Time-Resolved Fluorescence Energy Transfer Measurements Between Site-Specific Probes On The Ca-Atpase Of Sarcoplasmic Reticulum In Different Enzymatic States

Squier, Thomas C.; Bigelow, Diana J.; Ancos, Jorge Garcia de; Bishop, James E.; Inesi, Giuseppe

doi:10.1117/12.945424

Cited by 8 publications

(11 citation statements)

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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: 65%

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: 65%

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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“…SR vesicles (4 mg/ml SR protein) were digested with trypsin at a ratio of trypsin/SR protein of 1:100 up to 35 min at 25°C in 80 mM KCI, and 20 mM MOPS, pH 7.0 [27]. The reaction was stopped by addition of soybean trypsin inhibitor (type l-S, Sigma) at an inhibitor/trypsin ratio of 2:1.…”

Section: Limited Tryptic Digestionmentioning

confidence: 99%

“…The limited proteolysis of SR protein (containing both SERCAla and SERCA2a) yields subfragments A, A1, A2, and B [27]. Fig.…”

Section: The Localization Of Nitrotyrosinementioning

confidence: 99%

Accumulation of nitrotyrosine on the SERCA2a isoform of SR Ca‐ATPase of rat skeletal muscle during aging: a peroxynitrite‐mediated process?

Viner¹,

Ferrington²,

Hühmer³

et al. 1996

FEBS Letters

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132

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The SR Ca-ATPase in skeletal muscle SR vesicles isolated from young adult (5 months) and aged (28 months) rats was analyzed for nitrotyrosine. Only the SERCA2a isoform contained significant amounts with approximately one and four nitrotyrosine residues per young and old Ca-ATPase, respectively. The in vitro exposure of SR vesicles of young rats to peroxynitrite yielded selective nitration of the SERCA2a Ca-ATPase even in the presence of excess SERCAla. No nitration was observed during the exposure of SR vesicles to nitric oxide in the presence of 02. These data suggest the in vivo presence of peroxynitrite in skeletal muscle. The greater nitrotyrosine content of SERCA2a from aged tissue implies an age-associated increase in susceptibility to oxidation by this species.

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“…In addition, the overall octahedral or tetrahedral shape of the electronic distribution of Co2+ ions in aqueous solutions tends to minimize the effects of the orientation factor in energy transfer between each donor/acceptor pair. This is a major advantage with respect to pairs of donor and acceptor being large planar fluorescent labels, such as those used in [6,71. We have carried out a systematic study on the effect of Co2+ and Co2+ .…”

Section: Mg2+mentioning

confidence: 99%

Distances between functional sites of the Ca²⁺+ Mg²⁺‐ATPase from sarcoplasmic reticulum using Co²⁺ as a spectroscopic ruler

Cuenda¹,

Henao²,

Gutiérrez‐Merino³

1990

European Journal of Biochemistry

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Cobalt ion inhibits the Ca2+ + Mg2 +-ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co2+ appropriately as a spectroscopic ruler to map functional sites of the Ca2+ + Mg2+-ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies.1. The apparent Ko.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co2+ binding to the enzyme. Millimolar Ca2 from which we obtain a distance of 1.1 -1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites. Co2+. ATP can be used as a substrate by this enzyme with V,,, of 2.4 f 0.2 pmol ATP hydrolyzed min- Co2+ partially quenches, about 10The sarcoplasmic reticulum Ca2+ + Mg2 +-ATPase uses Mg2+ . ATP as its physiological substrate and couples ATP hydrolysis to the active transport of Ca" across this membrane [l]. Binding of two Ca2+ ions to high-affinity binding sites/ATPase molecule is step that precedes Ca2+ transport to the luminal space of sarcoplasmic reticulum [l, 21. To understand better the Ca2+ transport process coupled to ATP hydrolysis requires complementary experimental information obtained by using kinetic and structural studies of this enzyme. In this regard, the location of Ca2+ and ATP binding sites relative to each other is an important issue; there is a large range of values for the distance between these sites on ATPase reported by different groups of investigators, i. e. 1 -6 nm [3 -71. Forster energy transfer between different donor/acceptor pairs of fluorescent targets selectively bound to these sites has been used to obtain these estimations. On the other hand, Xray diffraction studies [8, 91 and image processing of electron microscopy data [lo, 111 have provided evidence for an overall shape of an ATPase unit protruding about 4.0 -6.0 nm from the lipid/water interface and having a cross-sectional diameter of the cytosolic lobule of approximately 8.0-9.0 nm in its dimeric form, which is probably the predominant state in

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Time-Resolved Fluorescence Energy Transfer Measurements Between Site-Specific Probes On The Ca-Atpase Of Sarcoplasmic Reticulum In Different Enzymatic States

Cited by 8 publications

References 0 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

Accumulation of nitrotyrosine on the SERCA2a isoform of SR Ca‐ATPase of rat skeletal muscle during aging: a peroxynitrite‐mediated process?

Distances between functional sites of the Ca²⁺+ Mg²⁺‐ATPase from sarcoplasmic reticulum using Co²⁺ as a spectroscopic ruler

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Time-Resolved Fluorescence Energy Transfer Measurements Between Site-Specific Probes On The Ca-Atpase Of Sarcoplasmic Reticulum In Different Enzymatic States

Cited by 8 publications

References 0 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

Accumulation of nitrotyrosine on the SERCA2a isoform of SR Ca‐ATPase of rat skeletal muscle during aging: a peroxynitrite‐mediated process?

Distances between functional sites of the Ca2++ Mg2+‐ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler

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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

Distances between functional sites of the Ca²⁺+ Mg²⁺‐ATPase from sarcoplasmic reticulum using Co²⁺ as a spectroscopic ruler