Protection of Scallop Sarcoplasmic Reticulum ATPase from Thermal Inactivation by Removal of Calcium from High-Affinity Binding Sites on the Enzyme

: Sarcoplasmic reticulum (SR) vesicles were isolated from scallop muscle by the method of Abe et al. (J. Biochem. 112, 822-827, 1992) and their thermolability was examined in the presence and absence of Ca2+. When SR was preincubated at 38 degrees C in the presence of 0.1 mM Ca2+, Ca2+ transport activity decreased as a function of time with a half-inhibition time of about 5 min. Activities of the Ca(2+)-dependent ATPase, phosphoenzyme (EP) formation and E2 to E1 transition were decreased by the heat treatment in parallel with the Ca2+ transport activity. In contrast, when SR was preincubated at 38 degrees C in the presence of 2-5 mM EGTA, all of these activities, except for the Ca2+ transport, were markedly protected from the heat inactivation. The uncoupling between Ca2+ transport and the ATPase reaction did not lead to a rise in the Ca2+ permeability of SR membrane. Plots of the ATPase activity or steady-state level of EP against pCa in the thermal incubation medium revealed a typical sigmoidal curve with a half-inhibition concentration and Hill number of about 0.5 microM and 1.80, respectively. These results suggest that 2 mol of Ca2+ must be removed from the high-affinity Ca2+ binding sites on the ATPase to stabilize the Ca(2+)-ATPase against heat inactivation. The protection from heat inactivation disappeared if SR was preincubated at 38 degrees C after having been solubilized with a nonionic detergent, but returned when the detergent was removed to reconstitute the SR membrane. These results suggest that the protection of ATPase from thermal inactivation in EGTA may require a membrane structure in which the ATPase molecules exist in an appropriate arrangement.