The binding of nucleotides and bivalent cations to the calcium-and-magnesium ion-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum

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Abstract
The binding of MgATP to purified Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all 3 processes demonstrated negative co-operativity, with half-saturation of all 3 parameters at a MgATP concentration of 40-50 .mu.M, and a Hill coefficient (h) of 0.8. The variation of binding constant with pH was measured and showed tighter binding of MgATP with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3 .mu.M. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.