Coupling of calcium transport with ATP hydrolysis in scallop sarcoplasmic reticulum.

Previously, we showed that incubation of the scallop sarcoplasmic reticulum (SR) with EGTA at above 37°C resulted in the uncoupling of ATP hydrolysis with Ca 2 + transport [Nagata et aL (1996) J. Biochem. 119, 1100-1105].We have extended this study by comparing the kinetic behavior of Ca 2 + release and binding to the uncoupled SR with that of intact scallop or rabbit SR. The change in the Ca 2 + concentration in the reaction medium, as determined as the absorption of APIII, was followed using a stopped flow system. Intact scallop SR was preincubated with Ca 2 + in the presence of a Ca 2 + ionophore, A23187, and then ATP was added to initiate the reaction. The Ca 2 + level in the medium increased to the maximum level in several seconds, and then slowly decreased to the initial low level. The rising and subsequent slow decay phases could be related to the dissociation and reassociation of Ca 2 + with the Ca-ATPase, respectively. When uncoupled scallop SR vesicles were preincubated with CaCl 2 in the absence of A23187 and then the reaction was initiated by the addition of ATP, a remarkable amount of Ca 2 + was released from the SR vesicles into the cytosolic solution, whereas, with intact scallop or rabbit SR, only a sharp decrease in the Ca 2 + level was observed. Based on these findings, we concluded that the heat treatment of scallop SR in EGTA may alter the conformation of the Ca-ATPase, thereby causing Ca 2 + to be released from the enzyme, during the catalytic cycle, at the cytoplasmic surface, but not at the lumenal surface of SR vesicles.