Modulation of calcium binding in sarcoplasmic reticulum adenosinetriphosphatase

Watanabe, T.; Lewis, David E.; Nakamoto, Robert K.; Kurzmack, M.; Fronticelli, Clara; Inesi, G.

doi:10.1021/bi00526a015

Cited by 131 publications

(65 citation statements)

References 29 publications

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“…Fig.2 shows that inhibition of MgE'-P formation by Ca2+ is qualitatively similar to inhibition observed for the vesicular protein [ 161, and in particular that it is a cooperative function of [Ca2']. The measured Hill coefficient (inset) is about 1.4, similar to that reported for equilibrium Ca2+ binding studies at pH 6 [22]. In terms of the scheme of fig.…”

Section: Methodssupporting

confidence: 78%

“…Our previous detailed study with leaky SR vesicles [18], in which both [Mg"] and [Pi] were varied (but [Mg"] was kept below the level at which inhibition sets in) gave I = 0.48. This result, with the aid of equations given previously ([ 181, see also [22]) demonstrates that the number of phosphorylation sites per protein molecule is unaltered by solubilization, but that the binding affinity for Pi is reduced. A similar result (same number of sites, but lower binding affinity) is obtained from a Scatchard plot based on the Mg*+-dependence of phosphorylation, using the data of fig.3 below 20 mM Mg*+, i.e., at Mg*+ concentrations too small for manifestation of the cooperative inhibitory effect.…”

Section: Methodssupporting

confidence: 63%

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Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Martin

Tanford

1984

FEBS Letters

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Phosphorylation (by inorganic phosphate) of sarcoplasmic reticulum Ca pump protein has been studied in a detergent solution in which the protein has been previously shown to exist as a monomer. The course of the reaction is qualitatively similar to that observed for membrane-bound (possibly oligomeric) protein.In particular, the results indicate that alternation between the two principal conformational states of the Ca pump protein persists in the monomeric state, which suggests that the machinery for coupling of ATP hydrolysis to Ca *+ transport is intact. There are quantitative differences between monomeric and membrane-bound protein with respect to phosphorylation, but they are not necessarily related to the state of association.

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Section: Methodssupporting

confidence: 78%

Section: Methodssupporting

confidence: 63%

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Martin

Tanford

1984

FEBS Letters

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“…(15), and dashed line is a fit using an alternate model that is identical to the proposed model except SERCA is assumed to sequentially bind three protons instead of four. Calculated dependence of various SERCA species concentration on free calcium concentration [Ca 2+ ] at fixed values of pH (6,7,8) and free Mg 2+ (0, 0.1 and 10 mM) in the solution; the calculated curves are obtained by using Eqs. (1)- (11) and parameter values listed in Table 2.…”

Section: Discussionmentioning

confidence: 99%

“…Binding is pH dependent [8,9] and occurs with high affinity and positive cooperativity. The two Ca 2+ binding sites reside within the membrane bound region of the ATPase [10][11][12].…”

mentioning

confidence: 99%

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Zafar¹,

Hussain²,

Liu³

et al. 2008

Archives of Biochemistry and Biophysics

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Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca 2+ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca 2+ binding isotherms obtained by equilibrium measurements in the presence of various H + and Mg 2+ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca 2+ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation. KeywordsCa 2+ ATPase; Ca 2+ binding; H + /Ca 2+ exchange; Statistical analysis Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. The characteristics of this binding are of utmost importance, as they determine catalytic activation, specificity and stoichiometry of transport and countertransport, and efficiency of ATP utilization. They also correspond to structural features of the enzyme protein with regard to binding affinity and specificity, as well as conformational consequences of binding resulting in catalytic activation. We provide here a detailed analysis of Ca 2+ binding to the Ca 2+ transport ATPase of sarco-endoplasmic reticulum (SERCA) [1,2]. The SERCA isoform of skeletal muscle is a well characterized enzyme [3,4] that utilizes the free energy of ATP for Ca 2+ transport against a concentration gradient. The functional unit is a protein monomer comprising 994 amino acid residues. The sequence is folded into a cluster of 10 segments forming a transmembrane region, and three relatively large domains ("N", "P" and "A") protruding from the cytosolic surface of the membrane [5,6]. The ATPase cycle begins with high affinity binding of Ca 2+ derived from the cytosolic medium ("outside"), followed by ATP utilization to form a phosphorylated enzyme intermediate. Isomerization of the phosphoenzyme intermediate is then coupled to active transport of the bound Ca 2+ across the membrane ("inside"). Hydrolytic cleavage of the phosphoenzyme is the final step that allows enzyme turnover. The high Ca 2+ affinity state of the enzyme is generally referred to as E 1 , and the low affinity as E 2 . Methods Ca 2+ binding and ATPase measurementsVesicular fragments of sarcoplasmic reticulum membrane were obtained from rabbit hind leg muscle as previously described [19]. SERCA accounts for 50-60% of total protein in this preparation. Total protein was determined by the Folin method, standardized with serum albumin.Calcium binding was measured under equilibrium conditions by molecular sieve chromatography [7,20] We now discuss the energies associated with the proposed SERCA species. The energy of an unoccupied species (E) is set as the reference and the energies of all other species are measured with respect to it. ...

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“…While there has been general agreement that there are twice as many Ca 2+-binding sites as there are phosphorylation sites or high affinity ATPase-binding sites Abbreviations: bicine, N,N-bis-(2-hydroxyethyl)-glycine: Ca2+-ATPase, calcium-and magnesium-dependent adenosine triphosphatase (EC 3.6.1.3); C 12E9, nona-ethyleneglycol dodecyl ether; FITC, Fluorescein 5 isothiocyanate (isomer I); FTC, fluorescein thiocarbamyl; SR, sarcoplasmic reticulum (4 nmol/mg SR protein), the estimated number of peptide chains is -6 nmol/mg [7,8], an intermediate figure, from which it is difficult to draw clear conclusions.…”

Section: Introductionmentioning

confidence: 99%

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum

et al. 1982

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Incorporation of 4.5 nmol fluorescein isothiocyanate/mg rabbit sarcoplasmic reticulum, or of 7.4 nmol/ mg purified ATPase, was sufficient to inhibit the activity completely. These results are not consistent with the suggestion (Pick, U. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta 626, 255-261) that 2 mol ATPase were inhibited by each mole of reagent incorporated. A single labelled peptide was purified from the inhibited ATPase and it was shown that Lys 3/190, 10 residues from the N-terminus of tryptic fragment B, was the reactive lysine residue. This site is close to a potential nucleotide-binding fold in the ATPase sequence. A similar peptide showing only 2 conservative replacements was isolated from the sarcoplasmic reticulum of the lobster. (Ca 2 + + Mg 2 + )A TPase inhibition Reactive lysine residue Fluorescein isothiocyanateIntegral membrane protein Nucleotide-binding fold (Sarcoplasmic reticulum)

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Modulation of calcium binding in sarcoplasmic reticulum adenosinetriphosphatase

Cited by 131 publications

References 29 publications

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum

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Modulation of calcium binding in sarcoplasmic reticulum adenosinetriphosphatase

Cited by 131 publications

References 29 publications

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca2+‐dependent adenosine triphosphatase of sarcoplasmic reticulum

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Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum