Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca²⁺-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca²⁺

Abstract

Isolated sarcoplasmic reticulum vesicles in the presence of Mg²⁺ and absence of Ca²⁺ retain significant ATP hydrolytic activity that can be attributed to the Ca²⁺-ATPase protein. At neutral pH and the presence of 5 mM Mg²⁺, the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg²⁺ or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca²⁺-free medium is increased by Mg²⁺ but decreased by vanadate when Mg²⁺ is present. It is suggested that MgATP hydrolysis in the absence of Ca²⁺ requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca²⁺-independent activity is operative at basal levels of cytoplasmic Ca²⁺ or when the Ca²⁺ binding transition is impeded.