The Nucleotide-Binding Site of the Sarcoplasmic Reticulum Ca−ATPase Is Conformationally Altered in Aged Skeletal Muscle†

Biochemistry ◽  
1999 ◽  
Vol 38 (45) ◽  
pp. 14887-14896 ◽  
Author(s):  
Baowei Chen ◽  
Terry E. Jones ◽  
Diana J. Bigelow
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site

scholarly journals Stimulation of the Ca2+-ATPase of sarcoplasmic reticulum by disulfiram

1996 ◽  
Vol 320 (1) ◽  
pp. 101-105 ◽  
Author(s):  
Anthony P STARLING ◽  
J. Malcolm EAST ◽  
Anthony G LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Binding Site ◽  
Equilibrium Constant ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Single Site ◽  
Stimulation Of

Disulfiram [bis(diethylthiocarbamoyl)disulphide] has been found to stimulate reversibly the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. At pH 7.2, 2.1 mM ATP and 25 °C, ATPase activity was found to double on addition of 120 µM disulfiram. Stimulation fitted to binding of disulfiram at a single site with a Kd of 61 µM. Disulfiram had no effect on the Ca2+ affinity of the ATPase or on the rate of phosphorylation of the ATPase by ATP, but increased the rate of dissociation of Ca2+ from the phosphorylated ATPase (the transport step) and increased the rate of dephosphorylation of the phosphorylated ATPase. It also decreased the level of phosphorylation of the ATPase by Pi, consistent with a 7.5-fold decrease in the equilibrium constant of the phosphorylated to non-phosphorylated forms (E2PMg/E2PiMg) at 80 µM disulfiram. Disulfiram had no significant effect on the concentration of ATP resulting in stimulation of ATPase activity, suggesting that it does not bind to the empty nucleotide-binding site on the phosphorylated ATPase. Studies of the effects of mixtures of disulfiram and jasmone (another molecule that stimulates the ATPase) suggest that they bind to separate sites on the ATPase.

scholarly journals Effects of Mg2+, anions and cations on the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 723-730 ◽  
Author(s):  
H I Stefanova ◽  
R M Napier ◽  
J M East ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Site ◽  
Conformational Transition ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Phosphorylated Form ◽  
Low Concentrations ◽  
Anions And Cations

In a previous paper [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227] we presented a kinetic model for the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum. Here we extend the model to account for the effects on ATPase activity of Mg2+, cations and anions. We find that Mg2+ concentrations in the millimolar range inhibit ATPase activity, which we attribute to competition between Mg2+ and MgATP for binding to the nucleotide-binding site on the E1 and E2 conformations of the ATPase and on the phosphorylated forms of the ATPase. Competition is also suggested between Mg2+ and MgADP for binding to the phosphorylated form of the ATPase. ATPase activity is increased by low concentrations of K+, Na+ and NH4+, but inhibited by higher concentrations. It is proposed that these effects follow from an increase in the rate of dephosphorylation but a decrease in the rate of the conformational transition E1′PCa2-E2′PCa2 with increasing cation concentration. Li+ and choline+ decrease ATPase activity. Anions also decrease ATPase activity, the effects of I- and SCN- being more marked than that of Cl-. These effects are attributed to binding at the nucleotide-binding site, with a decrease in binding affinity and an increase in ‘off’ rate constant for the nucleotide.

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