肌浆网可溶Ca2+-ATP酶正向和逆向反应中形成的磷酸化中间体动力学性质的差异。

Difference in kinetic properties of phosphorylated intermediates formed in the forward and backward reactions of solubilized Ca2+-ATPase of sarcoplasmic reticulum.

作者信息

Nakamura Y

出版信息

J Biochem. 1980 Jul;88(1):177-81.

Abstract

Solubilized Ca2+-ATPase (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The kinetic properties of the phosphorylated intermediate (EP) formed by the reaction of SSR with ATP were compared with those of EP formed by the reaction with Pi. The time course of decay of E32P formed with 4 microM AT32P in the presence of 19 mM CaCl2 and 10 mM MgCl2 (forward reaction) was measured by adding 0.4 mM unlabeled ATP and 10 mM Pi at pH 6.0 and 30 degrees C. The rate of E32P decay was accelerated by 0.4 mM ADP. On the other hand, when the time course of decay of E32P formed with 10 mM 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2 (backward reaction) was measured by adding 0.4 mM unlabeled ATP and 15 mM CaCl2, the rate of E32P decay was unaffected by 0.4 mM ADP. AT32P was produced on adding ADP to E32P formed with AT32P in the presence of 10 mM CaCl2 and 10 mM MgCl2, while no AT32P was produced on adding ADP to E32P formed with 32Pi in the presence of 5 mM EGTA and 10 mM MgCl2, even when 15 mM CaCl2 was added simultaneously with ADP.

摘要

通过用非离子去污剂（C12E8）溶解破碎的肌浆网（FSR），然后用吐温80置换去污剂，使用DEAE - 纤维素柱制备了可溶性Ca2 + - ATP酶（SSR）。将SSR与ATP反应形成的磷酸化中间体（EP）的动力学性质与与Pi反应形成的EP的动力学性质进行了比较。在19 mM CaCl2和10 mM MgCl2存在下，用4 microM AT32P形成的E32P的衰变时间进程（正向反应）通过在pH 6.0和30℃下加入0.4 mM未标记的ATP和10 mM Pi来测量。0.4 mM ADP加速了E32P的衰变速率。另一方面，当在5 mM EGTA和10 mM MgCl2存在下用10 mM 32Pi形成的E32P的衰变时间进程（反向反应）通过加入0.4 mM未标记的ATP和15 mM CaCl2来测量时，E32P的衰变速率不受0.4 mM ADP的影响。在10 mM CaCl2和10 mM MgCl2存在下，向由AT32P形成的E32P中加入ADP时产生AT32P，而在5 mM EGTA和10 mM MgCl2存在下，向由32Pi形成的E32P中加入ADP时，即使与ADP同时加入15 mM CaCl2，也不产生AT32P。