Phosphorylation of solubilized sarcoplasmic reticulum by orthophosphate and its thermodynamic characteristics. The dominant role of entropy in the phosphorylation.
A large fraction of the Ca-2plus- and Mg-2plus-dependent ATPase (EC 3.6.1.3) in sarcoplasmic reticulum membranes solubilized with Triton X-100 was phosphorylated with Pi. The phosphorylation required Mg-2plus but was strongly inhibited by low concentrations of Ca-2plus. A Ca-2plus ion concentration of 30 muM caused half-maximum inhibition in the presence of 50 mM MgCl2. The phosphorylated enzyme showed a rapid turnover and was in dynamic equilibrium with Pi in the medium. At equilibrium the amount of the phosphorylated enzyme increased markedly with increased in the reaction temperature. The apparent standard free energy change, the apparent standard enthalpy change, and the apparent standard entropy change in the formation of the phosphorylated enzyme from the enzyme-phosphate complex in the presence of excess Mg-2plus at 37 degrees and pH 7.0 were, respectively, 0.35 Cal per mol, 15.9 Cal per mol, and 50.2 e.u. per mol. The susceptibility of the acid-denatured phosphorylated enzyme to hydroxylamine showed that the phosphorylated enzyme is of an acyl phosphate type. The present results are consistent with the probability that the phosphorylation results from reversal of late steps in the Ca-2plus transport process. The results clearly show that the phosphorylated enzyme is stabilized by a great increase in entropy upon its formation from the enzyme-phosphate complex.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002151:Calorimetry; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000187:drug effects / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D012995:Solubility; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D006898:Hydroxylamines; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D011092:Polyethylene Glycols; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium