Functional properties of sarcoplasmic reticulum Ca(2+)-ATPase after proteolytic cleavage at Leu119-Lys120, close to the A-domain.

By measuring the phosphorylation levels of individual proteolytic fragments of SERCA1a separated by electrophoresis after their phosphorylation, we were able to study the catalytic properties of a p95C-p14N complex arising from SERCA1a cleavage by proteinase K between Leu(119) and Lys(120), in the loop linking the A-domain with the second transmembrane segment. ATP hydrolysis by the complex was very strongly inhibited, although ATP-dependent phosphorylation and the conversion of the ADP-sensitive E1P form to E2P still occurred at appreciable rates. However, the rate of subsequent dephosphorylation of E2P was inhibited to a dramatic extent, and this was also the case for the rate of "backdoor" formation of E2P from E2 and P(i). E2P formation from E2 at equilibrium nevertheless indicated little change in the apparent affinity for P(i) or Mg(2+), while binding of orthovanadate was weaker. The p95C-p14N complex also had a slightly reduced affinity for Ca(2+) and exhibited a reduced rate for its Ca(2+)-dependent transition from E2 to Ca(2)E1. Thus, disruption of the N-terminal link of the A-domain with the transmembrane region seems to shift the conformational equilibria of Ca(2+)-ATPase from the E1/E1P toward the E2/E2P states and to increase the activation energy for dephosphorylation of Ca(2+)-ATPase, reviving the old idea of the A-domain being a phosphatase domain as part of the transduction machinery.