Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.

Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.
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肌球蛋白诱导的 F-肌动蛋白变化:通过连接 Gln-41 的丹酰乙二胺对子结构域 2 进行荧光探测。

DOI:
10.1016/s0006-3495(96)79703-5
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发表时间:
1996
影响因子:
3.4
通讯作者:
Reisler,E
Reisler,E
中科院分区:
生物学3区
文献类型:
--
作者:
Kim,E;Miller,CJ;Motoki,M;Seguro,K;Muhlrad,A;Reisler,E

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用Dansyl乙二胺(DED)标记肌动蛋白Gln-41位点,通过转氨酶反应监测肌球蛋白亚片段1(S1)与F-actin 38-52环上His-40-Gly-42位点的相互作用。用枯草杆菌蛋白酶和胰蛋白酶对F-肌动蛋白进行蛋白水解,并对热处理的F-肌动蛋白进行acto-S1 ATP酶测量,结果表明Gln-41的标记对亚结构域2和肌动蛋白丝具有稳定作用。在体外运动实验中,DED对Gln-41的acto-S1 ATP酶的K(m)和Vmax值以及肌动蛋白丝的滑动速度没有影响。这表明S1不与肌动蛋白上的40-42位点结合,或者这种结合在功能上不重要。单克隆抗丹酰IgG与DED-F-肌动蛋白的结合在不存在核苷酸的情况下不影响acto-S1结合,这表明40-42位点对严格的acto-S1结合没有多大贡献。肌球蛋白诱导的肌动蛋白上的亚结构域2的变化表现为通过增加的荧光DED-F-肌动蛋白,减少的可访问性的探针碰撞淬灭剂,和部分位移的抗丹酰IgG从肌动蛋白由S1。有人提出,这些变化在38-52环的肌动蛋白起源于S1结合到其他肌球蛋白识别位点的肌动蛋白。
Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38–52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40–42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40–42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38–52 loop on actin originate from S1 binding to other myosin recognition sites on actin.
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