MacLean-Fletcher S D, Pollard T D
J Cell Biol. 1980 May;85(2):414-28. doi: 10.1083/jcb.85.2.414.
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.
我们使用低剪切落球试验研究了棘阿米巴冷提取物升温时发生的凝胶化过程的动力学。我们发现该反应至少有两个步骤，需要0.5 mM ATP和1.5 mM MgCl₂，并且受到微摩尔浓度Ca²⁺的抑制。最适pH为7.0，温度为25℃ - 30℃。用MgCl₂和ATP进行冷预孵育可提高反应速率。ATP的非水解类似物在这种“增强反应”或凝胶化过程中均不能替代ATP。两种纯化的棘阿米巴蛋白中的任何一种或四种部分纯化的蛋白中的任何一种都能使纯化的肌动蛋白交联形成凝胶，但没有一种能解释粗提取物中反应对Mg-ATP的依赖性或其受Ca²⁺的调节。这表明提取物中除了肌动蛋白交联蛋白外，还含有依赖Mg-ATP和Ca²⁺的因子，这些因子调节凝胶化过程。
