Trybus K M, Lowey S
Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254.
J Cell Biol. 1987 Dec;105(6 Pt 2):3007-19. doi: 10.1083/jcb.105.6.3007.
Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing.
当平滑肌肌球蛋白在低离子强度的焦磷酸盐或柠檬酸盐/三羟甲基氨基甲烷缓冲液中进行透析时，会形成小的双极细丝，即“微丝”。与在大约生理离子条件下形成的合成细丝不同，微丝在沉降速度实验中表现出超尖锐的边界，表明其具有均一性。电子显微镜和流体动力学技术被用于表明，20 - 22S的平滑肌肌球蛋白微丝长度为380纳米，由12 - 14个分子组成。通过改变溶剂，可以得到一系列15 - 30S范围内不同大小的聚合物。相比之下，骨骼肌肌球蛋白优先形成稳定的32S微丝（雷斯勒，E.，P. 张，和N. 博罗乔夫。1986年。《生物物理杂志》49:335 - 342），这表明两种肌球蛋白在组装特性上存在潜在差异。向平滑肌肌球蛋白微丝中添加盐会导致其单向生长成为更长的“侧极”型细丝，而骨骼肌肌球蛋白则始终形成双极细丝。与合成细丝一样，添加1 mM的MgATP会导致去磷酸化的微丝解离为折叠单体和二聚体的混合物。调节轻链的磷酸化阻止了核苷酸介导的解聚，尽管它对微丝的结构没有可检测到的影响。这些结果表明，磷酸化导致的细丝稳定性差异很大程度上是由于肌球蛋白头部发生的构象变化，而不是由于细丝堆积的差异。
