Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase

Stephens, Eileen M.; Grisham, Charles M.

doi:10.1021/bi00589a016

Cited by 71 publications

(20 citation statements)

References 38 publications

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“…This value is in agreement with that previously reported for various lanthanide ions by other authors [14,[23][24][25]. However, this value of the dissociation constant of Tb .…”

Section: Intrinsicfluorescence Of the Ca2 +-Atpasesupporting

confidence: 93%

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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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“…This value is in agreement with that previously reported for various lanthanide ions by other authors [14,[23][24][25]. However, this value of the dissociation constant of Tb .…”

Section: Intrinsicfluorescence Of the Ca2 +-Atpasesupporting

confidence: 93%

“…In another paper [4] it was proposed that this hydration change also affects the calcium transport sites. Thus, another way to pursue the investigation of the transport mechanism consists of the study of the hydration of the binding sites [6,14,24,39,40]. Also important is the determination of the distances between the binding sites.…”

Section: Discussionmentioning

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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“…It is very likely that binding to the high affinity sites of Ca2+-ATPase proceeds via a similar process. This idea is further substantiated in [25] where the electron spin relaxation of gadolinium ions bound to the high affinity sites of Ca'+-ATPase were measured. They suggested that the bound gadolinium ions are much less hydrated than in solution and that the binding sites have a reduced accessibility of solvent water.…”

Section: High Affinity Calcium Sitesmentioning

confidence: 87%

Is Ca²⁺‐ATPase a water pump?

Dupont

1983

FEBS Letters

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The mechanism of free energy coupling in active transport is discussed with special reference to the sarcoplasmic reticulum Ca '+-ATPase. In the current working schemes for cation transport ATPases, free energy transduction is nearly always based on enzyme conformational changes. The principal objective of the present article is to examine whether recent experimental results on Ca*+-ATPase may in fact be better explained by assuming the existence of a direct chemiosmotic process. In the scheme proposed, free energy transduction between ATP and calcium is based on a transfer of solvation water between the acylphosphate bond and the bound calcium ions.

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“…Probably phosphorylation of the enzyme is required for the occlusion to occur, although the phosphorylation level obtained with Eu3+ is only 25% of that observed in presence of Ca2+. Dupont [32] and Stephen and Grisham [5] have also proposed that in the phosphorylated enzyme the high-affinity Ca2+ binding sites are occluded.…”

Section: Eflect Of Atp On Eu3+-atpase Complexmentioning

confidence: 99%

Characterization of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum by laser-excited europium luminescence

Gangola¹,

Shamoo²

1987

Eur J Biochem

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The molecular environment of Ca2+ translocating sites of skeletal muscle sarcoplasmic reticulum (SR) (Ca2+ + Mg2+)-ATPase has been studied by pulsed-laser excited luminescence of Eu3+ used as a Ca2+ analogue.Interaction of Eu3+ with SR was characterized by investigating its effect on partial reactions of the Ca2+ transport cycle. In native SR vesicles, Eu3+ was found to inhibit CaZ+ binding, phosphoenzyme formation, ATP hydrolysis activity and Ca2+ uptake in parallel fashion. The non-specific binding of Eu3+ to acidic phospholipids associated with the enzyme was prevented by purifying (Ca2 + + Mg2 +)-ATPase and exchanging the endogenous lipids with a neutral phospholipid, dioleoylglycerophosphocholine. The results demonstrate that the observed inhibition of Ca2+ transport by Eu3+ is due to its binding to Ca2+ translocating sites.The 'Fo --* 5Do transition of Eu3+ bound to these sites was monitored. The non-Lorentzian nature of the excitation profile and a double-exponential fluorescence decay revealed the heterogeneity of the two sites. Measurement of fluorescence decay rates in H 2 0 / D 2 0 mixture buffers further distinguished the sites. The number of water molecules in the first co-ordination sphere of Eu3+ bound at transport sites were found to be 4 and 1.5. Addition of ATP reduced these numbers to zero and 0.6. These data show that the calcium ions in translocating sites are well enclosed by protein ligands and are further occluded down to zero or one water molecule of solvation during the transport process.The membrane-bound enzyme, (Ca2is the primary calcium pump protein for sequestration of Ca2+ in skeletal muscle sarcoplasmic reticulum [l, 21. The (Ca2+ + Mg2')-ATPase is a single polypeptide of relative molecular mass 110000. The enzyme contains two highaffinity Ca2+ binding sites which are presumed to be both the transport sites and the sites of Ca2+ activation of ATP hydrolysis. In a recent report from our laboratory it has been demonstrated that the two high-affinity sites are heterogeneous [3]. They differ in their sensitivity to temperature and tryptic digestion. Detailed information on the molecular environment of these Ca2 +-translocating sites would add to the understanding of the structure and functional relationship of the enzyme. [8-101 to probe the Ca2+ binding sites in (Ca2+ + Mgzf)-ATPase of SR. The method involves direct excitation of the 7Fo + 5Do transition between non-degenerate levels in the Eu3+ ion by means of an intense pulsed-laser source. The excitation profiles and fluorescence decay constants are highly sensitive to the environment of Eu3 + ions and are therefore used to characterize the distinct Eu3+ binding sites in the system. Fluorescence lifetime measurements in H 2 0 and D 2 0 allow the determination of the number of water molecules in the first coordination sphere of Eu3+ ion. This study was performed to obtain information regarding the molecular environment of Ca2+ translocating sites and the changes that occur during Ca2 + uptake associated with ATP hydrolysis. Preli...

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Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase

Cited by 71 publications

References 38 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

Is Ca²⁺‐ATPase a water pump?

Characterization of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum by laser-excited europium luminescence

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Lithium-7 nuclear magnetic resonance, water proton nuclear magnetic resonance and gadolinium electron paramagnetic resonance studies of the sarcoplasmic reticulum calcium ion transport adenosine triphosphatase

Cited by 71 publications

References 38 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

Is Ca2+‐ATPase a water pump?

Characterization of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum by laser-excited europium luminescence

Contact Info

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Resources

About

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

Is Ca²⁺‐ATPase a water pump?