Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors
Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors
复制标题
棘阿米巴提取物凝胶化的粘度分析及两种凝胶化因子的纯化
作者：
S. MacLean;T. Pollard
我们已经研究了凝胶化过程的动力学，发生在温暖的冷提取物的阿米巴使用低剪切落球测定。我们发现，该反应至少有两个步骤，需要0.5 mM ATP和1.5 mM MgCl 2，并被微摩尔Ca++抑制。最佳pH值为7.0，温度为25 ° C-30 ° C。通过用MgCl 2和ATP冷预孵育来增加反应速率。ATP的不可水解的类似物在这种“增强反应”或凝胶化过程中不会取代ATP。两种纯化的或四种部分纯化的阿米巴蛋白质中的任何一种都会交联纯化的肌动蛋白形成凝胶，但没有一种可以解释粗提物中的反应对Mg-ATP的依赖性或Ca++对其的调节。这表明，提取物中含有，除了肌动蛋白交联蛋白，因子依赖于Mg-ATP和Ca++，调节凝胶化过程。
We have studied the kinetics of the gelation process that occurs upon warming cold extracts of Acanthamoeba using a low-shear falling ball assay. We find that the reaction has at least two steps, requires 0.5 mM ATP and 1.5 mM MgCl2, and is inhibited by micromolar Ca++. The optimum pH is 7.0 and temperature, 25 degrees-30 degrees C. The rate of the reaction is increased by cold preincubation with both MgCl2 and ATP. Nonhydrolyzable analogues of ATP will not substitute for ATP either in this "potentiation reaction" or in the gelation process. Either of two purified or any one of four partially purified Acanthamoeba proteins will cross-link purified actin to form a gel, but none can account for the dependence of the reaction in the crude extract on Mg-ATP or its regulation by Ca++. This suggests that the extract contains, in addition to actin-cross-linking proteins, factors dependent on Mg-ATP and Ca++ that regulate the gelation process.