Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum.

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca(2+)-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca(2+)-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.