Regular paperHigh-affinity and low-affinity vanadate binding to sarcoplasmic reticulum Ca2+-ATPase labeled with fluorescein isothiocyanate
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Sarcoplasmic reticulum calcium ATPase interactions with decaniobate, decavanadate, vanadate, tungstate and molybdate
2012, Journal of Inorganic BiochemistryCitation Excerpt :It also suggests that V10 will bind to the calcium pump at the vanadate binding site, but at higher concentrations it will also bind to the nucleotide binding site, as previously found using fluorescence and ESR spectroscopy [63,64]. For monovanadate a kd in the order of μM− 1 was found, [65], using fluorescein isothiocyanate (FITC) fluorescence. The metal:protein ratio of 1:1 described above, was confirmed by applying the Job method, using several metal:protein ratios (not shown: supplementary Fig. 1).
Structural studies of a stabilized phosphoenzyme intermediate of Ca <sup>2+</sup>-ATPase
2005, Journal of Biological ChemistryCitation Excerpt :The fact that decavanadate actually binds to this species is documented by the slight decrease in fluorescence caused by decavanadate addition. This decrease is comparable, in relative terms, to that seen when decavanadate is added to the high fluorescence species (not shown, but see Ref. 30), and is not simply due to absorption of the excitation wavelength by decavanadate. We used [32P]AcP to quantify the amount of covalent phosphoenzyme formed under various conditions.
Calcium transport by sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase: Role of the a domain and its C-terminal link with the transmembrane region
2002, Journal of Biological ChemistryRemarkable stability of solubilized and delipidated sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase with tightly bound fluoride and magnesium against detergent-induced denaturation
2002, Journal of Biological ChemistryCitation Excerpt :Hydrogen bonding of F−with residues at or near the phosphorylation site in the Mg2+·F−·E2 complex likely contributes at least in part to stabilization of such compactly organized domain structure. Orthovanadate, a phosphate analog, binds at the phosphorylation site in the presence of Mg2+ and absence of Ca2+(25-27) and produces a compactly organized conformation, as Mg2+/F− does (6). On the other hand, the Ca2+-ATPase in the presence of Mg2+/vanadate and absence of Ca2+ was reported to be rapidly denatured by C12E8 (8).
Phosphorylated Ca<sup>2+</sup>-ATPase Stable Enough for Structural Studies
2001, Journal of Biological ChemistryCitation Excerpt :We found that the low fluorescence of this species remained stable in the presence of thapsigargin and decavanadate (Fig.1 a). The decavanadate-induced fast drop in FITC fluorescence (followed by a slower rise, because the added decavanadate also contained trace amounts of orthovanadate) is essentially due to true fluorescence quenching, as also described for non-phosphorylated FITC-labeled ATPase (6). Because the same phosphorylated form can be formed from Piand passively loaded vesicles, at low enough free Ca2+ (4,5), this made it possible to evaluate the stability of the above low fluorescence phosphorylated form on a much longer time scale.
A Remarkably Stable Phosphorylated Form of Ca<sup>2+</sup>-ATPase Prepared from Ca<sup>2+</sup>-loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles
2001, Journal of Biological ChemistryCitation Excerpt :Similarly, although the formation from Pi of this low fluorescence species was inhibited by thapsigargin, an inhibitor of Ca2+-dependent changes as well as of phosphorylation from Pi (15), once formed, this species was resistant to the addition of thapsigargin (Fig. 2, traces H and I). Addition of orthovanadate to a preformed low fluorescence species brought the fluorescence back to a high level, at a concentration-dependent rate (data not shown), but decavanadate could bind to this species without destabilizing it; about the same relative quenching was observed when decavanadate was added to control FITC-ATPase (16) or to a low fluorescence TG-stabilized FITC-ATPase species formed from AcP (data not shown).2 Traces J–N in Fig. 2 show how formation of the low fluorescence species was influenced by the final free Ca2+ concentration in the medium.