Browsing by Author "McIntosh, David B"

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    A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles
    (2001) Champeil, Philippe; Henao, Fernando; Lacapère, Jean-Jacques; McIntosh, David B
    After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism.
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    Asparagine 706 and Glutamate 183 at the Catalytic Site of Sarcoplasmic Reticulum Ca 2+ -ATPase Play Critical but Distinct Roles in E 2 States
    (2006) Clausen, Johannes D; McIntosh, David B; Woolley, David G; Anthonisen, Anne Nyholm; Vilsen, Bente; Andersen, Jens Peter
    Mutants with alteration to Asn(706) of the highly conserved (701)TGDGVND(707) motif in domain P of sarcoplasmic reticulum Ca(2+)-ATPase were analyzed for changes in transport cycle kinetics and binding of the inhibitors vanadate, BeF, AlF, and MgF. The fluorides likely mimic the phosphoryl group/P(i) in the respective ground, transition, and product states of phosphoenzyme hydrolysis (Danko, S., Yamasaki, K., Daiho, T., and Suzuki, H. (2004) J. Biol. Chem. 279, 14991-14998). Binding of BeF, AlF, and MgF was also studied for mutant Glu(183) --> Ala, where the glutamate of the (181)TGES(184) motif in domain A is replaced. Mutations of Asn(706) and Glu(183) have in common that they dramatically impede the function of the enzyme in E2 states, but have little effect in E1. Contrary to the Glu(183) mutant, in which E2P slowly accumulates (Clausen, J. D., Vilsen, B., McIntosh, D. B., Einholm, A. P., and Andersen, J. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2776-2781), E2P formation was not detectable with the Asn(706) mutants. Differential sensitivities of the mutants to inhibition by AlF, MgF, and BeF made it possible to distinguish different roles of Asn(706) and Glu(183). Hence, Asn(706) is less important than Glu(183) for gaining the transition state during E2P hydrolysis but plays critical roles in stabilization of E2P ground and E2.P(i) product states and in the major conformational changes associated with the Ca(2)E1P --> E2P and E2 --> Ca(2)E1 transitions, which seem to be facilitated by interaction of Asn(706) with domain A.
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    ATPase and Multidrug Transport Activities of the Overexpressed Yeast ABC Protein Yor1p
    (1998) Decottignies, Anabelle; Grant, Althea M; Nichols, J Wylie; de Wet, Heidi; McIntosh, David B; Goffeau, André
    The Saccharomyces cerevisiae genome encodes 15 full-size ATP binding cassette transporters (ABC), of which PDR5, SNQ2, and YOR1 are known to be regulated by the transcription factors Pdr1p and Pdr3p (pleiotropic drug resistance). We have identified two new ABC transporter-encoding genes, PDR10 and PDR15, which were up-regulated by the PDR1-3 mutation. These genes, as well as four other ABC transporter-encoding genes, were deleted in order to study the properties of Yor1p. The PDR1-3 gain-of-function mutant was then used to overproduce Yor1p up to 10% of the total plasma membrane proteins. Overexpressed Yor1p was photolabeled by [gamma-32P]2', 3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP (K0.5 = 45 microM) and inhibited by ATP (KD = 0.3 mM) in plasma membranes. Solubilization and partial purification on sucrose gradient allowed to detect significant Yor1p ATP hydrolysis activity (approximately 100 nmol of Pi.min-1.mg-1). This activity was phospholipid-dependent and sensitive to low concentrations of vanadate (I50 = 0.3 microM) and oligomycin (I50 = 8.5 microg/ml). In vivo, we observed a correlation between the amount of Yor1p in the plasma membrane and the level of resistance to oligomycin. We also demonstrated that Yor1p drives an energy-dependent, proton uncoupler-insensitive, cellular extrusion of rhodamine B. Furthermore, cells lacking both Yor1p and Pdr5p (but not Snq2p) showed increased accumulation of the fluorescent derivative of 1-myristoyl-2-[6-(NBD)aminocaproyl]phosphatidylethanolamine. Despite their different topologies, both Yor1p and Pdr5p mediated the ATP-dependent translocation of similar drugs and phospholipids across the yeast cell membrane. Both ABC transporters exhibit ATP hydrolysis in vitro, but Pdr5p ATPase activity is about 15 times higher than that of Yor1p, which may indicate mechanistic or regulatory differences between the two enzymes.
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    Characterisation of the reaction of 1,4-phenylenebismaleimide with Ca²⁺-ATPase and elucidation of the intramolecular crosslink site
    (1997) Seekoe, Tshepo W; McIntosh, David B
    The SR Ca²⁺-ATPase is an ATP driven pump that removes calcium from the sarcoplasm and myofibrils to allow muscle relaxation. The sulfhydryl crosslinker, 1,4- phenylenebismaleimide, reacts with Ca²⁺-ATPase (110 kD) to form a species with an apparent molecular weight of 125 kD, as well as dimers and high order oligomers, on SDS-P AGE. During the course of this study we have optimised and characterised the reaction of 1,4-phenylenebismaleimide with SR Ca²⁺-ATPase to produce the 125 kD species that is reminiscent of an E 125 species formed by intramolecular crosslink with glutaraldehyde. The glutaraldehyde crosslink involves the active site Lys 492 and Arg 678, in a zero distance link that overlaps with the ATP binding pocket, since it can be inhibited by nucleotides. It has been previously shown that the putative intramolecular crosslink with 1,4-phenylenebismaleimide is also sensitive to nucleotide binding. We show that the formation of the putative intramolecular crosslink of SR vesicles ( approximately 20 % of ATPase) with 1,4-phenylenebismaleimide is optimum at alkaline pH with micromolar concentrations of the crosslinker. The formation of ATPase dimers and high order oligomers, which were prominent in the reaction with SR vesicles, were eliminated by solubilising in Triton X-100. Under these conditions and in the presence of calcium, two intramolecular crosslinks are formed as seen in the formation of 125 and 130 kD species. The former seems to be in proximity of the y-phosphate and the latter in the β-phosphate region of the ATP binding site according to nucleotide protection studies. In the presence of detergent (Triton X-100) and absence of calcium, only the 125 kD species is formed and requires stabilisation by thapsigargin, a sesquiterpene lactone that binds the transmembrane α-helices. These conditions yield up to 60 % intramolecularly crosslinked ATPase. Trypsin digestion altered the apparent molecular weight of the 125 kD species to 135 kD, suggesting, in accordance with the results of glutaraldehyde crosslink, that the putative intramolecular crosslink 1s between tryptic fragments A and B. [¹⁴C]1,4-phenylenebismaleimide was synthesised to further characterise the reaction and to elucidate crosslinked amino acid residue following protein digestion, radioactive peptide purification, and sequencing. From filtration studies it was evident that a number of sulfhydryl residues were derivatized in both SR vesicles and solubilised Ca²⁺-ATPase. The results suggests that there is very fast reacting set of sulfhydryl groups, which could comprise of sulthydryls from Ca²⁺-ATPase and/or a minor contaminant protein as previous studies have indicated. Only this fast set was reduced by nucleotide binding. In Triton X-100, the total reactive residues increased two-fold and the biphasic nature of the curve showed that the intramolecular crosslink possibly involves a fast reacting sulfhydryl residue and a slow reacting one. Derivatization with [¹⁴C]1,4-phenylenebismaleimide followed by digestion and HPLC analysis revealed radio labelled peaks. Purification and sequencing of the adducts identified 8 reactive cysteines, namely Cys 12, Cys 344, Cys 364, Cys 471, Cys 498, Cys 636, Cys 670 and Cys 674. The cysteines involved in the putative intramolecular crosslink could not be identified but it is proposed that either Cys 471 or Cys 498 crosslink with Cys 670 or Cys 674.
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    Critical Interaction of Actuator Domain Residues Arginine 174, Isoleucine 188, and Lysine 205 with Modulatory Nucleotide in Sarcoplasmic Reticulum Ca 2+ -ATPase
    (2008) Clausen, Johannes D; McIntosh, David B; Woolley, David G; Andersen, Jens Peter
    ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.
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    Expression and purification of HGXPRT from Plasmodium falciparum and characterisation of its interaction with an effector chalcone
    (2008) Van der Bergh, Naadia; McIntosh, David B
    The aim of this study was to express and purify PƒHGXPRT and to explore three techniques, namely gel filtration HPLC, photolabelling, and Isothermal Titration Calorimetry (ITC) for ultimately characterising the interaction of the effector chalcones with the protein.
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    Importance of Conserved N-domain Residues Thr 441 , Glu 442 , Lys 515 , Arg 560 , and Leu 562 of Sarcoplasmic Reticulum Ca 2+ -ATPase for MgATP Binding and Subsequent Catalytic Steps: PLASTICITY OF THE NUCLEOTIDE-BINDING SITE
    (2003) Clausen, Johannes D; McIntosh, David B; Vilsen, Bente; Woolley, David G; Andersen, Jens Peter
    Nine single mutations were introduced to amino acid residues Thr441, Glu442, Lys515, Arg560, Cys561, and Leu562 located in the nucleotide-binding domain of sarcoplasmic reticulum Ca2+-ATPase, and the functional consequences were studied in a direct nucleotide binding assay, as well as by steady-state and transient kinetic measurements of the overall and partial reactions of the transport cycle. Some partial reaction steps were also examined in mutants with alterations to Phe487, Arg489, and Lys492. The results implicate all these residues, except Cys561, in high affinity nucleotide binding at the substrate site. Mutations Thr441 --> Ala, Glu442 --> Ala, and Leu562 --> Phe were more detrimental to MgATP binding than to ATP binding, thus pointing to a role for these residues in the binding of Mg2+ or to a difference between the interactions with MgATP and ATP. Subsequent catalytic steps were also selectively affected by the mutations, showing the involvement of the nucleotide-binding domain in these reactions. Mutation of Arg560 inhibited phosphoryl transfer but enhanced the E1PCa2 --> E2P conformational transition, whereas mutations Thr441 --> Ala, Glu442 --> Ala, Lys492 --> Leu, and Lys515 --> Ala inhibited the E1PCa2 --> E2P transition. Hydrolysis of the E2P phosphoenzyme intermediate was enhanced in Glu442 --> Ala, Lys492 --> Leu, Lys515 --> Ala, and Arg560 --> Glu. None of the mutations affected the low affinity activation by nucleotide of the phosphoenzyme-processing steps, indicating that modulatory nucleotide interacts differently from substrate nucleotide. Mutation Glu442 --> Ala greatly enhanced reaction of Lys515 with fluorescein isothiocyanate, indicating that the two residues form a salt link in the native protein.
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    Phosphorylated Ca 2+ -ATPase Stable Enough for Structural Studies
    (2001) Henao, Fernando; Delavoie, Franck; Lacapère, Jean-Jacques; McIntosh, David B; Champeil, Philippe
    The atomic structure of sarcoplasmic reticulum Ca(2+)-ATPase, in a Ca(2+)-bound conformation, has recently been elucidated (Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655). Important steps for further understanding the mechanism of ion pumps will be the atomic structural characterization of different key conformational intermediates of the transport cycle, including phosphorylated intermediates. Following our previous report (Champeil, P., Henao, F., Lacapère, J.-J. & McIntosh, D. B. (2000) J. Biol. Chem. 276, 5795-5803), we show here that it is possible to prepare a phosphorylated form of sarcoplasmic reticulum Ca(2+)-ATPase (labeled with fluorescein isothiocyanate) with a week-long stability both in membranes and in mixed lipid-detergent micelles. We show that this phosphorylated fluorescein isothiocyanate-ATPase can form two-dimensional arrays in membranes, similar to those that were used previously to reconstruct from cryoelectron microscopy images the three-dimensional structure of Ca(2+)-free unphosphorylated ATPase. The results also provide hope that crystals of phosphorylated Ca(2+)-ATPase suitable for x-ray crystallography will be achieved.
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    Photoaffinity labeling the nucleotide sites of the sarcoplasmic reticulum Ca²⁺-ATPase
    (1989) Seebregts, Christopher J; McIntosh, David B
    We have synthesized a new class of photoaffinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido-ATP, -ADP and -AMP (TNP- 8N₃ATP, -ADP and -AMP), and their radiolabeled derivatives, and characterized their interaction with the sarcoplasmic reticulum Ca²⁺-ATPase. The TNP-8N₃-nucleotides were synthesized from ATP in three steps involving bromination in the 8-position of the adenine ring followed by displacement with an azido group and then trinitrophenylation of the resulting 8N₃-nucleotide with TNBS. Inclusion of the oxidizing agent, DTNB, in the final reaction was found to be necessary to prevent reduction of the azido group by the released sulfite anion and also elevated the yield of trinitrophenylation to about 80%. Purity was determined spectrophotometrically, as well as by anion exchange TLC and reversed phase HPLC. In the dark, the compounds were found to display most of the features of the parent TNP-nucleotides and interacted with the Ca²⁺-ATPase in a similar way. When activated by illumination, the probes were specifically incorporated into SR vesicles with high efficiency at alkaline pH. The site of labeling was identified as being on the A₁ tryptic fragment.
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    The role of TNP-Nucleotides, LYS492 and CA²⁺chelators in the skeletal muscle sarcoplasmic reticulum CA²⁺atpase cycle
    (1998) Wichmann, Janine; McIntosh, David B; Berman, Mervyn C
    In the first part of this study, the kinetics of decay of TNP-nucleotide superfluorescence was investigated with a view to understanding the role of nucleotides and Lys492 in later steps in the catalytic cycle of the skeletal muscle Ca²⁺ATPase. It has been found previously, and verified here, that tethering TNP-8N₃-AMP to the Ca²⁺ATPase via Lys492 retarded the Ca²⁺ initiated decay of Pᵢ-induced superfluorescence 10-fold compared with untethered nucleotide. The rapidity of the decay upon addition of EDTA suggested that the E₂ ↔ E₁ → E₁Ca₂ steps were being monitored rather than dephosphorylation per se. Tethered diand triphospho species did not accelerate the decay. While monophasic kinetics was observed with untethered TNP-AMP and TNP-8N₃-AMP, complex kinetics were observed with the di- and triphospho TNP-nucleotides. This was shown to be due to the utilization of TNP-ADP and -ATP, and the azido derivatives, as coupled substrates of the Ca²⁺ATPase in the forward direction of catalysis in the presence of Ca²⁺. The hydrolysis rates of TNP-ADP, TNP-ATP, TNP-8N₃ -ADP, and TNP-8N₃ -ATP were 10, 5, 15 and 10 nomoles/min/mg of protein, respectively, at room temperature and pH 5.5. Ca²⁺ transport was supported by all four nucleotides. This is the first time that a diphosphonucleotide has been shown to support Ca²⁺ transport. A new nonhydrolysable triphospho TNPnucleotide, TNP-AMP-PCP was synthesized and shown to interact with the Ca²⁺ATPase in a similar way, in terms of superfluorescence, as the other TNP-nucleotides. It did not show the complex kinetics on inhibition of the Pcinduced superfluorescence by Ca²⁺, but neither did it accelerate the kinetics. It was concluded that TNP-nucleotides do not accelerate the E₂ ↔ E₁ transition under these conditions, possibly because of the presence of glycerol in the medium. In the second part of the study, it was shown that addition of small amounts of chelators EGTA, EDTA, BAPTA, DTPA, HEDTA and NTA to a Ca²⁺ transport assay in which the free Ca²⁺ concentration is monitored by Fluo-3 causes the Ca²⁺ATPase to pump to apparently lower levels as seen in the [Ca²⁺] lim fluorescence. Addition of chelator retards pump function in the sense that it takes longer for 50 nmols Ca²⁺ to be accumulated. Increased thermodynamic efficiency of the pump and contaminating heavy metal ions were considered as possible mechanisms. To some extend Zn²⁺ and Cd²⁺, but not Fe²⁺ and Cu²⁺, appeared to reverse the partial inhibition. While interpretation of the results is difficult, it is suggested that heavy metal ions interact with luminal loops of the Ca²⁺ATPase and enhance Ca²⁺ release under conditions of high luminal Ca²⁺ concentrations.
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    Roles of Conserved P Domain Residues and Mg 2+ in ATP Binding in the Ground and Ca 2+-activated States of Sarcoplasmic Reticulum Ca 2+-ATPase
    (2004) McIntosh, David B; Clausen, Johannes D; Woolley, David G; MacLennan, David H; Vilsen, Bente; Andersen, Jens Peter
    Residues in conserved motifs (625)TGD, (676)FARXXPXXK, and (701)TGDGVND in domain P of sarcoplasmic reticulum Ca(2+)-ATPase, as well as in motifs (601)DPPR and (359)NQR(/K)MSV in the hinge segments connecting domains N and P, were examined by mutagenesis to assess their roles in nucleotide and Mg(2+) binding and stabilization of the Ca(2+)-activated transition state for phosphoryl transfer. In the absence of Mg(2+), mutations removing the charges of domain P residues Asp(627), Lys(684), Asp(703), and Asp(707) increased the affinity for ATP and 2',3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine 5'-triphosphate. These mutations, as well as Gly(626)--> Ala, were inhibitory for ATP binding in the presence of Mg(2+) and for tight binding of the beta,gamma-bidentate chromium(III) complex of ATP. The hinge mutations had pronounced, but variable, effects on ATP binding only in the presence of Mg(2+). The data demonstrate an unfavorable electrostatic environment for binding of negatively charged nucleotide in domain P and show that Mg(2+) is required to anchor the phosphoryl group of ATP at the phosphorylation site. Mutants Gly(626) --> Ala, Lys(684) --> Met, Asp(703) --> Ala/Ser/Cys, and mutants with alteration to Asp(707) exhibited very slow or negligible phosphorylation, making it possible to measure ATP binding in the pseudo-transition state attained in the presence of both Mg(2+) and Ca(2+). Under these conditions, ATP binding was almost completely blocked in Gly(626) --> Ala and occurred with 12- and 7-fold reduced affinities in Asp(703) --> Ala and Asp(707) --> Cys, respectively, relative to the situation in the presence of Mg(2+) without Ca(2+), whereas in Lys(684) --> Met and Asp(707) --> Ser/Asn the affinity was enhanced 14- and 3-5-fold, respectively. Hence, Gly(626) and Asp(703) seem particularly critical for mediating entry into the transition state for phosphoryl transfer upon Ca(2+) binding at the transport sites.
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    Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase
    (2005) Stokes, David L; Delavoie, Franck; Rice, William J; Champeil, Philippe; McIntosh, David B; Lacapère, Jean-Jacques
    Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species.
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    Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase
    (2001) Seekoe, Tshepo; Peall, Susan; McIntosh, David B
    Thapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase. It binds the Ca(2+)-free E2 conformation in the picomolar range, supposedly resulting in a largely catalytically inactive species. We now find that thapsigargin has little effect on medium P(i) <--> HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me(2)SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me(2)SO on the four rate constants governing the reaction of P(i) with Ca(2+)-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k(-2)), whereas that of Me(2)SO is to greatly retard P(i) dissociation (k(-1)), accounting for its well known effect on increasing the apparent affinity for P(i). These effects persist when the agents are used in combination and substantially account for the activated oxygen exchange (v(exchange) = k(-2)[EP]). Kinetic simulations show that the overall rate constant for the formation of EP is very fast (approximately 300 s(-1)) when the exchange is maximal. Thapsigargin greatly stabilizes Ca(2+)-ATPase against denaturation in detergent in the absence of Ca(2+), as revealed by glutaraldehyde cross-linking, suggesting that the membrane helices lock together. It seems that the reactions at the phosphorylation site, associated with the activated exchange reaction, are occurring without much movement of the transport site helices, and we suggest that they may be associated solely with an occluded H+ state.