which precipitates out the γ-globulins leaving transferrin in the supernatant [19]. A solution of Rivanol was prepared by the addition of 0.375% (w/v) Rivanol and 10% (w/v) glycerol to the running buffer and made up to volume with de-ionised water. A 25l aliquot was removed from the serum mixture and 200l of Rivanol solution added. This mixture was centrifuged at 2,000g for 3 minutes and 20l of the supernatant was loaded onto the gel. The gel was then run at 80V overnight. Normal serum samples were iron loaded with FeNTA. The amount of FeNTA added was calculated so that the transferrin would not be completely saturated, thereby decreasing the likelihood of the presence of nontransferrin-bound iron (NTBI). The approximate saturation of transferrin in these experiments was 80% as determined by densitometry scanning of urea gels.

Percentage iron saturation of transferrin was calculated by determining the percentage of each of the four forms of transferrin, using densitometry scanning of urea gels and calculated using equation 2.

S t a g e 2 ( a ) P O S T -P R I N T

Percentage Iron Saturation = % Diferric Tf + ½ (% N-monoferric Tf) + ½ (% C-monoferric Tf)

.….…….( 2)

Iron removal from transferrin -ferrioxamine (FO) assay.

Diferric transferrin (32.5M) in 20mM MOPS (pH 7) was incubated with a combination of bidentate chelators and DFO at varying concentrations at 37C. Aliquots were removed at regular time intervals and ultrafiltered through 30kD filters (Whatman, Brentford, U.K.) for 5 minutes at 15,000g to remove the protein; the filtrate (50l) was then injected on to the HPLC column for analysis.

A metal-free HPLC system (Waters Bio-system 625 with 911 Photodiode Array detector) with PEEK (polyether-ethyl ketone) tubing was used for these measurements in order to prevent iron contamination of the samples. A Chrompak octadecylsilane (ODS) glass column was adopted and the flow rate was set at 0.4ml/min. The temperature of the injector was maintained at 4C

during the analysis to minimise any changes in the rate of reaction while the samples were in the autosampler. The detection wavelength was set at 430nm to detect FO. The mobile phase consisted of 20mM phosphate buffer pH 7, with 6% acetonitrile, yielding a retention time of 6 mins for FO. All samples were prepared in 20mM MOPS (pH 7). Concentrations of FO in the samples were determined by use of a standard curve of FO.

Speciation plots.

Speciation plots of the iron complexes of deferiprone and CP511 were generated using the computer programme Hyperquad Simulation and Speciation (HySS) [START_REF] Alderighi | Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species[END_REF]. The parameters incorporated into this model were logK(FeOH) = 2.563, logK(Fe(OH) 2 ) = 6.205, logK(Fe(OH) 4 ) = 21.883, logK(Fe 2 (OH) 2 ) = 2.843, logK(Fe 3 (OH) 4 ) = 6.054 [START_REF] Baes | The hydrolysis of cations[END_REF]. For deferiprone, logK 1 = 15.1, logK 2 = 11.51, logK 3 = 9.27 [START_REF] Motekaitis | Stabilities of the iron(III) chelates of 1,2dimethyl-3-hydroxy-4-pyridinone and related ligands[END_REF], pKa 1 = 3.68 and pKa 2 = 9.77 [START_REF] Liu | Synthesis, physicochemical characterization, and biological evaluation of 2-(1'Hydroxyalkyl)-3hydroxypyridin-4-ones: novel iron chelators with enhanced pFe 3+ values[END_REF]. For CP511, logK 1 = 14.5, logK 2 = 10.49, logK 3 = 7.47, pKa 1 = 2.32 and pKa 2 = 6.66 [START_REF] Piyamongkol | Novel synthetic approach to 2-(1'hydroxyalkyl)-and 2-amido-3-hydroxypyridin-4-ones[END_REF].

RESULTS.

Iron transfer between hydroxypyridinones and desferrioxamine.

An absorption trace recording iron transfer from CP358 to DFO is shown in figure 2. It can be seen that the transfer of iron from the bidentate chelator to DFO follows a biphasic pattern, with a relatively fast initial transfer followed by a slower phase. A similar pattern was observed for the rate of iron transfer from all the HPO chelators tested in this study. This observation can best be explained by a two-step iron transfer mechanism; the initial fast phase corresponding to the formation of a ternary Fe.HPO.DFO mixed ligand complex followed by the slower formation of the Fe-DFO complex (Figure 1). The initial rates of transfer of iron from various Fe-HPO complexes (10M:30M) to 10M DFO are presented in figure 3. It can be seen that there is an appreciable variation in the rate of iron transfer. CP95 was found to be the most effective at transferring iron under the experimental conditions adopted. The rate of iron transfer from deferiprone and CP40 to DFO was approximately 1.5-fold slower than that observed from CP95 to DFO. CP363 and CP375, which are higher affinity chelators, had rates of iron transfer similar to that of deferiprone. The slowest rates of iron transfer to DFO were observed with CP357, CP358, CP506 and CP511. These iron transfer experiments were repeated in the presence of 300M citrate, a physiological iron chelator present in the plasma and the rate of transfer of iron from the majority of the HPO chelators to DFO was reduced by approximately 1.5-fold.

However, the converse was noted in the case of iron transfer from CP357 and CP358 to DFO, the addition of citrate leading to a 2-fold increase in the rate of iron transfer to DFO (data not shown).

Under in vivo conditions, a major proportion of the administered dose of a chelator would be present in the unchelated form. It was therefore decided to examine the rate of iron transfer from HPO to DFO in the presence of 100M excess HPO chelator (Figure 4). The data is presented as the ratio of the rate of iron transfer to DFO in the presence of excess HPO chelator to the rate of iron transfer to DFO in the presence of a 1:3 ratio of iron to HPO (that is, no excess chelator). All of the rates are markedly reduced in the presence of excess chelator. Significantly, the rates of iron transfer from the lower affinity chelators CP95 and deferiprone were the least affected by the presence of excess HPO. The presence of excess HPO has the greatest effect on the transfer of iron from the chelators CP357, CP358, CP506 and CP511.

S t a g e 2 ( a ) P O S T -P R I N T

Iron transfer from the Fe-DFO complex, ferrioxamine (FO) to deferiprone was investigated at both pH 5.5 and pH 7.4 in order to verify whether FO will act as an efficient 'sink' and prevent the re-distribution of iron under both extracellular conditions and conditions found in the lysosome. At deferiprone concentrations less than 300M, there was no detectable transfer of iron to deferiprone at either pH 5.5 or 7.4. As the concentration of deferiprone was increased above 300M, transfer of iron occurred, with the transfer at pH 5.5 being greater than at pH 7.4

(Figure 5). However, these latter deferiprone concentrations would not be achieved under clinical conditions.

Iron exchange between transferrin and chelating agents.

Having established that the transfer of iron from the bidentate chelators to DFO will occur under clinically relevant chelator concentrations, it was of interest to examine whether this 'shuttling' of iron would result in enhanced scavenging of iron from transferrin. An example of a urea gel is shown in figure 6. Lane 1 shows a single band corresponding to diferric transferrin. Lanes 2 and 3 show the presence of 4 bands, namely, apotransferrin, C-monoferric transferrin, N-monoferric transferrin and diferric transferrin, following a 2h incubation of diferric transferrin with 200M and 600M deferiprone, respectively. The iron saturation of transferrin decreases with increased chelator concentration. A combination of bidentate chelators and DFO were incubated with transferrin and iron removal was monitored using urea polyacrylamide gel electrophoresis. Three chelators were chosen for this study, namely, deferiprone, CP358 and CP502, both CP358 and CP502 are analogues of deferiprone (Table 1). CP358 is a larger chelator, which has a bulky side-chain and is therefore not expected to be able to efficiently chelate iron from the ironbinding sites of transferrin. CP502 is a small, higher affinity iron chelator, which should be more 2). In all cases an increase in iron removal from transferrin was observed when DFO was combined with HPO compared to HPO alone, however statistical significance was not reached. When the concentration of HPO was further reduced to 50M, while maintaining the DFO concentration at 10M (Table 2), weakly enhanced scavenging of iron was seen when DFO was combined with deferiprone and CP358, but not with CP502.

The rate of iron removal from 80% saturated transferrin in serum was also investigated.

Combinations of HPO and DFO were incubated with the serum samples (Figure 7), the amount of iron removed by 100M deferiprone alone in the presence of serum was found to be comparable to that removed from purified transferrin (Table 2). Addition of 10M DFO did not result in an increase in the amount of iron removed from transferrin when compared to 100M HPO alone.

However, in the presence of equimolar concentrations of deferiprone and DFO, significant increases in iron removal from serum transferrin were observed when compared with deferiprone alone. The addition of 200M deferiprone did not result in any further increase in iron removal compared to 100M deferiprone. The combination of CP502/DFO did not result in enhanced scavenging activity even at high concentrations of chelator, however the combination of 100M

CP358 and 100M DFO did result in marginal synergistic effects (Figure 7).

Iron removal from iron-saturated transferrin was also monitored using the ferrioxamine (FO) assay (Figure 8). The addition of 10M DFO alone resulted in marginal (non significant) iron removal from transferrin. The addition of 30M deferiprone resulted in a large increase in the rate of iron removal. Addition of 100M deferiprone to 10M DFO resulted in slightly greater FO formation compared to that observed in the presence of 30M deferiprone and 10M DFO (data not shown). It is clear that the FO assay is more sensitive than the urea gel method for determining iron removal from transferrin at these lower chelator concentrations.

DISCUSSION

Although current chelation therapy, namely the use of either desferrioxamine or deferiprone is effective at increasing life expectancy of patients with transfusion-dependent anaemias, there are some disadvantages associated with their use. It has been hypothesised that the combined administration of these two chelators may increase the total amount of iron removed from the body, without the need for increasing chelator dose [START_REF] Grady | Iron chelation: combined therapy could be a better approach[END_REF]. Studies have been carried out which indicate that combined chelation therapy may not only prove to be effective at decreasing body iron burden, but may also decrease cost and increase compliance with these pharmaceuticals [START_REF] Gomber | Comparative efficacy of desferrioxamine, deferiprone and in combination on iron chelation in thalassemic children[END_REF].

It has been suggested that enhanced scavenging activity of combined chelation therapy is based on the transfer of iron from the smaller bidentate chelator to the hexadentate chelator (Figure 1)

and is therefore dependent on the rate of iron transfer between the two [START_REF] Grady | Iron chelation: combined therapy could be a better approach[END_REF]. In this work, the rate of iron transfer between a range of bidentate HPO chelators and DFO was determined using UVvisible spectroscopy (Figure 2). The lower affinity chelators deferiprone and CP95 (pFe 3+ values 19.4 and 19.9 respectively) were the most effective at iron transfer to DFO. The rates of iron transfer from CP357 and CP358 to DFO were approximately 2-fold lower than the rate of iron transfer from CP95. Both of these compounds possess long side-chains at the 2-position on the pyridinone ring. The presence of this side-chain could possibly interfere with the formation of the postulated DFO ternary complex (Figure 1). The high affinity chelators are the slowest iron donors to DFO, and this is probably due to a decrease in the rate of dissociation of the Fe(HPO) 3 complex to the intermediary Fe(HPO) 2 complex [27], which is suggested to be necessary for DFO chelation (Figure 1). From these data it appears that there is a limited relationship between the pFe 3+ value and the rate of transfer of iron to DFO, with an increase in pFe 3+ resulting in a decrease in the rate of transfer. However, other structural factors must also be involved in controlling the rate of transfer of iron from the bidentate chelator to DFO, since both CP95 and CP40, which have a similar pFe 3+ values to deferiprone have much slower rates of transfer. In the presence of citrate, the rate of transfer of iron from HPO to DFO was decreased by approximately 1.5-fold in most cases (data not shown). The high concentration of citrate could possibly encourage transfer of iron to citrate. Once formed, dimeric and oligomeric iron-citrate species [START_REF] Evans | Nature of non transferrin bound iron: studies on iron citrate complexes and thalassaemic sera[END_REF], which are not readily accessible to DFO, would be predicted to retard the rate of iron transfer to DFO. The marked retardation in rate of exchange in the presence of excess chelator (Figure 4) is almost certainly associated with a shift in equilibrium of the dissociation process (Figure 1). In the presence of excess chelator, the equilibrium will adjust, favouring the formation of the Fe(HPO) 3 complex, which will not donate iron to DFO, thereby reducing the rate of transfer. The lower affinity chelators such as deferiprone, CP40 and CP95 are not as greatly affected as the higher affinity chelators and the bulky chelators. Thus, even in the presence of excess chelator, the lower affinity chelators are more likely to dissociate than the higher affinity chelators and therefore the rate of transfer of iron from these chelators is likely to be greater. The relative proportions of the partially co-ordinated Fe(HPO) 2 complex under any given condition can be calculated from speciation plots. Comparing the speciation plots obtained for deferiprone and CP511 at pH 7.4 provides an insight into the reasons for the variation in rates of transfer of iron from these two chelators to DFO (Figure 9). The presence of a higher proportion of the Fe(deferiprone) 2 complex compared to the Fe(CP511) 2 complex, demonstrates that the higher pFe of CP511 results in a decrease in the dissociation of the Fe(HPO) 3 complex compared to deferiprone and thereby decreases the rate of iron transfer to DFO. Even in the presence of a 3fold molar excess of HPO compared to iron, a proportion of partially coordinated Fe(HPO) 2 will exist in solution, the exact concentration of which depending on the pFe of the chelator.

To determine the efficacy of DFO as a 'sink' molecule, the rate of transfer of iron from the Fe-DFO complex to deferiprone was investigated. The slow rates of transfer demonstrate that once chelated by DFO, it is highly improbable that the iron could be mobilised under clinically achievable HPO concentrations. Thus, although greater iron removal was observed at pH 5.5, the concentration of deferiprone required to mobilise iron was much higher than would be found under clinical conditions. Clearly DFO acts an effective 'sink' molecule and is a good candidate for combined chelation therapy with HPOs such as deferiprone.

Following on from the experiments demonstrating that transfer of iron from bidentate chelators to DFO occurs under clinically relevant concentrations of chelator, the interaction of chelators with transferrin was investigated. Transferrin iron is not readily accessible to DFO and so it was of interest to determine whether this iron could be chelated in the presence of both a bidentate HPO and DFO. At high deferiprone concentration the combination of deferiprone and DFO resulted in marginally greater iron removal from transferrin compared to deferiprone alone. Having demonstrated that enhanced scavenging activity is possible for iron removal from transferrin at high deferiprone concentrations, chelator concentrations were reduced to simulate conditions achievable in vivo (Table 2). In the absence of DFO, CP502 was found to be the most effective at removing iron from transferrin. CP502 has the highest affinity for iron of these three chelators and is also relatively small, thereby enabling access to the iron-binding sites on transferrin. CP358 would be restricted in its access to the iron-binding sites on transferrin due to the larger size of the molecule. In the presence of DFO there is also a variation in the enhancement in iron removal observed with each HPO chelator, with a greater increases observed with the combination of deferiprone and DFO (approximately 2.4 fold increase in iron removal, compared to deferiprone alone) and lower increases observed with the combination of CP502/DFO and CP358/DFO (approximately 1.3 fold and 1.7 fold increases in iron removal, respectively, compared to either HPO chelator alone). The reason for this discrepancy in scavenging activity between the HPO chelators is probably due to their relative abilities to transfer iron to DFO. The rate of iron transfer from CP502 and CP358 to DFO is slower when compared to deferiprone (Figure 3).

Although purified transferrin has been shown to be an effective model for iron removal studies, experiments were also carried out using iron-loaded serum samples (Figure 7). The total iron removed from transferrin in serum by 100M HPO alone was comparable to that achieved from purified transferrin. The addition of equimolar concentrations of deferiprone and DFO resulted in appreciable synergistic effects. In contrast, the combinations of CP358/DFO and CP502/DFO failed to demonstrate such an effect. The most probable reason for this marked difference in behaviour is that both CP358 and CP502 do not donate iron(III) to DFO as readily as deferiprone (Figures 3 and4).

It was not possible to monitor iron removal from transferrin in the presence of low concentrations of bidentate chelator (for example, 30M) using urea gels due to the insensitivity of the method, therefore, an alternative procedure based on the quantification of FO was adopted to monitor iron removal from transferrin at lower concentrations of bidentate chelator. The use of this method provided a time-course for the removal of iron. Any increase in FO formation in the presence of deferiprone must occur due to shuttling of iron from transferrin to DFO by the bidentate chelator, as DFO is unable to directly remove iron from transferrin under these conditions. Iron removal using a combination of 30M deferiprone and 10M DFO results in faster iron removal compared to DFO alone (Figure 8). The experiments described in this work demonstrate that the combination of the bidentate chelator, deferiprone, and hexadentate chelator, DFO, results in enhanced scavenging activity of iron from transferrin at clinically achievable levels of both chelators. In the presence of DFO, this mobilised iron will be donated to the hexadentate ligand (Figure 3 and4). Thus, it is possible to predict that the simultaneous administration of the two ligands will lead to more efficient removal of iron from iron-overloaded patients. Furthermore, it is suggested that the combination of a chelator such as deferiprone with a 'sink' molecule, such as DFO, could further improve the efficacy of chelation of tissue iron by ligand exchange of iron. It should be noted, however, that enhanced scavenging activity of iron may also lead to increased toxicity due to enhanced iron removal from essential iron-containing enzymes. HPO chelators have been shown to inhibit lipoxygenase activity in vitro [START_REF] Abeysinghe | The environment of the lipoxygenase iron-binding site explored with novel hydroxypyridinone iron chelators[END_REF], an effect, which may be accentuated in the presence of a 'sink' molecule such as DFO. Inhibition of ribonucleotide reductase [START_REF] Cooper | The relationship of intracellular iron chelation to the inhibition and regeneration of human ribonucleotide reductase[END_REF] and tyrosine and tryptophan hydroxylases [START_REF] Waldmeier | Inhibition of catechol-omethyltransferase (COMT) as well as tyrosine and tryptophan hydroxylase by the orally active iron chelator, 1,2-dimethyl-3-hydroxypyridin-4-one (L1,CP20), in rat brain in vivo[END_REF] have also been shown to occur in the presence of HPO chelators. Limited information exists regarding the toxic effects of combined chelation therapy.

Combined chelation therapy with deferiprone and DFO has been demonstrated to enhance iron scavenging activity in vivo in a number of clinical cases and has been shown to be especially beneficial in decreasing cardiac iron burden [START_REF] Fabio | Reversal of cardiac complications by deferiprone and deferoxamine combination therapy in a patient affected by a severe type of juvenile hemochromatosis (JH)[END_REF]. This phenomenon has been attributed to the 'shuttling' of iron from the bidentate chelator to the hexadentate chelator [START_REF] Grady | Iron chelation: combined therapy could be a better approach[END_REF][START_REF] Link | Exploring the "iron shuttle" hypothesis in chelation therapy: Effects of combined deferoxamine and deferiprone treatment in hypertransfused rats with labeled iron stores and in iron-loaded rate heart cells in culture[END_REF]. It has previously been established that deferiprone removes iron from transferrin, however the present study is the first to conclusively demonstrate that in the presence of both deferiprone and DFO in vitro, the chelated iron is 'shuttled' onto DFO to form the FO complex, which, in the absence of deferiprone does not form. This phenomenon demonstrates the possibility of 'shuttling' iron from a bidentate chelator to a hexadentate chelator and the subsequent removal of iron from a source unavailable to DFO alone.

In summary, the results presented here demonstrate that in the presence of a bidentate and hexadentate chelator enhanced iron scavenging activity occurs, at clinically relevant chelator concentrations and that amongst the HPO chelators, deferiprone would appear to be particularly well suited for such applications. The fully co-ordinated Fe(HPO) 3 complex exists in equilibrium with the partially co-ordinated Fe(HPO) 2 complex in solution. DFO is able to form a ternary complex with Fe(HPO) 2 and subsequently displace the remaining HPO molecules from iron.

S t a g e 2 (

 2 a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society chelator to the other can be monitored by UV-visible spectroscopy. In most cases the absorbance was monitored at a wavelength of 455nm. The rates of iron transfer were calculated using a method proposed by Rose in 1964 [18].

S t a g e 2 (

 2 a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  effective at iron removal from transferrin when compared to deferiprone. No iron removal was observed in the presence of 100M DFO alone. Transferrin was incubated with a range of deferiprone concentrations in the presence and absence of 100M DFO (data not shown). At deferiprone concentrations greater than 1mM, a large percentage of the iron was removed from transferrin (approximately 60%). At this deferiprone concentration, the further addition of 100M DFO resulted in a synergistic increase in iron removal with approximately 75% iron removal from transferrin. As the concentration of deferiprone was increased up to a concentration of 5mM almost all the iron was removed from transferrin, but the further addition of DFO had virtually no effect. Following a 25mg/kg dose of deferiprone in man, the maximal S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society concentration of the drug in the plasma is approximately 100M [24]. The steady state concentration of DFO during a 40mg/kg intravenous infusion is 10M [25]. Iron removal from transferrin was therefore investigated in the presence of a range of HPO chelators (100M) in the presence and absence of 10M DFO (Table

S t a g e 2 (

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S t a g e 2 (

 2 a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society 13

S t a g e 2 (

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  The complete conversion of DFO to FO takes approximately S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society 5h. A similar effect was observed with 100M deferiprone. This observation confirms the findings previously reported, relating to the iron removal from transferrin by deferiprone [9].

S t a g e 2 (

 2 a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 .

 1 Figure 1. The proposed mechanism of iron transfer from Fe-HPO to DFO.

Figure 2 .

 2 Figure 2. Rate profile of iron transfer from CP358 to DFO.

Figure 3 .

 3 Figure 3. Rate of iron transfer from Fe-HPO to DFO.

Figure 4 .

 4 Figure 4. Ratio of the rate of iron transfer from Fe-HPO in the presence of excess HPO to DFO compared to the rate of iron transfer from a 1:3 molar concentration of Fe:HPO to DFO.

Figure 5 . 5 (

 55 Figure 5. Rate of iron transfer from DFO to deferiprone

Figure 6 .

 6 Figure 6. A urea gel showing the four forms of transferrin.

Figure 7 .

 7 Figure 7. Iron removal from serum transferrin.

Figure 8 .

 8 Figure 8. Iron removal from transferrin measured by FO formation following incubation with deferiprone and DFO.

Figure 9 .

 9 Figure 9. Speciation plots of iron-deferiprone and iron-CP511 complexes.

Figure 1 Fe

 1 Figure 1

  Figure 2

Figure 3 0

 3 Figure 3

Figure 4 0

 4 Figure 4

Figure 5 Rate 32 Figure 6

 5326 Figure 5

  Figure 7

  Figure 8

Table 1 . Structure of hydroxypyridin-4-onesTable 2 . Percentage iron removal from transferrin in presence of 50M HPO  10M DFO

 12 Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

	and 100M HPO  10M DFO.
	32.5M diferric transferrin was incubated with 50/100M HPO in the presence and absence of
	10M DFO for 2h at 37C in 50mM MOPS (pH 7.4). Results are expressed as mean  s.d (n=3).
	No statistical significance was observed between the values for iron removal from HPO alone
	compared with HPO and DFO using the Students t test. Percentage iron removed was calculated
	using densitometry scanning.

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 1 .

 1

		O
			OH
				R 1	R 2	R 6	Ref
	R 6	N	R 2
		R 1
		Deferiprone	CH 3	CH 3	H	[12]
		CP40		CH 2 CH 2 OH	CH 3	H	[12]
		CP95		CH 2 CH 2 CH 3	C 2 H 5	H	[12]
		CP357		CH 3	CH 2 NHCOCH 2 CH 2 NHCOCH 3	CH 3	[13]
		CP358		CH 3	CH 2 NHCOCH 2 CH 2 CONHC 3 H 7	CH 3	[13]
		CP363		CH 3	CH(CH 3 )OCH 3	CH 3	[14]
		CP375		CH 3	CH(C 2 H 5 )OCH 3	CH 3	[15]
		CP502		CH 3	CONHCH 3	CH 3	[12]
		CP511		H	CONHCH 3	CH 3	[16]

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 .

 2

			50M HPO +		100M HPO +
		50M HPO		100M HPO
			10M DFO		10M DFO
	Deferiprone	4.7  2.6	6.0  2.1	5.8  1.9	13.9  5.2
	CP502	5.5  1.5	5.7  1.1	13.5  5.2	17.5  8.0
	CP358	2.8  1.7	4.8  1.4	8.3  6.2	14.0  2.2

S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 8 Oct 2007 as manuscript BJ20070823 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070823 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements:

LDD received a PhD studentship from the National Institutes of Health (grant number 1R01DK57645-01). This work was also supported by a component grant from the MRC (grant number G0000187).

S t a g e 2 ( a ) P O S T -P R I N T


Oluseye A Ogunbayo

Francesco Michelangeli
email: f.michelangeli@bham.ac.uk

THE WIDELY UTILIZED BROMINATED FLAME RETARDANT, TETRABROMOBISPHENOL A (TBBPA) IS A POTENT INHIBITOR OF THE SERCA Ca 2+ PUMP

Tetrabromobisphenol A (TBBPA) is currently the most widely used type of brominated flame retardant (BFR), employed to reduce the combustibility of a large variety of electronic and other manufactured products. Recent studies have indicated that BFRs, including TBBPA, are bioaccumulating within humans at relatively high concentrations. BFRs, including TBBPA have also been shown to be cytotoxic and potentially endocrine disrupting in a variety of cell types.

Furthermore, TBBPA has specifically been shown to cause disruption of Ca 2+ homeostasis within cells, which may be the underlying cause of its toxicity. In this study, we have demonstrated that TBBPA is a potent non-isoform specific inhibitor of the SERCA Ca 2+ -ATPase (apparent Ki 0.46 -2.3µM), thus, we propose that TBBPA inhibition of Ca 2+ ATPase contributes, at least in part, to Ca 2+ signalling disruption. TBBPA binds directly to the ATPase without the need to partition into the phospholipid bilayer. From activity data and Ca 2+ -induced conformational data, it appears that the major effect of TBBPA is to decrease the Ca 2+ ATPase affinity for Ca 2+ binding (increasing the K d from approx. 1µM to 30µM in the presence of 10µM TBBPA). Low concentrations of TBBPA can quench the tryptophan fluorescence of the ATPase and this quenching can be reversed by 2,5-di-tertbutylhydroquinone (BHQ) and 4-n-nonylphenol, but not thapsigargin, indicating that TBBPA and BHQ may be binding to similar regions in the Ca 2+ -ATPase.

INTRODUCTION

Ca 2+ plays an important role in many cellular signalling processes. Indeed, inadequate or prolonged elevations of intracellular [Ca 2+ ] levels may lead to deleterious effects, ranging from failure in signal transduction to apoptotic processes within and between cells [START_REF] Berridge | Calcium--a life and death signal[END_REF]. Therefore, Ca 2+ homeostasis in the cytosol is very crucial to cell viability and function, with the potential for serious consequences arising when normal control is disturbed. The sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) has long been implicated as playing a central role in the mechanism of Ca 2+ transport across the membrane, from the cell cytosol into the endoplasmic reticulum and this is one of the major mechanisms by which low levels of free cytosolic Ca 2+ concentration are maintained in the cell.

SERCA Ca 2+ pumps belong to the P-type family of ion transporters [START_REF] Toyoshima | Structural basis of ion pumping by Ca 2+ -ATPase of the sarcoplasmic reticulum[END_REF] and three isoforms of this pump is known to exist which have a tissue-specific distribution [START_REF] Wu | Localization and quantification of endoplasmic reticulum Ca 2+ -ATPase isoform transcripts[END_REF].

The activities of SERCA Ca 2+ pumps have been found to be altered by a wide spectrum of lipophilic molecules, including a number of environmental endocrine disrupters [START_REF] Michelangeli | Mechanism of inhibition of the (Ca 2+ -Mg 2+ -ATPase by Nonylphenol[END_REF][START_REF] Brown | Characterisation of a novel Ca 2+ pump inhibitor (bis-phenol) and its effects on intracellular Ca 2+ mobilization[END_REF][START_REF] Hughes | Estrogenic alkylphenols induce cell death by inhibiting testis endoplasmic reticulum Ca 2+ pumps[END_REF]. While the majority of these molecules have non-specific mechanistic effects on these pumps, some of these hydrophobic molecules interact specifically at distinct sites on the protein and in some cases these molecules have proved invaluable in elucidating mechanistic steps within the Ca 2+ transport processes [START_REF] Longland | The mechanism of inhibition of the Ca 2+ -ATPase by mastoparan. Mastoparan abolishes cooperative Ca 2+ binding[END_REF][START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF][START_REF] Wootton | The effects of the phenylalanine 256 to valine mutation on the sensitivity of sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA) Ca 2+ pump isoforms 1, 2, and 3 to thapsigargin and other inhibitors[END_REF][START_REF] Wictome | The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca 2+ , Mg 2+ -ATPase[END_REF].

Tetrabromobisphenol A (TBBPA) is a highly lipophilic halogenated aromatic molecule and currently the most widely used type of brominated flame retardant (BFR) [START_REF] De Wit | An overview of brominated flame retardants in the environment[END_REF]. TBBPA and other BFRs are employed as additives in the manufacture of printed circuit boards and polymers used to make electrical equipment, textiles, adhesives, foam furniture and insulating foams, in order to reduce the risk of combustion [START_REF] De Wit | An overview of brominated flame retardants in the environment[END_REF].

Due to their wide spread use and abundance, recent studies have shown that BFRs exist in the environment far from their location of production and / or use; and that the concentrations of some of the BFRs, both in the environment and in humans, are rapidly increasing [START_REF] Alaee | The significance of brominated flame retardants in the environment: current understanding, issues and challenges[END_REF]. Concentrations of various BFRs have been found at levels as high as 1 mg/ kg of dried sewage slurry [START_REF] Law | Levels and trends of brominated flame retardants in the European environment[END_REF] and up to 160 ng/g in some samples of human serum lipid (equivalent to ≈ 0.3µM) [14]. More specifically, TBBPA was found to be as high as 3.8 ng/ g lipid in serum samples from electronics workers [START_REF] Thomsen | Brominated flame retardants in plasma samples from three different occupational groups in Norway[END_REF].

In-vitro studies have demonstrated that TBBPA is toxic to a variety of mammalian cell lines [START_REF] Strack | Cytotoxicity of TBBPA and effects on proliferation, cell cycle, MAPK pathways in mammalian cells[END_REF], as well as primary cells including; hepatocytes [START_REF] Nakagawa | Biotransformation and cytotoxicity of a brominated flame retardant tetrabromobisphenol A and its analogues in rat hepatocytes[END_REF], splenocytes [START_REF] Pullen | The flame retardants tetrabromobisphenol A and tetrabromobisphenol A-bisallylether suppress the induction of interleukin-2 receptor alpha chain (CD25) in murine splenocytes[END_REF] and cerebella granule cells [START_REF] Fonnum | Molecular mechanisms involved in the toxic effects of polychlorinated biphenyls and brominated flame retardants[END_REF][START_REF] Reistad | In vitro toxicity of tetrabromobisphenol-A on cerebellar granule cells: cell death, free radical formation, calcium influx and extracellular glutamate[END_REF]. In addition, TBBPA also shows estrogenic activity since it enhances the estrogen-dependent proliferation of MtT/E-2 cells [START_REF] Kitamura | Thyroid activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A[END_REF]. Furthermore, TBBPA at low µM concentrations has been shown to modulate a number of cell signalling processes such as the activities of tyrosine kinases, MAP kinases, protein kinase C and also elevate levels of reactive oxygen species and cytosolic Ca 2+ , when added to neutrophils [START_REF] Reistad | The effect of a brominated flame retardant, tetrabromobisphenol-A, on free radical formation in human neutrophil granulocytes: the involvement of the MAP kinase pathway and protein kinase C[END_REF]. A recent study has also shown that low concentrations of TBBPA can also increase cytosolic Ca 2+ levels in cerebellar granule cells leading to cell death [START_REF] Reistad | In vitro toxicity of tetrabromobisphenol-A on cerebellar granule cells: cell death, free radical formation, calcium influx and extracellular glutamate[END_REF].

The widespread production and use of BFRs and the strong evidence of increased contamination of the environment and humans by these chemicals has led us to investigate the effects of TBBPA; the most commonly used BFR, on SERCA Ca 2+ pumps as a mechanism of disrupting Ca 2+ signalling pathways.

MATERIALS AND METHODS

Tetrabromobisphenol A was purchased from Acrōs Organics (purity given as 97%). All other reagents were of analytical grade. TBBPA was dissolved in dimethylsulfoxide to give a stock solution of 20mM and the solvent was never more than 1.0% (v/ v) in the assays performed. The pKa of TBBPA was determined in aqueous solution by the method described in Bilmen et al, (2003) [START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF].

Native sarcoplasmic reticulum (SR) was prepared from rabbit skeletal muscle as described by [START_REF] Michelangeli | Methods of reconstitution of the purified sarcoplasmic reticulum (Ca 2+ -Mg 2+ )-ATPase using bile salt detergents to form membranes of defined lipid to protein ratios or sealed vesicles[END_REF] [START_REF] Michelangeli | Methods of reconstitution of the purified sarcoplasmic reticulum (Ca 2+ -Mg 2+ )-ATPase using bile salt detergents to form membranes of defined lipid to protein ratios or sealed vesicles[END_REF] and typically had a purity of ≈80% Ca 2+ -ATPase. Porcine cerebella microsomes were prepared as described in [START_REF] Brown | The opening of the inositol 1,4,5-trisphosphate-sensitive Ca2+ channel in rat cerebellum is inhibited by caffeine[END_REF].

Ca 2+ -ATPase Activity

Ca 2+ -ATPase activity in cerebella microsomes was performed employing the phosphate liberation assay as described by [START_REF] Longland | Biochemical mechanisms of calcium mobilisation induced by mastoparan and chimeric hormone-mastoparan constructs[END_REF] [START_REF] Longland | Biochemical mechanisms of calcium mobilisation induced by mastoparan and chimeric hormone-mastoparan constructs[END_REF].

The effects of TBBPA on SR Ca 2+ -ATPase activity was investigated as a function of [Ca 2+ ], [ATP] and at different pH values, employing a coupled enzyme assay as previously described in [START_REF] Michelangeli | Methods of reconstitution of the purified sarcoplasmic reticulum (Ca 2+ -Mg 2+ )-ATPase using bile salt detergents to form membranes of defined lipid to protein ratios or sealed vesicles[END_REF][START_REF] Longland | Biochemical mechanisms of calcium mobilisation induced by mastoparan and chimeric hormone-mastoparan constructs[END_REF].

Typically, between 11-22 µg/ml of ATPase protein was added to a buffer containing 40mM Hepes/KOH (pH 7.2 or pH 6.0), 5mM MgSO 4 , 0.42mM phosphoenolpyruvate, 0.15mM NADH, 8.0 U pyruvate kinase, 20U lactate dehydrogenase, 1.01mM EGTA and 2.1mM ATP. In all experiments, unless otherwise stated, a free [Ca 2+ ] of 6µM (pCa 5.2) was used. Free Ca 2+ concentrations were calculated as described in [START_REF] Gould | A S t a g e 2 ( a ) P O S T -P R I N T kinetic model for the Ca 2+ -Mg 2+ -activated ATPase of sarcoplasmic reticulum[END_REF].

Phosphorylation studies

Phosphorylation of ATPase by [γ-32 P]ATP was carried out at 25 o C as described by Michelangeli et al., 1990 [4,7,8]. ATPase was diluted to 0.1 mg/ ml in 20mM Hepes/Tris (pH 7.2) containing 100mM KCl, 5mM MgSO 4 and 1mM CaCl 2 in a total volume of 1ml. 0.5 and 5mM stocks of ATP with specific activities of 100 or 10Ci/ mol, respectively were made in the above buffer, to cover the range Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. between 0 and 25µM. Reaction was started by the addition of [γ-32 P]ATP and stopped by the addition of ice-cold 40% (w/ v) TCA after 15secs. The assay was then placed on ice for about 30mins prior to filtration through Whatman GF/C filters. The filters were washed with 30ml of 12% (w/ v) TCA/ 0.2M H 3 PO 4 and left to dry. The filters were placed in scintillant and counted.

TBBPA binding to SERCA

The binding of TBBPA to SERCA was measured by monitoring its ability to quench the tryptophan fluorescence of the Ca 2+ -ATPase. The protein was diluted to 1 µM in a buffer containing 40mM Hepes/KOH (pH 7.2 or 6.0), 1mM EGTA and 100mM NaCl. Fluorescence intensity was measured in a Perkin-Elmer LS50B Fluorescence Spectrophotometer at 25°C (excitation 280nm, emission 340nm). In other experiments, some inhibitors of Ca 2+ -ATPase {i.e. Thapsigargin (TG), 2,5-di-tertbutylhydroquinone (BHQ) and Nonylphenol (NP)} were added separately after 1 µM TBBPA, in order to assess whether they could displace TBBPA binding and therefore reverse TBBPA-induced fluorescence quenching.

The rate constants for TBBPA binding to the Ca 2+ -ATPase and to phospholipid bilayers were assessed by monitoring either the rate of tryptophan fluorescence quenching of the Ca 2+ ATPase or of the membrane probe, pyrene dodecanonic (PDA) incorporated into phospholipid bilayers [START_REF] Ogunbayo | The interaction of the brominated flame retardant: Tetrabromobisphenol A with phospholipid membranes[END_REF]. Rapid kinetic fluorescence measurements of TBBPA binding to the Ca 2+ ATPase was performed using a stopped-flow Fluorescence spectrophotometer (Applied Photophysics, model SX17 MV). Briefly, the sample handling unit possesses two syringes, A and B (drive ratio 10:1), which are driven by a pneumatic ram. Tryptophan fluorescence quenching of the protein was monitored at 25°C when 1µM Ca 2+ -ATPase ( in 20mM Hepes, 100mM KCl, 5mM MgSO 4 , pH 7.2 ) was rapidly mixed with 10µM (final concentrations) of TBBPA in DMSO, exciting at 280 nm and measuring the emission above 320 nm using a cut-off filter. In order to assess the binding of TBBPA to phospholipid membranes, unilammellar liposomes of dioleylphosphatidylcholine containing PDA at a molar ratio of 500: 1 were prepared in the hepes buffer as above, followed by probe sonication and a freeze-thaw cycle. The time course of TBBPA quenching of PDA within DOPC liposomes was measured using a Hi-Tech SFA-12 stopped-flow accessory attached to a Perkin-Elmer LS50B fluorimeter by mixing 90 µM liposomes (equivalent concentration of lipids found associated with 1µM SR Ca 2+ ATPase within the SR vesicles) with 10 µM TBBPA. Excitation was at 342 nm and emission at 395 nm.

Intrinsic tryptophan fluorescence measurements to monitor Ca 2+ -induced conformational changes of Ca 2+ ATPase

The conformational change induced by the addition of Ca 2+ to the ATPase was monitored by observing the change in intrinsic tryptophan fluorescence [as described in [START_REF] Michelangeli | Mechanism of inhibition of the (Ca 2+ -Mg 2+ -ATPase by Nonylphenol[END_REF][START_REF] Longland | The mechanism of inhibition of the Ca 2+ -ATPase by mastoparan. Mastoparan abolishes cooperative Ca 2+ binding[END_REF][START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF]. 50µg/ml ATPase (in the absence and presence of 3.5µM TBBPA) was added to a buffer containing 20mM Hepes/Tris, 100mM MgSO 4 , (pH 7.0) and the induced-conformational change was measured as a percentage

Measurement of the transient kinetics of the conformational changes associated with Ca 2+binding and dissociation

Rapid kinetic fluorescence measurements were performed using a stopped-flow Fluorescence spectrophotometer as described in [START_REF] Longland | The mechanism of inhibition of the Ca 2+ -ATPase by mastoparan. Mastoparan abolishes cooperative Ca 2+ binding[END_REF][START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF]. In the Ca 2+ binding experiments, syringe A, containing the Ca 2+ -ATPase (1µM) in 20mM Hepes/Tris (pH7.2), 100mM KCl, 5mM MgSO 4 , 50µM EGTA was rapidly mixed with syringe B, containing 1mM Ca 2+ (final concentration). In Ca 2+ disscociation experiments, syringe A, containing the Ca 2+ ATPase (1µM) in 20mM Hepes/Tris (pH7.2), 100mM KCl, 5mM MgSO 4 , 100µM Ca 2+ was rapidly mixed with syringe B, containing 2mM EGTA (final concentration). Tryptophan fluorescence was monitored at 25°C by exciting the Ca 2+ -ATPase (rapidly mixed with buffers containing either Ca 2+ or EGTA) at 280nm and measuring the emission above 320nm using a cut off filter.

Molecular Modelling

The program Molegro Virtual Docker 2007 (with the standard default settings) was used in order to predict the ligand-protein interactions of the energy minimised conformational structure of TBBPA to the Ca 2+ -ATPase. The atomic coordinates for the E2 (TG + BHQ) form of the Ca 2+ -ATPase (PDB code 2AGV) was used for this virtual docking procedure. Figures were produced using the Swiss-pdbViewer.

RESULTS

Fig. 1 shows that TBBPA is a potent inhibitor of the skeletal muscle SERCA 1a isoform of the Ca 2+ -ATPase and that this inhibition is pH sensitive. At pH 7.2 the apparent inhibition constant (app. Ki: concentration causing 50% inhibition under these experimental conditions) was determined to be 1.7±0.3µM, while at pH 6.0 the potency increased significantly (app. Ki, 0.46±0.05µM). As low pH favours the E2 state of the Ca 2+ ATPase [START_REF] Michelangeli | Effect of pH on the activity of the Ca 2+ Mg 2+ -activated ATPase of sarcoplasmic reticulum[END_REF], this might indicate that TBBPA preferentially binds to and stabilizes the ATPase in this conformation. This pH effects is, however, unlikely to be due to protonation of the phenol groups on TBBPA since the pKa for TBBPA was determined to be 9.8±0.3, (typical of other phenol groups) and which would indicate that TBBPA is essentially fully protonated at both pH6 and pH 7.2. Cerebellum is known to express both SERCA2b and SERCA3. Fig. 1 (inset) shows that TBBPA can also inhibit the non-striated muscle SERCA isoforms SERCA2b and SERCA3, since it inhibits the cerebellar microsomal Ca 2+ -dependent ATPase activity with an app. Ki of 2.33±0.45µM at pH 7.2 (i.e. similar to that determined for SERCA 1a). Therefore it would seem Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

that TBBPA is a non-selective SERCA isoform inhibitor, unlike some other inhibitors previously examined [START_REF] Wootton | The effects of the phenylalanine 256 to valine mutation on the sensitivity of sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase (SERCA) Ca 2+ pump isoforms 1, 2, and 3 to thapsigargin and other inhibitors[END_REF]. Complete inhibition, in all cases, was observed at or below 10µM TBBPA. In comparison, we have also shown that the related, non-brominated, compound; bisphenol A, a known endocrine disrupter which is used as a plasticising agent, is a very much weaker inhibitor of the SR Ca 2+ ATPase with a Ki of 233±30 µM at pH 7.2 [START_REF] Hughes | Estrogenic alkylphenols induce cell death by inhibiting testis endoplasmic reticulum Ca 2+ pumps[END_REF][START_REF] Kirk | Environmental endocrine disrupters dysregulate estrogen metabolism and Ca2+ homeostasis in fish and mammals via receptorindependent mechanisms[END_REF]. (high affinity) form, increasing turnover rate, while the inhibitory phase, is associated, in part, with the binding of Ca 2+ to the E2 (low affinity) form of the enzyme. In the absence of TBBPA, the results gave V max of 5.0±0.2 I.U/mg, with a K m value of 0.62±0.06µM for the stimulatory phase and a K m value of 0.62±0.06mM for the inhibitory phase. Whereas, in presence of TBBPA (between 1-4 µM), the data showed both a decrease in the maximal activity and an increase in the K m for the stimulatory phase. At 1 µM TBBPA, the V max was slightly decreased to 4.81±0.40 I.U/mg, while the K m 's for the stimulatory and inhibitory phases increased to 1.04±0.1µM and 0.68±0.07mM, respectively. The changes became more apparent at 2.0µM TBBPA, as the V max decreased to 2.94±0.15 I.U/mg, while the K m 's increased further to 1.96±0.15µM and >1mM for the stimulatory and inhibitory phases, respectively. Furthermore, in the presence of 4µM TBBPA, V max decreased to 3.33±0.25 I.U/mg while both K m 's increased significantly (11.0±0.10µM, for the stimulatory and >1mM, for the inhibitory).

These results suggest that TBBPA may be decreasing the binding affinity of Ca 2+ -ATPase for Ca 2+ . Fig. 2B shows the activity of Ca 2+ -ATPase at various [ATP] in the absence and presence of 1.5µM TBBPA. The complex biphasic profiles have previously been modeled assuming a bi-Michaelis-Menton process described in [START_REF] Brown | Characterisation of a novel Ca 2+ pump inhibitor (bis-phenol) and its effects on intracellular Ca 2+ mobilization[END_REF][START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF][START_REF] Kosk-Kosicka | Kinetic characterization of detergentsolubilized sarcoplasmic reticulum adenosinetriphosphatase[END_REF], where it is assumed that, two independent ATP binding events occur during enzymatic turnover, designated as the high affinity catalytic site and the lower affinity regulatory site. ATP binds and phosphorylates the ATPase at the high affinity catalytic site while, the low affinity regulatory site accelerate ATPase cycle rate. In the absence of TBBPA, the data could be fitted assuming a catalytic K m and V max of 0.5µM and 1.0IU/mg, respectively; and a regulatory K m and V max of 1.0mM and 1.8IU/mg, respectively. In the presence of 1.5µM TBBPA, only the catalytic V max appeared substantially different compared to control, while all other parameters showed little or no differences (i.e. cat. K m = 0.5µM, cat. V max = 0.2 IU/mg; reg. K m = 0.4mM, reg. V max = 1.4IU/mg).

In order to assess the possible effect of TBBPA on the phosphorylation of the ATPase by ATP, experiments were performed at the optimal ATP concentration (25 µM) and varied concentrations of of TBBPA with a K i of 2.0±0.5 µM. As changes in the affinity of ATP binding to the ATPase can alter the level of phosphorylation by affecting the K ATP (concentration of ATP required to achieve 50% of the E-P max ), the effects of ATP concentration in the absence and presence of 2.5µM TBBPA were further investigated (fig. 3B). As shown in fig 3B, E-P max and K ATP values of 9.9±0.9 nmol/mg and 0.9±0.5 µM, respectively were obtained for the control, whereas in the presence of 2.5µM TBBPA, the data gave E-P max and K ATP values of 6.7±0.6 nmol/mg and 2.3±0.8 µM, respectively. Thus, TBBPA caused a decrease in the maximum phosphorylation level of the ATPase (fig. 3A) but had only relatively modest effects on the ATP affinity (ie K ATP ). competed for the same site on the ATPase as TBBPA. This was achieved by determining whether the quenching caused by 1µM TBBPA could be reversed by displacing it from its binding site. Fig. 4B shows fluorescence traces of both TBBPA quenching followed by the addition of a variety of inhibitors. It can be clearly seen that thapsigargin (2 x 1µM), showed no reversibility of the TBBPA quenching, however, both BHQ (2 x 5µM) and nonylphenol (2 x 5µM) inhibitors could significantly reverse TBBPA quenching upon their addition, indicating competitive binding. The displacement of TBBPA by Cyclopiazonic acid (CPA), another commonly used SERCA inhibitor was also assessed; but as CPA caused substantial tryptophan fluorescence quenching itself, its ability to displace TBBPA from the ATPase could not be determined by this method. For the sake of completeness, Fig. 4B In order to assess whether TBBPA binds directly to the Ca 2+ -ATPase or indirectly, via the phospholipid bilayer, the rate of TBBPA binding to the Ca 2+ ATPase and to DOPC unilammellar liposomes (at the same lipid concentration found within the SR vesicles) was investigated. Fig. 5A shows the rate of quenching of tryptophan fluorescence within the Ca 2+ ATPase. Quenching was completed within less than 50 ms and the quenching process could be fitted to a simple first order decay process with a rate constant of 90s -1 . The rate of TBBPA quenching of PDA within the DOPC membranes was considerably slower i.e. requiring about 10 s for completion (rate constant 0.24s -1 ; fig. 5B). These results would suggest that TBBPA binds directly to the Ca 2+ -ATPase, without the need to partition first, into the phospholipid bilayer.

Fig 6A shows the effect of 3.5 µM TBBPA on the tryptophan fluorescence changes of Ca 2+ -ATPase upon the addition of a range of free Ca 2+ concentrations, which is often used to assess the affinity of Ca 2+ binding to the ATPase [START_REF] Longland | The mechanism of inhibition of the Ca 2+ -ATPase by mastoparan. Mastoparan abolishes cooperative Ca 2+ binding[END_REF][START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF][START_REF] Fernandez-Belda | A comparative study of calcium transients by isotopic tracer, metallochromic indicator, and intrinsic fluorescence in sarcoplasmic reticulum ATPase[END_REF]. In the absence of TBBPA, the addition of Ca 2+ gave a ∆F max value of 9.1±0.3% and an app. K d of 0.7±0.1µM, whereas in the presence of 3.5µM TBBPA, there was a decrease in the maximal fluorescence change (∆F max value of 5.21±0.04%) and a substantial in increase in app. K d . This data also suggests that TBBPA decreases the affinity of Ca 2+ -ATPase for Ca 2+ . Fig 6B illustrates the effect of varying [TBBPA] on tryptophan fluorescence changes of Ca 2+ -ATPase upon the addition of Ca 2+ at pH 7.2. In the absence of TBBPA, addition of Ca 2+ to the ATPase caused a 9.09 +/-0.31% decrease in tryptophan fluorescence, whereas in the presence of 10µM TBBPA, a 3.76 +/-0.66 % decrease in tryptophan fluorescence resulted. The IC 50 for TBBPA under these conditions is about 3.5µM. It is also apparent from the data in fig. 6A that there is also a small increase in fluorescence over the 10nM to 10µM free [Ca 2+ ] concentration range in the presence of 3.5µM TBBPA. However, since under these experimental conditions, only a proportion of the Ca 2+ -ATPase is affect by TBBPA (fig. 6B), this would suggest that this component of the fluorescence change is due to the proportion of ATPase unaffected by TBBPA (thus having an app K d similar to control). At pH 7.2, the rate constants for the conformational changes associated with either Ca 2+ binding to ATPase or its dissociation from ATPase were measured in the absence and presence of TBBPA by following the Ca 2+ -induced changes in tryptophan fluorescence using stopped-flow spectrofluorimetry. In Fig 7A & B, all the data could be fitted to a mono-exponential equation (rate constants given in table 1) as this is the simplest relationship which gave good fits to the data obtained in these experiments. As shown in table 1, the rate constant for Ca 2+ binding decreased whereas the rate constant for Ca 2+ dissociation increased, with increasing [TBBPA]. Consequently, the dissociation S t a g e 2 ( a ) P O S T -P R I N T constants K d for Ca 2+ binding (calculated from the on and off rate constants) increased proportionally with increasing [TBBPA] from 1.4µM in the absence of TBBPA to almost 30µM in the presence of 10µM TBBPA (table 1).

DISCUSSION

Brominated flame retardants are becoming prevalent throughout our environment, and many of these chemicals have been shown to bio-accumulate within humans to alarmingly high levels [START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF][START_REF] De Wit | An overview of brominated flame retardants in the environment[END_REF][START_REF] Alaee | The significance of brominated flame retardants in the environment: current understanding, issues and challenges[END_REF][START_REF] Law | Levels and trends of brominated flame retardants in the European environment[END_REF].

Some recent studies have shown that in some individuals, levels in the 1000's ng/g lipid in human breast milk have been found [START_REF] Meironyte | Analysis of polybrominated diphenyl ethers in Swedish human milk. A time-related trend study, 1972-1997[END_REF]. Toxicological studies have shown that a number of these BFRs are both neurotoxic and can act as endocrine disrupters [START_REF] Reistad | In vitro toxicity of tetrabromobisphenol-A on cerebellar granule cells: cell death, free radical formation, calcium influx and extracellular glutamate[END_REF][START_REF] Kitamura | Thyroid activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A[END_REF][START_REF] Meerts | In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PDBEs, and polybrominated bisphenol A compounds[END_REF] and several studies have shown that TBBPA can in fact disturb Ca 2+ signalling pathways at low µM concentrations [START_REF] Reistad | In vitro toxicity of tetrabromobisphenol-A on cerebellar granule cells: cell death, free radical formation, calcium influx and extracellular glutamate[END_REF][START_REF] Reistad | The effect of a brominated flame retardant, tetrabromobisphenol-A, on free radical formation in human neutrophil granulocytes: the involvement of the MAP kinase pathway and protein kinase C[END_REF]. One obvious possibility for this is that, TBBPA can modulate the activity of Ca 2+ transporters such as SERCA Ca 2+ pumps.

Based on the Ca 2+ -ATPase activities data from this study, TBBPA proved to be a very potent inhibitor of the Ca 2+ -ATPase and affects SERCA isoforms: type 1a and the non striated muscle isoforms type 2b & 3, found in cerebellar microsomes, to similar extents. The inhibition of ATPase activity by TBBPA is also pH-sensitive, with a low pH favouring increased inhibition. The fact that under optimal conditions the Ki for inhibition is only 2-3 times higher than the molarity of the ATPase in these assays would likely indicate that the Ca 2+ -ATPase has one (or only a few binding) site(s) for TBBPA which when occupied, inhibits activity. It is also clear that there is likely to be a number of sites of differing affinities for TBBPA on the ATPase as determined from fluorescence quenching studies, however, only occupation of the high affinity site(s) is / are required to elicit enzyme inhibition. These sites are also likely to be accessible directly from the aqueous phase, rather than via the lipid bilayer.

From the activity versus [ATP] and the phosphorylation data, it is unlikely that the major factor causing inhibition is due to TBBPA effects on ATP binding or phosphoryl-transfer. However, what does appear to be the major factor for inhibition is the observation that, TBBPA dramatically reduces the affinity of the ATPase for Ca 2+ binding and this reduction in binding affinity is due to both a decrease in the rate of binding and an increase in the rate of Ca 2+ dissociation from the ATPase. The calculated affinity for Ca 2+ binding was shown to be dramatically reduced from (1.4 µM to 30 µM) in the presence of TBBPA. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Binding site competition studies, utilizing the displacement of TBBPA (which quenches tryptophan fluorescence of the Ca 2+ -ATPase) with a variety of other Ca 2+ -ATPase inhibitors has shown that the quenching could be substantially reversed by BHQ and nonylphenol, both of which have been shown to stabilize the Ca 2+ ATPase in an E2 conformational state [START_REF] Michelangeli | Mechanism of inhibition of the (Ca 2+ -Mg 2+ -ATPase by Nonylphenol[END_REF][START_REF] Wictome | The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca 2+ , Mg 2+ -ATPase[END_REF]. The fact that TBBPA is a more potent inhibitor at low pH (which favours the E2 form [START_REF] Michelangeli | Effect of pH on the activity of the Ca 2+ Mg 2+ -activated ATPase of sarcoplasmic reticulum[END_REF], and the added fact that TBBPA decreases the Ca 2+ affinity of the ATPase (again consistent with being in an E2 state), would lead us to propose that TBBPA also inhibits the Ca 2+ ATPase by stabilizing it in a E2 conformation. However, as TBBPA quenching of the ATPase is not reversed by thapsigargin (which again stabilizes the E2 form [START_REF] Wictome | The inhibitors thapsigargin and 2,5-di(tert-butyl)-1,4-benzohydroquinone favour the E2 form of the Ca 2+ , Mg 2+ -ATPase[END_REF]), this would indicate that the site occupied by thapsigargin is distinct from that of TBBPA. Indeed, recent crystallographic studies have shown that thapsigargin and BHQ do bind to distinct sites within the Ca 2+ -ATPase [START_REF] Obara | Structural role of countertransport revealed in Ca(2+) pump crystal structure in the absence of Ca(2+)[END_REF], with BHQ binding to a hydrophobic pocket of the E2 state within the transmembrane regions encompassed by M1, M2, M3 and M4. Using the high accuracy molecular docking program Molegro Virtual Docker 2007, two potential binding sites for TBBPA on the E2 form of the Ca 2+ -ATPase were predicted (figure 8A). These two sites are distinct from the thapsigargin binding site and are both located within a large cavity encompassed by the transmembrane helices M1, M2, M3 and M4, and enclosed at the top of the pocket by regions of the phosphorylation domain.

Figure 8B shows the predicted TBBPA binding site 1, which is in an identical position to where BHQ binds within the crystal structure [START_REF] Obara | Structural role of countertransport revealed in Ca(2+) pump crystal structure in the absence of Ca(2+)[END_REF], i.e. in a hydrophobic region at the bottom of the cavity encircled by; Asp 59, Leu 61 and Val 62 (within M1); Leu 253 (within M3); and Pro 308, Leu 311 and Pro 312 (within M4). This region within M4 is close to the Ca 2+ binding site II and could well explain the effect of TBBPA on the affinity for Ca 2+ binding. Figure 8C shows details of the predicted binding site 2 found towards the top of this cavity, consisting of the top of transmembrane helix M3 (Asp 245, Thr 247 and Gln 250); the start of the phosphorylation domain at Thr 341 and Pro337 (within helix P1), and at Glu 715 and Glu 732 also within the phosphorylation domain (at the ends of strands P6 and P7, respectively). Binding at this site may account, in part, for the effect of TBBPA reducing ATP-dependent phosphorylation. Our experimental data probably favours site 1, because BHQ can reverse TBBPA quenching, however they are not necessarily mutually exclusive, as TBBPA may conceivably bind to both sites. Although there are no tryptophan residues close to these binding sites, it seems clear that since TBBPA can bind directly to the Ca 2+ -ATPase from the aqueous phase, it most likely enters into this cavity by passing close to the Trp 107 (at the top of M2). As binding of TBBPA to the ATPase is dynamic, at any given time within some ATPases, TBBPA must therefore be in close proximity to Trp 107, for fluorescence quenching to take place.

The fact that this cavity has also been implicated as being a Ca 2+ entry pathway, allowing Ca 2+ to enter into specific binding sites buried deep within the transmembrane region [START_REF] Moncoq | The molecular basis for cyclopiazonic acid inhibition of the sarcoplasmic reticulum calcium pump[END_REF], is also supported by our data that shows that TBBPA reduces Ca 2+ binding to the ATPase. It is, however, interesting to note that from activity, mutagenesis and modelling data obtained for 2-aminoethoxydiphenyl borate association and dissociation [START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF]. We deduced from that study that it bound on the adjacent side of the transmembrane bundle of the SERCA protein within a hydrophobic pocket defined by M3, M5 and M6 [START_REF] Bilmen | Inhibition of SERCA Ca 2+ pumps by 2-aminoethoxydiphenyl borate (2-APB). 2-APB reduces both Ca 2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca 2+binding sites[END_REF], and also occluded the Ca 2+ entry pathway. Now from this study with TBBPA, we suggest that the cavity encompassed by M1, M2, M3 and M4 forms the pathway for Ca 2+ entry, since it also reduces the affinity for Ca 2+ binding by affecting both the on and off rates for Ca 2+ binding. As we know that there are two Ca 2+ binding sites on SERCA and that a number of potential Ca 2+ entry pathway sites have been proposed by several groups [START_REF] Toyoshima | Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution[END_REF][START_REF] Lee | What the structure of a calcium pump tells us about its mechanism[END_REF][START_REF] Zhang | The role of the M6-M7 loop (L67) in stabilization of the phosphorylation and Ca 2+ binding domains of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA)[END_REF][START_REF] Zhang | Detailed characterization of the cooperative mechanism of Ca 2+ binding and catalytic activation in the Ca 2+ transport (SERCA) ATPase[END_REF][START_REF] Menguy | The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca 2+ of sarcoplasmic reticulum Ca 2+ -ATPase[END_REF] as to the exact route for Ca 2+ to enter and bind, it would be tempting to speculate that each of the Ca 2+ , enters their respective binding sites via a distinct pathway, located on adjacent sides of the transmembrane helix bundle. One piece of evidence which may support this view is the fact that when the Ca 2+ ATPase is reconstituted with short chain length phospholipids it reduces the stochiometry for Ca 2+ binding from 2 to 1 without significantly affecting its affinity or the phosphorylation level [START_REF] Michelangeli | Effects of phospholipids on binding of calcium to (Ca 2+ -Mg 2+ -ATPase[END_REF]. Thus this indicates that the 2 Ca 2+ binding events can, under some circumstances, be made distinct.

In summary, the most commonly used BFR, TBBPA, which has been shown to be highly cytotoxic and cause disturbances of intracellular Ca 2+ homeostasis within cells, may elicit its effects, at least in part, by inhibiting the SERCA Ca 2+ pumps. the experimental data assuming a 1 st order process, with the parameters given in table 1. The wavy appearances of some of the traces are due to the over-sampling function on the stopped-flow, being employed to reduce the signal-to-noise levels. -------------------------------------------------------------------------------------------[TBBPA]µM k on (µM -1 s -1 ) k off (s -1 ) K d (k off / k on ) (µM)

LEGENDS

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  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070843 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070843

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070843 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070843Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  change in total tryptophan fluorescence, over a range of free [Ca 2+ ] (10 -100µM), was measured at 25°C (excitation 285nm, emission 325nm).

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070843 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070843

Fig. 2A shows

  Fig. 2A shows the effect of TBBPA on ATPase activity as a function of free [Ca 2+ ]. Ca 2+ -ATPase activity exhibited a classical 'bell-shaped' profile with respect to free [Ca 2+ ] in the absence and presence of TBBPA. The stimulatory phase is associated with Ca 2+ binding to the ATPase in an E1

  TBBPA. As shown in Fig 3A, the level of Ca 2+ -ATPase phosphorylation was reduced in the presence S t a g e 2 ( a ) P O S T -P R I N T

Fig

  Fig 4A shows the binding of TBBPA to Ca 2+ -ATPase, monitored by tryptophan fluorescence quenching, at pH 7.2 and pH 6.0. Both sets of data could be reasonably fitted using a simple one-site binding relationship where the K d values were determined to be 16 and 27 µM, for the pH 6 and pH 7.2 data, respectively (statistical analysis of goodness-of-fit as assessed by chi 2 tests were determined to be around 0.97, shown by the dotted lines). Slightly better fits to the data could, however, be achieved assuming a two-site binding relationship (chi 2 ≥ 0.992). Therefore our data would favour the possibility that fluorescence quenching of the ATPase by TBBPA could be best represented assuming a high and low affinity binding sites. The solid lines presented in fig 4A for the pH 6.0 data was achieved assuming K d values of 1.0 µM and 19 µM, while the fit to the pH 7.2 data was achieved assuming K d values for the two sites of 2.2 µM and 44 µM, for the high and low affinity sites, respectively. These results would therefore indicate that the high affinity K d values obtained here probably relates to the binding sites on the ATPase which are associated with inhibition, since they are comparable with the Ki values. The fluorescence quenching of the Ca 2+ -ATPase at low concentrations of TBBPA (1µM) was used to determine whether other commonly used hydrophobic inhibitors

  also shows the effects of these inhibitors on the Ca 2+ -ATPase tryptophan fluoresence in the absence of TBBPA. As can be seen these inhibitors do not themselves cause an increase in tryptophan fluorescence of the Ca 2+ -ATPase in the absence of TBBPA, although BHQ and TG did cause a slight S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070843 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070843 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

( 1 -

 1 2%) quenching in fluorescence.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070843 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070843

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070843 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070843 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

( 2 -

 2 APB) (a low affinity SERCA inhibitor) which also reduced Ca 2+ binding by affecting both Ca 2+

Figure 1 :

 1 Figure 1: The effects of TBBPA on Ca 2+ -ATPase activity.Ca 2+ -ATPase activity at a range of TBBPA concentrations was measured in rabbit skeletal muscle SR membranes at 25°C in a buffer at pH 7.2 ( ■ ) or pH 6.0 ( ▲ ). Inset shows the effects of TBBPA on the Ca 2+ -ATPase activity in cerebellar microsomes measured at pH7.2 and 37°C. The data points represent the mean ± SD of between 3-6 determinations

Figure 2 :

 2 Figure 2: The effects of TBBPA on ATPase activity as a function of [Ca 2+ ] and [ATP]. (A) The effects of varying [TBBPA] and free [Ca 2+ ] on SR Ca 2+ -ATPase activity measured at pH7.2, 25°C. The concentrations of TBBPA used were; No TBBPA ( ■ ), 1µM ( ▲ ), 2µM ( ▼ ) and 4µM ( ♦ ). (B) The effects of varying [ATP] on Ca 2+ ATPase activity measured at pH7.2 and 25°C, in the presence of no TBBPA ( ■ ) and 1.5µM TBBPA ( ▲ ). The lines represent the best fits to the data assuming a bi-Michaelis-Menton equation, with the parameters given in the text. The data points represent the mean ± SD of between 3-6 determinations.

Figure 3 :

 3 Figure 3: Effects of TBBPA on phosphorylation of the Ca 2+ -ATPase by ATP. (A) Shows the effects of TBBPA (0-20µM) on the phosphorylation of the Ca 2+ -ATPase in the presence of Ca 2+ (1mM) by [ 32 P]ATP (25µM), at pH7.2 and 25°C. (B) shows how ATP-dependent phosphorylation of the Ca 2+ -ATPase over a range of [ 32 P] ATP concentrations (0-25µM) is affected in the absence ( ■ ) and presence ( ▲ ) of 2.5µM TBBPA. The data represent the mean ± SD of 3 determinations.

Figure 4 :

 4 Figure 4: The quenching effects of TBBPA on tryptophan fluorescence of the Ca 2+ -ATPase. (A) Shows the effects of TBBPA on tryptophan fluorescence of the Ca 2+ -ATPase measured at pH 7.2 (■) and pH6.0 (▲), at 25°C. The solid lines represent the best fit to the data assuming two-site binding (parameters given in text), while the dotted lines represent the best fits to the data assuming one-site binding (parameters given in text). The data points represent the mean values ± SD of 3 determinations. (B) Shows traces of tryptophan fluorescence levels being quenched by the addition of 1µM TBBPA. Following this, two additions of thapsigargin (1µM each) were made. In subsequent experiments, the quenching of 1µM TBBPA was reversed by either two additions of BHQ (5µM each), or of nonylphenol (5µM each). Control traces in which TG, (1µM), BHQ (5µM) and NP (5µM) were added in the absence of TBBPA are also shown. Each trace is representative of 3 identical experiments.

Figure 5 :

 5 Figure 5: Kinetics of TBBPA binding to the Ca 2+ -ATPase and to phospholipid bilayers. (A) Shows the rate at which the tryptophan fluorescence of the Ca 2+ -ATPase (1µM, final concentration, which is equivalent to 90 µM phospholipid) is quenched by the addition of TBBPA S t a g e 2 ( a ) P O S T -P R I N T

(

  10µM, final concentration), at pH 7.2, 25°C. The solid line represents the best fit to the experimental data assuming a 1 st order decay process (k obs = 90s -1 ). The data points represent the mean of 8 accumulated runs. (B) Shows the rate of TBBPA (10 µM) quenching of the membrane bound PDA probe within DOPC unilamellar liposomes (90µM, final concentration). The fitted line represents the best fit to the experimental data, assuming a 1 st order decay process (k obs = 0.24s -1 ).

Figure 6 :

 6 Figure 6: The effects of TBBPA on the Ca 2+ -induced tryptophan fluorescence change of the Ca 2+ -ATPase. (A) Shows changes in the Ca 2+ -ATPase tryptophan fluorescence induced by variations in free [Ca 2+ ] in the absence (▲) or presence ( ■ ) of 3.5µM TBBPA, measured at pH 7.0, 25°C. (B) Shows the effect of varied [TBBPA] on the maximal tryptophan fluorescence change induced by the addition of 100µM free [Ca 2+ ]. The data points are the mean ± SD of 3 or more determinations.

Figure 7 :

 7 Figure 7: The effects of TBBPA on the kinetics of the Ca 2+ -induced tryptophan fluorescence change of the Ca 2+ -ATPase. (A) shows the effects of various TBBPA concentrations (traces from top to bottom of 0, 2, 4 and 10µM) on Ca 2+ binding conformational change monitored by following the increase in tryptophan fluorescence of the Ca 2+ -ATPase upon addition of Ca 2+ . (B) Shows the effects of various concentrations of TBBPA (from bottom to top of 0, 2, 4 and 10µM) on the conformational change following Ca 2+ dissociation from the Ca 2+ -ATPase, monitored as a decrease in tryptophan fluorescence. The traces are the means of 8-10 accumulated runs and the solid lines are the best fits to

Figure 8 :

 8 Figure 8: Predicted binding sites for TBBPA. Molegro Virtual Docker 2007 was used to predict potential TBBPA binding sites on the E2 (TG + BHQ) SERCA1a crystal structure (PBD code 2AGV). (A) shows the two potential TBBPA binding sites (labelled site 1 and site 2). In addition, the thapsigargin binding site is also shown. The transmembrane helices are colour code accordingly: M1 (red), M2 (orange), M3 (yellow), M4 (green), M5 (cyan), M6 (blue), M7 (purple), M8 (pink), M9 (dark grey), M10 (white). (B) shows a close up of TBBPA predicted binding site 1, specifically highlighting amino acids within 4Å of TBBPA and therefore able to potentially form either hydrogen bonds or Van der Waals interactions. (C) shows a close up of TBBPA predicted binding site 2. S t a g e 2 ( a ) P O S T -P R I N T

Table1:

  The kinetic parameters of Ca 2+ binding and dissociation from the Ca 2+ -ATPase in the presences of TBBPA -

  -----------------------------------------------------------------------------------------These values are presented as means± SEM. Data presented is a result of an average of 8 -10 individual experiments.

FIG 4 S

 4 FIG 4

  FIG 5A

FIG 6 S

 6 FIG 6

  FIG 7

FIG 8 S

 8 FIG 8 S t a g e 2 ( a ) P O S T -P R I N T

© 2007 The Authors Journal compilation © 2007 Biochemical Society

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070843 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements: Dr Jon Ride is thanked for helping with the Molegro virtual docking program. We would like to thank Houdini Ho Tin Wu for preliminary experiments. The college of Medicine, University of Ibadan, Nigeria and the School of Biosciences, University of Birmingham, UK are thanked for partial scholarship support to O.A.O. A grant from the Medical Faculty of the University of Birmingham was also awarded to undertake this work. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 5 Sep 2007 as manuscript BJ20070843 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070843

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.