REVERSIBLE INACTIVATION OF THE SARCOPLASMIC-RETICULUM CA2-ATPASE COUPLED TO REARRANGEMENT OF CYTOPLASMIC PROTEIN DOMAINS AS REVEALED BY CHANGES IN TRYPSINIZATION PATTERN()

Repetitive homogenization of skeletal muscle sarcoplasmic reticulum (S R) membranes in the presence of chelating agents at low ionic strength leads to the loss of the Ca-ATPase activity. This inactive state of t he enzyme is coupled to an extensive rearrangement of the cytosolic do mains as visualized by a completely different trypsinization pattern o f the enzyme. In addition to the primary cleavage site (Arg 505), a no vel trypsinization site (Arg 334), just N-terminal of the phosphorylat ion domain and localized on the primary tryptic fragment A, becomes ex posed. Cleavage at the latter site yields a soluble fragment of M(r) 2 0 117 and the membrane-bound N-terminal one-third of the ATPase of M(r ) 35 279. Two additional trypsinization sites C-terminal of the nucleo tide binding domain become exposed in the inactive Ca2+-ATPase conform ation. Rapid cleavage at these sites yields two soluble fragments of a bout 15 and 10 kDa. All together, the three soluble fragments comprise most of the large cytosolic loop of the Ca2+-ATPase. The inactivation and the change in trypsinization pattern can be reversed by rehomogen ization of the extracted membranes in the presence of divalent cations . The results suggest the presence of an occluded site for divalent ca tions which can be depleted or refilled during application of sheer fo rces. Occupation of this site is essential to confer to the enzyme an active conformation.