Weber A, Pennise C R, Babcock G G, Fowler V M
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
J Cell Biol. 1994 Dec;127(6 Pt 1):1627-35. doi: 10.1083/jcb.127.6.1627.
Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.
许多蛋白质已被证明能够封闭肌动蛋白丝快速生长（带刺）的末端，但尚未发现有蛋白质能阻止其在缓慢生长（钝端）的末端进行延伸和解聚。原肌球蛋白调节蛋白是一种最初从红细胞中分离出来的原肌球蛋白结合蛋白，通过免疫荧光染色已定位到骨骼肌细肌丝钝端或其附近的位点（福勒，V.M.，M.A.，苏斯曼，P.G.米勒，B.E.弗卢彻，和M.P.丹尼尔斯。1993年。《细胞生物学杂志》120: 411 - 420）。我们的实验表明，原肌球蛋白调节蛋白与原肌球蛋白一起是一种钝端封闭蛋白：在与丝末端浓度化学计量比的浓度下（解离常数Kd≤1纳摩尔），它能完全阻止含原肌球蛋白的肌动蛋白丝钝端的延伸和解聚。在没有原肌球蛋白的情况下，原肌球蛋白调节蛋白起“渗漏”帽的作用，部分抑制钝端丝末端的延伸和解聚（抑制延伸的解离常数Kd = 0.1 - 0.4微摩尔）。因此，原肌球蛋白调节蛋白可以直接在丝的钝端与肌动蛋白结合。原肌球蛋白调节蛋白还使纯肌动蛋白丝钝端的临界浓度增加一倍，而不影响带刺丝末端的聚合速率或程度，这表明原肌球蛋白调节蛋白不会隔离肌动蛋白单体。我们的实验提供了直接的生化证据，表明原肌球蛋白调节蛋白在钝端丝末端与末端原肌球蛋白和肌动蛋白分子都结合，并且是长期以来寻找的钝端封闭蛋白。我们提出，原肌球蛋白调节蛋白在维持横纹肌和红细胞中稳定的、含原肌球蛋白的肌动蛋白丝的窄长度分布中起作用。
