Inhibition of the (Ca2+)ATPase from sarcoplasmic reticulum by dicyclohexylcarbodiimide: evidence for location of the Ca2+ binding site in a hydrophobic region.
Dicyclohexylcarbodiimide (DCCD) inhibits the (Ca2+)ATPase, Ca2+ uptake by sarcoplasmic reticulum vesicles and Ca2+ binding to the (Ca2+)ATPase from sarcoplasmic reticulum. Ca2+ (at micron concentrations) specifically protects against DCCD inhibition. The inhibition can, therefore, be readily demonstrated only in the presence of Ca2+ chelating agents such as EGTA. 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 molecular weight of 100 000 for the ATPase monomer, about 1 nmol of DCCD inhibits 4 nmol of ATPase. This result 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. We propose that DCCD reacts with the Ca2+-binding site of the ATPase.
D000818:Animals; D001665:Binding Sites; D002118:Calcium; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002234:Carbodiimides / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D010446:Peptide Fragments; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin
D002234:Carbodiimides; D010446:Peptide Fragments; D004533:Egtazic Acid; D004024:Dicyclohexylcarbodiimide; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium