Titration of the nucleotide binding sites of Sarcoplasmic Reticulum Ca2+-ATPase with 2′, 3′-0-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate and 5′-diphosphate

Dupont, Yves; Chapron, Yves; Pougeois, Richard

doi:10.1016/0006-291x(82)91250-5

Cited by 71 publications

(47 citation statements)

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“…As previously described [20], Tb-ATP binding was determined by filtration on Millipore filters using Tb-[I4C]ATP. Tb-[32P]ATP hydrolysis was evaluated by measuring the 32P production, after centrifugation into tubes containing charcoal in 0.1 M HCI [20].…”

Section: Methodsmentioning

confidence: 99%

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Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca²⁺‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP

Girardet

Dupont

Lacapère³

1989

European Journal of Biochemistry

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Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3 +-binding sites have been found: a first class of low-affinity (Kd = 10 pM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity ( < 0.1 pM) corresponds to the calcium transport sites, their occupancy by terbium induces the El to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP.Substitution of H 2 0 by D 2 0 shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site.Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2 + El transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the El -E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.The molecular mechanism by which plasma-membranetransport ATPases convert chemical energy of ATP into osmotic energy has been a challenging subject to investigation for many years. The state of our knowledge of these ATPases varies from one system to another, the reaction mechanism of the Na+/K+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase being by far the best understood.The most precise transport schemes used so far are the mechano-chemical schemes, where a major conformational change is thought to cause the ion binding site to be alternately exposed to one side and the other of the membrane. This scheme has been adopted by the vast majority of scientists working on the Ca2+-ATPase and Na'/K+-ATPase [1, 21.Another scheme, close to that advocated by Mitchell, invokes a direct interaction between the phosphorylated group and the transported ions [3,4]. Results of deMeis et al. [5] and Dupont and Pougeois [6] have: highlightened the role of the water activity in the active site of the Ca2'-ATPase, which may be extremely different from that of standard conditions. Enzymes. Ca*+-ATPase (EC 3.6.1.38); Na'/K+-ATPase (EC 3.6.1 .3 7).In this work we have investigated the interaction between the binding sites of the sarcoplasmic reticulum Ca2 '-ATPase, by using two fluorescent compounds: terbium ions as a substitute for calcium and Tb-FTP as an analogue of Mg-ATP.All the lanthan...

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Section: Methodsmentioning

confidence: 99%

“…Tb-[32P]ATP hydrolysis was evaluated by measuring the 32P production, after centrifugation into tubes containing charcoal in 0.1 M HCI [20]. All the experiments were performed at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca²⁺‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP

Girardet

Dupont

Lacapère³

1989

European Journal of Biochemistry

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“…To complement the preceding data and also to test CPA concentrations above the stoichiometric level, we took advantage of the fluorescent probe TNP-ATP (22,23). The binding of TNP-ATP to the enzyme in the absence of Ca 2ϩ is accompanied by a specific fluorescence increase.…”

Section: Ca 2ϩ -Atpase Inhibition By Cyclopiazonic Acidmentioning

confidence: 99%

On the Inhibition Mechanism of Sarcoplasmic or Endoplasmic Reticulum Ca2+-ATPases by Cyclopiazonic Acid

Plenge-Tellechea¹,

Soler

Fernández-Belda

1997

Journal of Biological Chemistry

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Ca 2؉-ATPase inhibition by stoichiometric and substoichiometric concentrations of cyclopiazonic acid was studied in sarcoplasmic reticulum preparations from rabbit fast-twitch muscle. The apparent affinity of the nonphosphorylated enzyme for ATP showed a K d of ϳ3 M in the absence of cyclopiazonic acid and ϳ28 M in the presence of the drug. Fractional saturation of the enzyme by cyclopiazonic acid was accompanied by the appearance of two ATP-binding populations (enzyme with and without drug) and a progressive increase in the half-maximal concentration for saturating the ATPbinding sites. Enzyme turnover in the presence of stoichiometric concentrations of cyclopiazonic acid displayed lower apparent affinity for ATP and lower maximal hydrolytic activity than in the absence of the drug. When cyclopiazonic acid is in the substoichiometric range, the observed kinetic parameters will correspond to the simultaneous contribution of two different reaction cycles sustained by the enzyme with and without drug. The inhibition could be elicited by adding ATP to allow the enzyme turnover when cyclopiazonic acid was preincubated with the enzyme in the presence of Ca 2؉. The onset of inhibition during enzyme cycling was observed over a period of seconds, revealing the existence of a low inhibition rate constant. It is concluded that cyclopiazonic acid decreases enzyme affinity for ATP in non-turnover conditions by approximately one order of magnitude. This allows enzyme cycling after drug binding, provided that a high ATP concentration is used. Cyclopiazonic acid and ATP do not compete for the same binding site.

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“…crystallography | ion pump | nucleotide derivatives T rinitrophenyl (TNP)-nucleotides (1) are often used for probing the structure of ATP-binding sites and conformational changes arising from nucleotide binding (2,3), and for measuring the affinity of ATP by competition experiments (2,4). It is a preferred ATP analogue for photochemical crosslinking with azide derivatives (5).…”

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“…For instance, TNP-ATP is a high affinity (nM) antagonist of P2X receptors, which have IC 50 for ATP (or AMPPCP) in the μM range (11). The affinity is at least one order of magnitude higher in the E2 states of Ca 2þ -ATPase (3,12) and Na þ , K þ -ATPase (4), representative P-type ATPases. Furthermore, TNP-AMP binds to Ca 2þ -ATPase similarly to or even more strongly than TNP-ATP (12), in marked contrast to AxPs.…”

mentioning

confidence: 99%

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca ²⁺ -ATPase in the absence of Ca ²⁺

Toyoshima

Yonekura

Tsueda

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Trinitrophenyl derivatives of adenine nucleotides are widely used for probing ATP-binding sites. Here we describe crystal structures of Ca 2þ -ATPase, a representative P-type ATPase, in the absence of Ca 2þ with bound ATP, trinitrophenyl-ATP, -ADP, and -AMP at better than 2.4-Å resolution, stabilized with thapsigargin, a potent inhibitor. These crystal structures show that the binding mode of the trinitrophenyl derivatives is distinctly different from the parent adenine nucleotides. The adenine binding pocket in the nucleotide binding domain of Ca 2þ -ATPase is now occupied by the trinitrophenyl group, and the side chains of two arginines sandwich the adenine ring, accounting for the much higher affinities of the trinitrophenyl derivatives. Trinitrophenyl nucleotides exhibit a pronounced fluorescence in the E2P ground state but not in the other E2 states. Crystal structures of the E2P and E2 ∼ P analogues of Ca 2þ -ATPase with bound trinitrophenyl-AMP show that different arrangements of the three cytoplasmic domains alter the orientation and water accessibility of the trinitrophenyl group, explaining the origin of "superfluorescence." Thus, the crystal structures demonstrate that ATP and its derivatives are highly adaptable to a wide range of site topologies stabilized by a variety of interactions.crystallography | ion pump | nucleotide derivatives T rinitrophenyl (TNP)-nucleotides (1) are often used for probing the structure of ATP-binding sites and conformational changes arising from nucleotide binding (2, 3), and for measuring the affinity of ATP by competition experiments (2, 4). It is a preferred ATP analogue for photochemical crosslinking with azide derivatives (5). These applications utilize the enhancement of fluorescence or absorption of visible light of the TNP group upon binding to a protein (6). Because of its sensitivity, competition with ATP/ADP has been a valuable means for examining mutational effects on nucleotide affinity (7). Thus, TNP nucleotides have been widely used with F1 (8), myosin (1), and P-type ATPases (2-5, 7, 9, 10), among others.Nonetheless, whether TNP derivatives are good mimics of authentic adenine nucleotides (AxPs) may be questionable. In several proteins TNP nucleotides have much higher affinities than the genuine AxPs. For instance, TNP-ATP is a high affinity (nM) antagonist of P2X receptors, which have IC 50 for ATP (or AMPPCP) in the μM range (11). The affinity is at least one order of magnitude higher in the E2 states of Ca 2þ -ATPase (3, 12) and Na þ , K þ -ATPase (4), representative P-type ATPases. Furthermore, TNP-AMP binds to Ca 2þ -ATPase similarly to or even more strongly than TNP-ATP (12), in marked contrast to AxPs. Thus, a substantially different binding mode of TNP derivatives is suggested. Although more than 20 entries are registered in the Protein Data Bank (PDB) for Ca 2þ -ATPase (reviewed in ref. 13), no structure with a bound TNP nucleotide exists. In fact, only three crystal structures have been published with bound TNP nucleotides. They are a bacterial h...

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Titration of the nucleotide binding sites of Sarcoplasmic Reticulum Ca2+-ATPase with 2′, 3′-0-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate and 5′-diphosphate

Cited by 71 publications

References 17 publications

Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca²⁺‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP

Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca²⁺‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP

On the Inhibition Mechanism of Sarcoplasmic or Endoplasmic Reticulum Ca2+-ATPases by Cyclopiazonic Acid

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca ²⁺ -ATPase in the absence of Ca ²⁺

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Titration of the nucleotide binding sites of Sarcoplasmic Reticulum Ca2+-ATPase with 2′, 3′-0-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate and 5′-diphosphate

Cited by 71 publications

References 17 publications

Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca2+‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP

Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca2+‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP

On the Inhibition Mechanism of Sarcoplasmic or Endoplasmic Reticulum Ca2+-ATPases by Cyclopiazonic Acid

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca 2+ -ATPase in the absence of Ca 2+

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Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca²⁺‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP

Evidence of a calcium‐induced structural change in the ATP‐binding site of the sarcoplasmic‐reticulum Ca²⁺‐ATPase using terbium formycin triphosphate as an analogue of Mg‐ATP

Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca ²⁺ -ATPase in the absence of Ca ²⁺