Miller L, Phillips M, Reisler E
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
J Biol Chem. 1988 Feb 5;263(4):1996-2002.
The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.
在解聚的G-肌动蛋白缓冲液中，研究了来自同一来源的肌球蛋白亚片段1（S-1）对兔骨骼肌肌动蛋白的聚合作用。在广泛的肌动蛋白/S-1摩尔比范围内，通过光散射实验监测聚合反应。与KCl和Mg2+引发的肌动蛋白组装中清晰分辨的成核-延伸步骤不同，在S-1存在下肌动蛋白的缔合在聚合反应中未显示出任何滞后现象。肌动蛋白与S-1的光散射滴定以及反之亦然的滴定表明，在肌动蛋白与S-1化学计量比为1:1时，聚合反应达到饱和。超速离心实验证实，即使在高肌动蛋白/S-1比率下，也只有化学计量的肌动蛋白被掺入1:1的肌动蛋白-S-1聚合物中。通过电子显微镜、光散射测量以及使用用N-（1-芘基）碘乙酰胺共价标记的肌动蛋白时观察到的荧光变化判断，这些聚合物与S-1与F-肌动蛋白的标准复合物没有区别。由S-1引发G-肌动蛋白聚合得到的F-肌动蛋白在10 mM KCl和0.5 mM MgCl2存在下可引发G-肌动蛋白的快速组装，并显示出肌球蛋白对MgATPase水解的正常激活作用。
