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The steady state kr, .:tics of the Ca2+-dependent adenosine triphosphatase reaction of 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 [+2PlATP to form an acid-stable phosphoenzyme.Evidence is presented that this phosphoenzyme represents a reaction intermediate of the ATP hydrolysis observed under these conditions.Because contaminating Na+ and K+ concentrations in the complete reaction medium could not account for the residual adenosine triphosphatase activity, the activity observed in the absence of added alkali metal salts is concluded to be alkali metal ion-independent.Ca*+ stimulated the rate of ATP hydrolysis in both 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 (-0.1 mu) was much higher than in KC1 (-10 FM).The rate of phosphoenzyme hydrolysis, estimated as the ratio (VIEP) between the rate of ATP hydrolysis (V) and the phosphoenzyme level (EP) at the steady state, also was maximal at 0.1 to 0.2 mu Ca2+ in the absence of alkali metal salts.Increasing MgCl, in the range between 0.1 mu and 10 mu stimulated V in the absence of added alkali metal salts.The optimal MgCl, concentration in the presence of KCI, however, was approximately 1 order of magnitude lower, and high MgClz concentrations (>2.0 mu) inhibited V. Qua!itatively similar differences in the effects of MgCI, were observed in the case of VlEP in the presence and absence of alkali metal salts.In the absence of added alkali metal salts, increasing ATP concentration above approximately 1 PM, which saturated the phosphorylation site, increased V without increasing EP.Thus, ATP appears to accelerate the rate constant of phosphoenzyme hydrolysis
Shigekawa et al. (Wed,) studied this question.