Cooperative calcium binding and calmodulin regulation in the calcium-dependent adenosine triphosphatase purified from the erythrocyte membrane.

Calcium binding to calcium-dependent ATPase purified from erythrocyte membrane was assessed by measurements of the ATPase intrinsic fluorescence. Calcium-binding isotherms obtained by fluorescence titration are identical to curves representing the Ca2+-concentration dependence of ATPase activity, and demonstrate that cooperativity is in fact a feature of the binding mechanism rather than an apparent effect of enzyme kinetics. Loss of cooperativity and a reduction of the ATPase affinity for calcium is observed at very low enzyme concentrations. This effect of enzyme dilution is prevented by calmodulin at 37 degrees C but not at 25 degrees C. It is suggested that calcium binding by erythrocyte-membrane ATPase is influenced by hydrophobic interactions of binding domains, exhibiting a dissociation constant between 10(-7) and 10(-8) M in the absence of calmodulin, at 37 degrees C and in a specific set of experimental conditions. The dissociation constant is decreased by calmodulin.