Kinetics of the cooperativity of the Ca2+-transporting adenosine triphosphatase of sarcoplasmic reticulum and the mechanism of the ATP interaction.

The extent of the negative cooperativity with MgATP of the Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum has been studied with various membrane preparations and under various conditions. Preparations studied were fragmented sarcoplasmic reticulum vesicles, deoxycholate-solubilized and fractionated ATPase, triton extracted reticulum, vesicles reconstituted from either detergent, and limited trypsin digests of the reticulum. Conditions studied were suboptimal, optimal, and inhibitory Ca²⁺ concentrations; temperatures from 13 to 46 °C; 1 or 5 mm MgCl₂; 0.1 m KCl, 0.1 m NaCl, or no added salt; and Triton or deoxycholate present in the assay. With preparations in which vesicles could accumulate Ca²⁺ ion, the ionophore A23187 was added to prevent inhibition by internal Ca²⁺ ions. Under all circumstances, the negative cooperativity of MgATP was present (Hill coefficient of 0.2 to 0.8), indicating the persistence of the properties of the enzyme molecule and its lipid environment giving rise to kinetic negative cooperativity. Attempts to measure the number of ATP sites by protection against N-ethylmaleimide inactivation and by binding of an analog suggested, but did not prove, that there was only one specific, active ATP binding site below 0.5 mm. These results are interpreted to be consistent with either of two mechanisms for ATP cooperativity of the Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum: (a) a single, high affinity ATP active site and a second, lower affinity “allosteric” activator site; or (b) a single ATP site which demonstrates two affinities through some kinetic mechanism such as a substrate-induced, slow transition.