Dissecting the Hydrolytic Activities of Sarcoplasmic Reticulum ATPase in the Presence of Acetyl Phosphate

Abstract

Sarcoplasmic reticulum vesicles and purified Ca$^{2+}$-ATPase hydrolyze acetyl phosphate both in the presence and absence of Ca$^{2+}$. The Ca$^{2+}$-independent activity was fully sensitive to vanadate, insensitive to thapsigargin, and proceeded without accumulation of phosphorylated enzyme. Acetyl phosphate hydrolysis in the absence of Ca$^{2+}$ was activated by dimethyl sulfoxide. The Ca$^{2+}$-dependent activity was partially sensitive to vanadate, fully sensitive to thapsigargin, and associated with steady phosphoenzyme accumulation. The Ca$^{2+}$/P(i) coupling ratio at neutral pH sustained by 10 mm acetyl phosphate was 0.57. Addition of 30% dimethyl sulfoxide completely blocked Ca$^{2+}$ transport and partially inhibited the hydrolysis rate. Uncoupling induced by dimethyl sulfoxide included the accumulation of vanadate-insensitive phosphorylated enzyme. When acetyl phosphate was the substrate, the hydrolytic pathway was dependent on experimental conditions that might or might not allow net Ca$^{2+}$ transport. The interdependence of both Ca$^{2+}$-dependent and Ca$^{2+}$-independent hydrolytic activities was demonstrated.