Effect of temperature and added ligands on the susceptibility of Ca2+,Mg2+-adenosine triphosphatase of the sarcoplasmic reticulum to trypsin.
Sarcoplasmic reticulum membranes were subjected to limited proteolysis by trypsin under various conditions and the products were analyzed with SDS-polyacrylamide gel electrophoresis. Besides the fragments well characterized in the literature, i.e. A, B, A1, and A2 [Thorley-Lawson, D.A. & Green, N.M. (1977) Biochem. J., 167, 739] of Mr 55,000, 45,000, 30,000, and 20,000, respectively, two closely related fragments derived from fragment A1, and another migrating a little faster than these (Mr 27,000-28,000), were produced at 35 degrees C. Appearance of these smaller fragments was accompanied by the disappearance not only of A1 but also of A2. This subfragmentation was prevented at 0 degrees C, so that a large amount of A1 fragment was accumulated. Ca2+ and AMP-P(NH)P, alone or in combination, also affected the susceptibility of the ATPase protein to tryptic digestion, resulting in a marked stabilization of A1 and A2 fragments. These findings afforded us a basis of devising a procedure for efficient production of these fragments, which would serve as useful starting materials for protein chemical analyses of this enzyme. The observed changes in susceptibility to proteolytic digestion are consistent with our current knowledge on the conformational changes of this ATPase.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000032:analysis; D013696:Temperature; D014357:Trypsin / Q000378:metabolism*
D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D014357:Trypsin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D005033:Ethylmaleimide