Purification of Limited Tryptic Fragments of Ca₂₊, Mg²⁺-Adenosine Triphosphatase of the Sarcoplasmic Reticulum and Identification of Conformation-Sensitive Cleavage Sites

抄録

Sarcoplasmic reticulum membranes were treated with trypsin, and samples enriched with A_1a, A_1b, and C fragments (Saito, K. et al. (1984) J. Biochem. 95, 1297-1304), respectively, were prepared. A_1b, and C fragments were purified to apparent homogeneity, and an approximately equimolar mixture of A₁(Met₁-Arg₁₉₈), A_1a, and A_1b fragments free from other contaminants was also obtained through gel permeation and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate. N- and C-terminal amino acid sequence analyses of these peptid es were carried out in order to identify the tryptic cleavage sites responsible for the formation of these fragments. Both A_1a and A_1b fragments had the same C-terminal sequence as A₁ fragment. Single cleavage of A₁ at T3a (Lys₂₁₈-A1a₂₁₉) yielded A_1a, while a cleavage between either Lys₂₃₄-11e₂₃₅ or Arg₂₃₆-Asp₂₃₇ (collectively designated as T_3b) resulted in Alb fragment. Thus, A_1a and A_1b fragments differed from A₁ fragment only by their loss of short stretches corresponding to the N-terminal region of the latter. On the other hand, C fragment represented the C-terminal half of B fragment (Ala₅₀₆- Gly₉₉₄). It had the same C-terminal sequence as B fragment and was produced by cleavage at T₄ (Lys₇₂₈-Thr₇₂₉). Cleavages at T_3a and T_3b profoundly affected the catalytic properties of SR-ATPase (Imamura, Y. and Kawakita, M. (1986) J. Biochem. 100, 133-141), and it was suggested that the segment of the ATPase molecule including the region between Ala₁₉₉ and Arg₂₃₆ is important in mediating the coupling between ATP splitting and Ca²⁺-transport.