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Reaction mechanism of Ca2+-dependent ATP hydrolysis by skeletal muscle sarcoplasmic reticulum in the absence of added alkali metal salts. I. Characterization of steady state ATP hydrolysis and comparison with that in the presence of KCl



Reaction mechanism of Ca2+-dependent ATP hydrolysis by skeletal muscle sarcoplasmic reticulum in the absence of added alkali metal salts. I. Characterization of steady state ATP hydrolysis and comparison with that in the presence of KCl



Journal of Biological Chemistry 253(5): 1442-1450



The steady state kinetics of the Ca2+-dependent ATPase reaction of rabbit skeletal muscle sarcoplasmic reticulum was compared in the presence and absence of added alkali metal salts. During steady state ATP hydrolysis in the absence of added alkali metal salts, the ATPase protein was phosphorylated by [.gamma.-32P]ATP to form an acid-stable phosphoenzyme. This phosphoenzyme apparently represents a reaction intermediate of the ATP hydrolysis observed under these conditions. Because contaminating Na+ and K+ concentrations in the complete reaction medium did not account for residual ATPase activity, the activity observed in the absence of added alkali metal salts probably was alkali metal ion-independent. Ca2+ stimulated rate of ATP hydrolysis in the presence and absence of added alkali metal salts, whereas excess Ca2+ inhibited ATP hydrolysis. The optimum free Ca2+ concentration in the absence of added alkali metal salts (.apprx. 0.1 mM) was much higher than in KCl (.apprx. 10 .mu.M). Rate of phosphoenzyme hydrolysis, estimated as the ratio (V/EP) between rate of ATP hydrolysis (V) and phosphoenzyme level (EP) at the steady state, also was maximal at 0.1-0.2 mM Ca2+ in the absence of alkali metal salts. Increasing MgCl2 in the range between 0.1 mM and 10 mM stimulated V in the absence of added alkali metal salts. Optimal MgCl2 concentration in the presence of KCl, however, was approximately 1 order of magnitude lower, and high MgCl2 concentrations (> 2.0 mM) inhibited V. Qualitatively similar differences in effects of MgCl2 were observed in the case of V/EP in the presence and absence of alkali metal salts. In the absence of added alkali metal salts, increasing ATP concentration above approximately 1 .mu.M, which saturated the phosphorylation site, increased V without increasing EP. ATP apparently accelerated the rate constant of phosphoenzyme hydrolysis under these conditions. This finding is in contrast to the previous observation that stimulation of V by high ATP concentrations in the presence of KCl was not associated with an increased rate constant for phosphoenzyme hydrolysis. ADP inhibited V and EP in the presence and absence of added alkali metal salts, but ADP decreased V/EP to a greater extent when alkali metal salts were not added. Kinetic properties of the phosphorylated intermediate formed at the steady state in the absence of added alkali metal salts probably differed significantly from those of the corresponding phosphoenzyme formed in the presence of KCl.

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Accession: 068532278

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PMID: 627548


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