Abstract

Conformational states in sarcoplasmic reticulum Ca2+-ATPase have been examined by tryptic and chymotryptic cleavage. High affinity Ca2+ binding (E1 state) exposes a peptide bond in the A fragment of the polypeptide chain to trypsin. Absence of Ca2+ (E2 state) exposes bonds in the B fragment, which are protected by binding of Mg2+ or ATP. After phosphorylation from ATP the tryptic cleavage pattern depends on the predominant phosphoenzyme species present. ADP-sensitive E1P and ADP-insensitive E2P have cleavage patterns identical to those of unphosphorylated E1 and E2, respectively, indicating that two major conformational states are involved in Ca2+ translocation. The transition from E1P to E2P is inhibited by secondary tryptic splits in the A fragment, suggesting that parts of this fragment are of particular importance for the energy transduction process. The tryptic cleavage patterns of phosphorylated forms of detergent solubilized monomeric Ca2+-ATPase were similar to those of the membrane-bound enzyme, indicating that Ca2+ translocation depends mainly on structural changes within a single peptide chain. On the other hand, the protection of the second cleavage site as observed after vanadate binding to membranous Ca2+-ATPase could not be achieved in the soluble monomeric enzyme. Shielding of this peptide bond may therefore be due to protein-protein interactions in the semicrystalline state of the vanadate-bound Ca2+-ATPase in membranous form.