Pardee J D, Spudich J A
J Cell Biol. 1982 Jun;93(3):648-54. doi: 10.1083/jcb.93.3.648.
The assembly of highly purified actin from Dictyostelium discoideum amoebae and rabbit skeletal muscle by physiological concentrations of KCI proceeds through successive stages of (a) rapid formation of a distinct monomeric species referred to as KCI-monomer, (b) incorporation of KCI-monomers into an ATP-containing filament, and (c) ATP hydrolysis that occurs significantly after the incorporation event. KCI-monomer has a conformation which is distinct from that of either conventional G- or F-actin, as judged by UV spectroscopy at 210-220 nm and by changes in ATP affinity. ATP is not hydrolyzed during conversion of G-actin to KCI-monomer. KCI-monomer formation precedes filament formation and may be necessary for the assembly event. Although incorporation of KCI-monomers into filaments demonstrates lagphase kinetics by viscometry, both continuous absorbance monitoring at 232 nm and rapid sedimentation of filaments demonstrate hyperbolic assembly curves. ATP hydrolysis significantly lags the formation of actin filaments. When half of the actin has assembled, only 0.1 to 0.2 mole of ATP are hydrolyzed per mole of actin present as filaments.
通过生理浓度的氯化钾从盘基网柄菌变形虫和兔骨骼肌中组装高度纯化的肌动蛋白，经历以下连续阶段：(a) 快速形成一种独特的单体物种，称为氯化钾单体；(b) 氯化钾单体掺入含ATP的细丝中；(c) 在掺入事件后显著发生ATP水解。通过210 - 220 nm的紫外光谱和ATP亲和力的变化判断，氯化钾单体的构象与传统的G - 肌动蛋白或F - 肌动蛋白的构象不同。在G - 肌动蛋白转化为氯化钾单体的过程中，ATP不被水解。氯化钾单体的形成先于细丝的形成，可能是组装事件所必需的。尽管通过粘度测定法将氯化钾单体掺入细丝显示出滞后相动力学，但在232 nm处的连续吸光度监测和细丝的快速沉降均显示出双曲线组装曲线。ATP水解明显滞后于肌动蛋白细丝的形成。当一半的肌动蛋白组装完成时，每摩尔以细丝形式存在的肌动蛋白仅水解0.1至0.2摩尔的ATP。
