Conformational Change of Ca2+,Mg2+-Adenosine Triphosphatase of Sarcoplasmic Reticulum upon Binding of Ca2+ and Adenyl-5′-yl-Imidodiphosphate as Detected by Trypsin Sensitivity Analysis1

Abstract

Ca²⁺-Mg²⁺-ATPase of sarcoplasmic reticulum was subjected to tryptic digestion under various conditions and the cleavage patterns were compared. The first tryptic cleavage to yield the NH₂-terminal A-fragment (Mr≃55,000) and COOH-terminal B-fragment (Mr≃45,000) [Thorley-Lawson, D.A. & Green, N.M. (1977) Biochem. J. 167, 739–748] was little affected by adding ligands such as Ca²⁺ and AMP-P(NH)P. On the other hand, subsequent splitting of A-fragment into A₁ (Mr≃30,000) and A₂ (Mr≃20,000), and further cleavages giving rise to three smaller fragments of Mr≃27,000–28,000 (A_1a, A_1b, and C) [Saito, K., et al. (1984) J. Biochem. 95, 1297] were profoundly affected by these ligands. A difference in cleavage sites was noted depending on Ca²⁺ ion concentration; thus, A_1b and C were the major components remaining after digestion in the presence and absence of Ca²⁺, respectively. AMP-P(NH)P markedly stabilized both A¹ and A₂ fragments, but the effect was much more prominent when Ca²⁺ was simultaneously present on the transport site. These findings suggest that conformational changes of the ATPase molecule upon binding of Ca²⁺, AMP-P(NH)P, or both are accompanied by corresponding changes in the susceptibility to tryptic digestion. Fragments A₁ and A₂ were both quite stable and fragmentation did not proceed beyond A₁ when sarcoplasmic reticulum membranes were treated with trypsin at 0°C. Significant further fragmentation of A₁ was observed only above 20°C, suggesting a conformational transition of the ATPase protein around that temperature.