Matsuo Naoko, Nagata Yoshiaki, Nakamura Jun, Yamamoto Taibo
Department of Biology, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
J Biochem. 2002 Mar;131(3):375-81. doi: 10.1093/oxfordjournals.jbchem.a003112.
Previously, we showed that incubation of the scallop sarcoplasmic reticulum (SR) with EGTA at above 37 degrees C resulted in the uncoupling of ATP hydrolysis with Ca2+ transport [Nagata et al. (1996) J. Biochem. 119, 1100-1105]. We have extended this study by comparing the kinetic behavior of Ca2+ release and binding to the uncoupled SR with that of intact scallop or rabbit SR. The change in the Ca2+ 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 Ca2+ in the presence of a Ca2+ ionophore, A23187, and then ATP was added to initiate the reaction. The Ca2+ 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 Ca2+ with the Ca-ATPase, respectively. When uncoupled scallop SR vesicles were preincubated with CaCl2 in the absence of A23187 and then the reaction was initiated by the addition of ATP, a remarkable amount of Ca2+ was released from the SR vesicles into the cytosolic solution, whereas, with intact scallop or rabbit SR, only a sharp decrease in the Ca2+ 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 Ca2+ to be released from the enzyme, during the catalytic cycle, at the cytoplasmic surface, but not at the lumenal surface of SR vesicles.
此前，我们发现，在37摄氏度以上用乙二醇双四乙酸（EGTA）孵育扇贝肌浆网（SR）会导致ATP水解与Ca2+转运解偶联[Nagata等人，（1996年）《生物化学杂志》119卷，第1100 - 1105页]。我们通过比较解偶联的扇贝SR与完整的扇贝或兔SR中Ca2+释放和结合的动力学行为，扩展了这项研究。使用停流系统跟踪反应介质中Ca2+浓度的变化，该变化通过测定APIII的吸光度来确定。完整的扇贝SR在Ca2+离子载体A23187存在的情况下用Ca2+进行预孵育，然后加入ATP启动反应。介质中的Ca2+水平在几秒钟内升至最高水平，然后缓慢降至初始低水平。上升阶段和随后的缓慢衰减阶段可能分别与Ca2+与Ca - ATP酶的解离和重新结合有关。当在没有A23187的情况下用CaCl2对解偶联的扇贝SR囊泡进行预孵育，然后通过添加ATP启动反应时，大量的Ca2+从SR囊泡释放到胞质溶液中，而对于完整的扇贝或兔SR，仅观察到Ca2+水平急剧下降。基于这些发现，我们得出结论，在EGTA中对扇贝SR进行热处理可能会改变Ca - ATP酶的构象，从而导致在催化循环过程中，Ca2+在SR囊泡的细胞质表面而非腔表面从该酶上释放出来。
