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.
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/10(6) g protein) for at least two days in the presence of 5 mM Ca2+, 0.5 M 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 5 mM 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.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D010758:Phosphorus / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D012995:Solubility; D013501:Surface-Active Agents / Q000494:pharmacology*
D003902:Detergents; D013501:Surface-Active Agents; D010758:Phosphorus; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000244:Adenosine Diphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium