Wang F, Chen L, Arcucci O, Harvey E V, Bowers B, Xu Y, Hammer J A, Sellers J R
Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2000 Feb 11;275(6):4329-35. doi: 10.1074/jbc.275.6.4329.
Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V(max) and a low K(ATPase). Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s(-1) under conditions where the V(max) was 3.3 s(-1), indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.
小鼠肌球蛋白V是一种双头非常规肌球蛋白，其颈部延伸，可结合六个钙调蛋白。小鼠肌球蛋白V的双头（重酶解肌球蛋白样）和单头（亚片段1样）片段在Sf9细胞中表达，完整的肌球蛋白V从小鼠脑中纯化得到。组织纯化的肌球蛋白V及其表达片段的肌动蛋白激活的MgATP酶具有高V(max)和低K(ATP酶)。钙调节完整肌球蛋白V的MgATP酶，但不调节片段的MgATP酶。MgATP酶活性和体外运动性对离子强度均不敏感。肌球蛋白V及其片段在非常低的肌球蛋白表面密度下使肌动蛋白移位。ADP显著抑制肌动蛋白激活的MgATP酶活性和体外运动性。在V(max)为3.3 s(-1)的条件下，ADP以约11.5 s(-1)的速率从肌球蛋白V亚片段1解离，这表明尽管ADP解离并非完全限速，但接近限速步骤。对肌动蛋白的高亲和力和ADP释放的缓慢速率有助于肌球蛋白头部在每个ATP酶循环的大部分时间内保持与肌动蛋白结合，并允许肌动蛋白丝在体外仅由少数肌球蛋白V分子移动。
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