Haeberle J R, Trybus K M, Hemric M E, Warshaw D M
Department of Physiology and Biophysics, University of Vermont, Burlington 05405.
J Biol Chem. 1992 Nov 15;267(32):23001-6.
The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.
使用体外运动测定法，研究了重组细肌丝在硫代磷酸化平滑肌肌球蛋白固定表面上的运动。重组细肌丝包含肌动蛋白、原肌球蛋白，以及纯化的鸡砂囊钙调蛋白或纯化的钙调蛋白COOH末端肌动蛋白结合片段。对照肌动蛋白-原肌球蛋白丝以2.3±0.5微米/秒的速度移动。当用10 mM二硫苏糖醇处理使完整的钙调蛋白和COOH末端片段保持单体形式时，它们对丝速度均无影响；然而，二者都是肌动蛋白激活的肌球蛋白ATP酶活性的有效抑制剂，这表明如Chalovich等人所报道的，钙调蛋白主要抑制肌球蛋白结合（Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85 - 99）。然而，在存在通过二硫键交联钙调蛋白的条件下，观察到了丝运动的抑制。为了确定单体钙调蛋白是否能如Chalovich等人所提出的那样，通过形成肌动蛋白-钙调蛋白-肌球蛋白复合物将肌动蛋白丝“系”在肌球蛋白表面，我们在细丝与肌球蛋白结合较弱且通常看不到运动的条件（80 mM KCl）下，寻找依赖钙调蛋白的细丝结合和运动。在钙调蛋白浓度≥0.26 microM时，肌动蛋白丝结合增加，并且观察到丝运动（2.6±0.6微米/秒）。加入高达26 microM的COOH末端片段时，未观察到完整钙调蛋白所呈现的增强运动。此外，COOH末端片段的摩尔过量竞争性地逆转了完整钙调蛋白所呈现的增强结合。这些结果表明，通过形成肌动蛋白-钙调蛋白-肌球蛋白复合物将肌动蛋白丝系在肌球蛋白上，增强了有效的肌动蛋白-肌球蛋白相互作用，而不会对移动的细丝施加显著的机械负荷。
