Department of Biophysics, Faculty of Medicine, University of Pécs, Hungary.
Eur Biophys J. 2011 May;40(5):619-26. doi: 10.1007/s00249-010-0659-y. Epub 2011 Jan 4.
During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P(i) release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P(i) from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P(i). The effect of both toxins can be sufficiently explained by local and mechanical blockade of P(i) dissociation.
在肌动蛋白聚合过程中，结合的 ATP 发生两步连续水解：高能核苷酸的化学断裂和 γ-磷酸盐的缓慢释放。在这项研究中，在肌动蛋白丝形成过程中监测了鬼笔环肽和 Jasplakinolide 对 P(i)释放动力学的影响。基于酶联测定的分光光度技术用于跟踪无机磷酸盐的释放。已经验证 Jasplakinolide 以与鬼笔环肽相同的方式减少 P(i)释放。通过从 F-肌动蛋白释放 P(i)，不可能证明毒素的长程变构效应。ATP 水解产物通过肌动蛋白丝的变性释放。对样品的 HPLC 分析表明，毒素结合区域中的 ATP 完全水解成 ADP 和 P(i)。两种毒素的作用都可以通过 P(i)解离的局部和机械阻断得到充分解释。
