Desensitization to Mg²⁺ of the Phosphoenzyme in Sarcoplasmic Reticulum Ca²⁺-ATPase by the Addition of a Nonionic Detergent and the Restoration of Sensitivity after Its Removal

Abstract

Sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle were solubilized with a high concentration of dodecyl octaethyleneglycol monoether (C₁₂E₈) and the kinetic properties of the Ca²⁺, Mg²⁺-dependent ATPase [EC 3. 6. 1. 3] were studied. The following results were obtained:
1. SR ATPase solubilized in C₁₂E₈ retains high ability to form phosphoenzyme ([EP]=4-5 mol/10⁶g protein) for at least two days in the presence of 5mM Ca²⁺, 0.5M KCl, and 20% glycerol at pH 7.55.
2. The ATPase activity was dependent on both Mg²⁺ and Ca²⁺. However, the rate of E³²P decay after the addition of unlabeled ATP was independent of Mg²⁺.
3. Most of the EP formed in the absence of Mg²⁺ was capable of reacting with ADP to form ATP in the backward reaction. However, in the presence of 5mM Mg²⁺, the amount of ATP formed was markedly reduced without loss of the reactivity of the EP with ADP.
4. The removal of C₁₂E₈ from the ATPase by the use of Bio-Beads resulted in the full restoration of the Mg²⁺ dependency of the EP decomposition.
5. These results strongly suggest that in the case of SR solubilized with a high concentration of C₁₂E₈ the decomposition of phosphoenzyme is Mg²⁺ independent and ATP is mainly hydrolyzed through Mg²⁺-dependent decomposition of an enzyme-ATP complex, which is in equilibrium with phosphoenzyme and ADP.