Champeil P, le Maire M, Andersen J P, Guillain F, Gingold M, Lund S, Møller J V
J Biol Chem. 1986 Dec 15;261(35):16372-84.
We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and Møller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.
我们之前已对肌浆网膜与非增溶浓度的C12E8（非离子洗涤剂八甘醇单十二烷基醚）之间相互作用的结构特征进行了表征（安德森，J.P.，勒迈尔，M.，克拉格 - 汉森，V.，尚佩尔，P.，以及莫勒，J.V.（1983年）《欧洲生物化学杂志》134卷，205 - 214页）。本研究特别对洗涤剂诱导的扰动的功能方面及其对理解ATP酶功能的影响进行了表征。扰动性洗涤剂降低了Vmax，但使Ca2 +转运保持完整。从多混合器猝灭实验判断，洗涤剂的掺入既不影响由ATP进行的钙依赖性磷酸化，也不影响从动力学分辨的双波长测量结果判断的两种磷酸化酶形式（Ca2E1P到E2P）之间的钙释放转变，该测量使用了对钙敏感的染料安替比拉佐III。然而，Vmax的降低是由于酶去磷酸化速率降低了3 - 4倍，而非磷酸化酶形式（E2到Ca2E1）之间的Ca2 +依赖性转变增强了近10倍。从钒酸盐反应性和胰蛋白酶降解模式实验中获得了C12E8使E2构象向E1状态转变以解释受扰动反应的证据。对于ATP加速Ca2E1P到E2P转变，获得了直接证据和稳态证据，这可能解释了核苷酸对酶周转的低亲和力调节作用。动力学数据表明，C12E8使ATP水解减少与E2P去磷酸化成为限速步骤的条件（高ATP浓度、低pH、无钾）相吻合。否则，Ca2E1P到E2P转变被推断为ATP酶循环的限速步骤，而通过调节E2到Ca2E1转变来控制循环速率的可能性非常小。只有在特殊情况下（无钾、高温以及使用ITP作为底物），这种转变才会成为限速步骤，并受到洗涤剂扰动使整个循环速率增强的影响。
