Iwane A H, Kitamura K, Tokunaga M, Yanagida T
Faculty of Engineering Science, Osaka University, Toyonaka, Japan.
Biochem Biophys Res Commun. 1997 Jan 3;230(1):76-80. doi: 10.1006/bbrc.1996.5861.
The sliding velocity of actin filaments propelled by chicken skeletal myosin subfragment-1 (S1) was measured when the tail end of S1 was specifically bound to the glass surface. To achieve the specific binding, a regulatory light chain was replaced by a recombinant fusion protein of biotin-dependent transcarboxylase (BDTC) and chicken gizzard smooth muscle regulatory light chain (cgmRLC). The BDTC-cgmRLC of S1 was then attached to the glass surface using a biotin-avidin system. The velocity of actin filaments caused by S1 bound to the surface in this manner was 6.8 +/- 0.6 microm/sec at 29 degrees C, which was 3.5-fold greater than that (1.9 +/- 0.3 microm/sec) when bound directly to the surface as in previous studies, but similar to that caused by native chicken skeletal myosin (6.5 +/- 0.6 microm/sec). The actin-activated Mg-ATPase activity was similar to that of S1 before the RLC of S1 was exchanged for BDTC-cgmRLC. The results indicate that S1 can produce a normal fast movement of actin filaments as well as hydrolyse ATP and generate force.
当鸡骨骼肌肌球蛋白亚片段-1（S1）的尾端特异性结合到玻璃表面时，测量了由其推动的肌动蛋白丝的滑动速度。为实现特异性结合，将调节轻链替换为生物素依赖性转羧酶（BDTC）与鸡砂囊平滑肌调节轻链（cgmRLC）的重组融合蛋白。然后使用生物素-抗生物素蛋白系统将S1的BDTC-cgmRLC附着到玻璃表面。在29℃时，以这种方式结合到表面的S1引起的肌动蛋白丝速度为6.8±0.6微米/秒，这比之前研究中直接结合到表面时的速度（1.9±0.3微米/秒）快3.5倍，但与天然鸡骨骼肌肌球蛋白引起的速度（6.5±0.6微米/秒）相似。在S1的RLC被BDTC-cgmRLC替换之前，肌动蛋白激活的Mg-ATP酶活性与S1的相似。结果表明，S1能够使肌动蛋白丝产生正常的快速移动，同时水解ATP并产生力。
