scholarly journals Inactivation of sarcoplasmic-reticulum Ca2+-ATPase in low-frequency-stimulated muscle results from a modification of the active site

1992 ◽  
Vol 285 (1) ◽  
pp. 303-309 ◽  
Author(s):  
S Matsushita ◽  
D Pette
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Active Site ◽  
Low Frequency ◽  
Fluorescein Isothiocyanate ◽  
Tryptophan Fluorescence ◽  
Atp Binding ◽  
Binding Characteristics ◽  
Intrinsic Tryptophan Fluorescence ◽  
Inactive Form ◽  
Induced Changes

Molecular changes underlying the partial inactivation of the sarcoplasmic-reticulum (SR) Ca(2+-) ATPase in low-frequency-stimulated fast-twitch muscle were investigated in the present study. The specific Ca(2+)-ATPase activity, as well as the ATP- and acetyl phosphate-driven Ca2+ uptakes by the SR, were reduced by approx. 30% in 4-day-stimulated muscle. Phosphoprotein formation of the enzyme in the presence of ATP or Pi was also decreased to the same extent. Measurements of ATP binding revealed a 30% decrease in binding to the enzyme. These changes were accompanied by similar decreases in the ligand-induced (ATP, ADP, Pi) intrinsic tryptophan fluorescence. A decreased binding of fluorescein isothiocyanate (FITC) corresponded to the lower ATP binding and phosphorylation of the enzyme. Moreover, Pi-induced changes in fluorescence of the FITC-labelled enzyme did not differ between SR from stimulated and contralateral muscles, indicating that Ca(2+)- ATPase molecules which did not bind FITC were responsible for the decreased Pi-dependent phosphorylation, and therefore represented the inactive form of the enzyme. No differences existed between the Ca(2+)-induced changes in the intrinsic fluorescence of SR from stimulated and contralateral muscles which fit their similar Ca(2+)-binding characteristics. Taking the proposed architecture of the Ca2(+)-ATPase into consideration, our results suggest that the inactivation relates to a circumscribed structural alteration of the enzyme in sections of the active site consisting of the nucleotide-binding and phosphorylation domains.

scholarly journals The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum

1986 ◽  
Vol 234 (2) ◽  
pp. 363-371 ◽  
Author(s):  
I Jona ◽  
A Martonosi
Keyword(s):  
Membrane Potential ◽  
Sarcoplasmic Reticulum ◽  
Fluorescence Intensity ◽  
Binding Sites ◽  
Fluorescein Isothiocyanate ◽  
Tryptophan Fluorescence ◽  
Lanthanide Ions ◽  
Positive Potential ◽  
High Affinity ◽  
Ion Substitution

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.

Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

1995 ◽  
Vol 15 (5) ◽  
pp. 317-326 ◽  
Author(s):  
Tohru Kanazawa ◽  
Hiroshi Suzuki ◽  
Takashi Daiho ◽  
Kazuo Yamasaki
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Tryptophan Fluorescence ◽  
Cytoplasmic Domain ◽  
Catalytic Cycle ◽  
Atp Binding ◽  
Atp Binding Site

Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps.

The Influence of Detergents on the Ca2+-and Mg2+-Dependent Adenosine Triphosphatase of the Sarcoplasmic Reticulum

1982 ◽  
Vol 37 (3-4) ◽  
pp. 299-307 ◽  
Author(s):  
Hans Lüdi ◽  
Bernhard Rauch ◽  
Wilhelm Hasselbach
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Ions ◽  
Inorganic Phosphate ◽  
Adenosine Triphosphatase ◽  
Tryptophan Fluorescence ◽  
The Other ◽  
Phosphorylated Protein ◽  
Intrinsic Tryptophan Fluorescence ◽  
Low Concentrations ◽  
Calcium Affinity

Abstract During the stepwise solubilization of sarcoplasmic reticulum vesicles with detergents, the following changes in the structural and enzymatic properties of the preparation are observed: 1. The viscosity of the vesicular suspension initially rises. This change is accompanied by the formation of elongated tubules. Subsequently the membranes are completely desintegrated, resulting in a considerable reduction of the viscosity. 2. A decrease in the activity of the Ca2+-dependent ATPase, which is restored after complete solubilization. 3. A decrease in the change of intrinsic tryptophan-fluorescence on removal of calcium ions, which is also restored after complete solubilization. 4. A decrease of the calcium affinity of the ATPase. 5. A decrease in the amount of phosphorylated protein formed by the incorporation of inorganic phosphate. On the other hand, the amount of phosphoprotein formed from ATP is not affected during solubilization. 6. The dependence of the initial rates of phosphoprotein formation from inorganic phosphate on either magnesium or inorganic phosphate at low concentrations of the respective ligand changes from an S-shape profile to a normal hyperbolic profile after solubilization.

scholarly journals Cytoplasmic interactions between phospholamban residues 1–20 and the calcium-activated ATPase of the sarcoplasmic reticulum

2001 ◽  
Vol 355 (3) ◽  
pp. 699-706 ◽  
Author(s):  
Parveen SHARMA ◽  
Valerie B. PATCHELL ◽  
Yuan GAO ◽  
James S. EVANS ◽  
Barry A. LEVINE
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Tryptophan Fluorescence ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Fluorescence Studies ◽  
Cytoplasmic Region ◽  
Spectral Changes ◽  
Conformational Effects ◽  
Intrinsic Tryptophan Fluorescence ◽  
Nmr Study ◽  
Phosphorylated Peptide

Phospholamban regulates the activity of the calcium-activated ATPase (CaATPase) of cardiac sarcoplasmic reticulum. Equilibrium fluorescence studies have shown that the N-terminal cytoplasmic region of phospholamban (residues 1–20, domain 1) causes a decrease in the intrinsic tryptophan fluorescence of the CaATPase. The interaction of phospholamban residues 1–20 with the CaATPase also results in spectral changes for the extrinsic chromophore FITC covalently attached to the cytoplasmic region of the calcium pump. The fluorescence changes for both reporter groups correlate with a dissociation constant of ≈ 40µM for the complex between phospholamban residues 1–20 and the CaATPase. Complex formation is notably weaker when phospholamban 1–20 is titrated into the CaATPase in the presence of calcium, with altered conformational effects resulting from binding. The interaction of domain 1 of phospholamban with the CaATPase is also reduced upon phosphorylation of phospholamban 1–20 at Ser-16. This region of phospholamban 1–20 is shown by isotope-edited NMR study to be involved in interaction with the CaATPase. Binding of the phosphorylated peptide is not abolished, however, indicating that phospholamban 1–20 remains associated with the CaATPase even after phosphorylation. The data provide direct evidence for the interaction between the cytoplasmic regions of phospholamban and the pump, and are discussed in the context of the mechanism for inhibition of cardiac CaATPase activity by phospholamban.

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