Inhibition of the calcium(2+) ion-dependent ATPase from sarcoplasmic reticulum by dicyclohexylcarbodiimide: evidence for location of the calcium(2+) ion binding site in a hydrophobic region

Abstract

Dicyclohexylcarbodiimide (DCCD) inhibits the (Ca2+)ATPase [equine muscle], Ca2+ uptake by sarcoplasmic reticulum vesicles and Ca2+ binding to the (Ca2+)ATPase from sarcoplasmic reticulum. Ca2+ (at .mu.M concentrations) specifically protects against DCCD inhibition. The inhibition can therefore be readily demonstrated only in the presence of Ca2+ chelating agents, e.g., EGTA [ethylene glycol tetraacetic acid]. In the presence of EGTA, the ionophore A-23187 increased the sensitivity to DCCD. The ionophore also increased the phosphorylation of the enzyme by inorganic phosphate in the presence of Mg2+. These results indicate that tightly bound Ca2+ is located in a hydrophobic region of the enzyme which is not accessible to EGTA. Complete inhibition of the (Ca2+)ATPase is accompanied by binding of 4-5 nmol of [14C]DCCD per mg of ATPase protein in the absence of Ca2+ compared with 2 nmol bound per mg in the presence of Ca2+ with no ATPase inhibition. Assuming a MW of 100,000 for the ATPase monomer, about 1 nmol of DCCD inhibits 4 nmol of ATPase. This suggests that the minimal functional unit of the enzyme is a tetramer. Following trypsin digestion of the [14C]DCCD-labeled ATPase, most of the radioactivity appears in the 20,000 dalton fragment. DCCD probably reacts with the Ca2+-binding site of the ATPase.