来自兔骨骼肌的肌球蛋白亚片段1与芘标记的细肌丝之间相互作用的动力学。

The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle.

作者信息

Geeves M A, Halsall D J

出版信息

Proc R Soc Lond B Biol Sci. 1986 Oct 22;229(1254):85-95. doi: 10.1098/rspb.1986.0076.

DOI:10.1098/rspb.1986.0076

Abstract

We have used actin labelled in Cys-374 with N-(1-pyrenyl)iodoacetamide to monitor the dynamics and equilibria of the interaction between myosin subfragment 1 and the actin-troponin-tropomyosin complex in the presence of calcium. These results are compared with those obtained for pure actin and myosin subfragment 1. The sensitivity of this fluorescent label allowed us to measure the binding affinity of myosin subfragment 1 for actin directly by fluorescence titration. The affinity of subfragment 1 for actin is increased sixfold by troponin-tropomyosin in the presence of calcium. Kinetic studies of the interaction of subfragment 1 and actin have revealed an isomerization of the actin-subfragment 1 complex from a state in which actin is weakly bound (Ka = 5.9 X 10(4) M-1) to a more tightly bound complex (Ka = 1.7 X 10(7) M-1) (Coates, Criddle & Geeves (1985) Biochem. J. 232, 351). Results in the presence of troponin-tropomyosin show the same isomerization. The sixfold increase in affinity of subfragment 1 for actin is shown to be due to a decrease in the rate of dissociation of actin from the weakly bound complex.

摘要

我们使用用N-(1-芘基)碘乙酰胺标记半胱氨酸-374位点的肌动蛋白，来监测在有钙存在的情况下肌球蛋白亚片段1与肌动蛋白-肌钙蛋白-原肌球蛋白复合物之间相互作用的动力学和平衡。将这些结果与纯肌动蛋白和肌球蛋白亚片段1的结果进行比较。这种荧光标记的灵敏度使我们能够通过荧光滴定直接测量肌球蛋白亚片段1对肌动蛋白的结合亲和力。在有钙存在的情况下，肌钙蛋白-原肌球蛋白使亚片段1对肌动蛋白的亲和力增加了六倍。对亚片段1与肌动蛋白相互作用的动力学研究表明，肌动蛋白-亚片段1复合物存在异构化，从肌动蛋白弱结合的状态（Ka = 5.9×10⁴ M⁻¹）转变为结合更紧密的复合物（Ka = 1.7×10⁷ M⁻¹）（科茨、克里德尔和吉夫斯（1985年）《生物化学杂志》232卷，351页）。在有肌钙蛋白-原肌球蛋白存在的情况下也观察到了同样的异构化。亚片段1对肌动蛋白亲和力增加六倍的原因是肌动蛋白从弱结合复合物中解离的速率降低。