caa a22 4500 39755625X CHVBK 20180308164803.0 cr unu---uuuuu 161202e199606 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021354 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021354 Protection of Scallop Sarcoplasmic Reticulum ATPase from Thermal Inactivation by Removal of Calcium from High-Affinity Binding Sites on the Enzyme [Elektronische Daten] [Yoshiaki Nagata, Jun Nakamura, Taibo Yamamoto] 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 Ca+. When SR was preincubated at 38 •C in the presence of 0.1 mM Ca+, Ca2+ transport activity decreased as a function of time with a half-inhibition time of about 5 min. Activities of the Ca+-dependent ATPase, phosphoenzyme (EP) formation and E2 to El transition were decreased by the heat treatment in parallel with the Ca+ transport activity. In contrast, when SR was preincubated at 38•C in the presence of 2-5 mM EGTA, all of these activities, except for the Ca+ transport, were markedly protected from the heat inactivation. The uncoupling between Ca+ transport and the ATPase reaction did not lead to a rise in the Ca+ 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 μM and 1.80, respectively. These results suggest that 2 mol of Ca+ must be removed from the high-affinity Ca2+ binding sites on the ATPase to stabilize the Ca+-ATPase against heat inactivation. The protection from heat inactivation disappeared if SR was preincubated at 3°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. © by the Journal of Biochemistry Regular Papers nationallicence Ca+-ATPase nationallicence chemiosmotic uncoupling nationallicence heat inactivation nationallicence high-affinity Ca+ binding nationallicence scallop muscle sarcoplasmic reticulum nationallicence Nagata Yoshiaki Faculty of Science Osaka University Toyonaka, Osaka 560 aut Nakamura Jun Department of Biological Science, College of General Education, Tohoku University Kawauchi, Sendai, Miyagi 980 aut Yamamoto Taibo Faculty of Science Osaka University Toyonaka, Osaka 560 aut The Journal of Biochemistry Oxford University Press 119/6(1996-06), 1100-1105 0021-924X 119:6<1100 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021354 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021354 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Nagata Yoshiaki Faculty of Science Osaka University Toyonaka, Osaka 560 aut NATIONALLICENCE 700 1- Nakamura Jun Department of Biological Science, College of General Education, Tohoku University Kawauchi, Sendai, Miyagi 980 aut NATIONALLICENCE 700 1- Yamamoto Taibo Faculty of Science Osaka University Toyonaka, Osaka 560 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/6(1996-06), 1100-1105 0021-924X 119:6<1100 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford