Purification of limited tryptic fragments of Ca2+,Mg2+-adenosine triphosphatase of the sarcoplasmic reticulum and identification of conformation-sensitive cleavage sites.
Sarcoplasmic reticulum membranes were treated with trypsin, and samples enriched with A1a, A1b, and C fragments (Saito, K. et al. (1984) J. Biochem. 95, 1297-1304), respectively, were prepared. A1b and C fragments were purified to apparent homogeneity, and an approximately equimolar mixture of A1(Met1-Arg198), A1a, and A1b 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 peptides were carried out in order to identify the tryptic cleavage sites responsible for the formation of these fragments. Both A1a and A1b fragments had the same C-terminal sequence as A1 fragment. Single cleavage of A1 at T3a (Lys218-Ala219) yielded A1a, while a cleavage between either Lys234-Ile235 or Arg236-Asp237 (collectively designated as T3b) resulted in A1b fragment. Thus, A1a and A1b 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 T3a and T3b 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.
Imamura
Y
Y
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo.
Kawakita
M
M
D000595:Amino Acid Sequence; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000302:isolation & purification*; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003840:Deoxycholic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin
D010455:Peptides; D003840:Deoxycholic Acid; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases