Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP

Ca2+ transport and UTP hydrolysis catalyzed by sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle was studied. A passive Ca2+ load inside microsomal vesicles clearly decreased the net uptake rate and the final accumulation of Ca2+ but not the UTP hydrolysis rate, causing energy uncoupling. In the absence of passive leak, the Ca2+/P-i coupling ratio was 0.7-0.8. UTP hydrolysis did not maintain a rapid component of Ca2+ exchange between the cytoplasmic and lumenal compartments as occurs with ATP. The uncoupling process in the presence of UTP is associated with: (i) the absence of a steady state accumulation of ADP-insensitive phosphoenzyme; (ii) the cytoplasmic dissociation of Ca2+ bound to the ADP-sensitive phosphoenzyme; and (iii) the absence of enzyme inhibition by cyclopiazonic acid. All these characteristics confirm the lack of enzyme conformations with low Ca2+ affinity and point to the existence of an uncoupling mechanism mediated by a phosphorylated form of the enzyme. Suboptimal coupling values can be explained in molecular terms by the proposed functional model.