Niederländer Nicolas, Raynaud Fabrice, Astier Catherine, Chaussepied Patrick
CRBM-CNRS, Montpellier, France.
Eur J Biochem. 2004 Nov;271(22):4572-81. doi: 10.1111/j.1432-1033.2004.04429.x.
Titin is known to interact with actin thin filaments within the I-band region of striated muscle sarcomeres. In this study, we have used a titin fragment of 800 kDa (T800) purified from striated skeletal muscle to measure the effect of this interaction on the functional properties of the actin-myosin complex. MALDI-TOF MS revealed that T800 contains the entire titin PEVK (Pro, Glu, Val, Lys-rich) domain. In the presence of tropomyosin-troponin, T800 increased the sliding velocity (both average and maximum values) of actin filaments on heavy-meromyosin (HMM)-coated surfaces and dramatically decreased the number of stationary filaments. These results were correlated with a 30% reduction in actin-activated HMM ATPase activity and with an inhibition of HMM binding to actin N-terminal residues as shown by chemical cross-linking. At the same time, T800 did not affect the efficiency of the Ca(2+)-controlled on/off switch, nor did it alter the overall binding energetics of HMM to actin, as revealed by cosedimentation experiments. These data are consistent with a competitive effect of PEVK domain-containing T800 on the electrostatic contacts at the actin-HMM interface. They also suggest that titin may participate in the regulation of the active tension generated by the actin-myosin complex.
已知肌联蛋白可与横纹肌肌节 I 带区域内的肌动蛋白细肌丝相互作用。在本研究中，我们使用了从横纹肌骨骼肌中纯化得到的 800 kDa 肌联蛋白片段（T800）来测量这种相互作用对肌动蛋白 - 肌球蛋白复合物功能特性的影响。基质辅助激光解吸电离飞行时间质谱显示，T800 包含完整的肌联蛋白 PEVK（富含脯氨酸、谷氨酸、缬氨酸和赖氨酸）结构域。在原肌球蛋白 - 肌钙蛋白存在的情况下，T800 增加了肌动蛋白丝在重酶解肌球蛋白（HMM）包被表面上滑动的速度（平均值和最大值），并显著减少了静止丝的数量。这些结果与肌动蛋白激活的 HMM ATP 酶活性降低 30%以及化学交联显示的 HMM 与肌动蛋白 N 端残基结合受到抑制相关。同时，沉降实验表明，T800 不影响 Ca(2+) 控制的开/关开关的效率，也不改变 HMM 与肌动蛋白的整体结合能。这些数据与含 PEVK 结构域的 T800 对肌动蛋白 - HMM 界面静电接触的竞争作用一致。它们还表明，肌联蛋白可能参与调节肌动蛋白 - 肌球蛋白复合物产生的主动张力。
