Horiuchi K Y, Chacko S
Department of Pathobiology, University of Pennsylvania, Philadelphia 19104, USA.
J Muscle Res Cell Motil. 1995 Feb;16(1):11-9. doi: 10.1007/BF00125306.
The effect of smooth muscle myosin at different levels of light chain phosphorylation on caldesmon-mediated movement of actin filaments was investigated using an in vitro motility assay. Myosin at different levels of phosphorylation was obtained by mixing different proportions of fully phosphorylated and unphosphorylated myosin in monomeric form, while keeping the total myosin concentration constant. The average velocity of actin filaments containing tropomyosin was 1.20 +/- 0.046 microns s-1 at 30 degrees C with fully phosphorylated myosin. This velocity was not altered when the percentage of unphosphorylated myosin coated on the nitrocellulose surface was increased to 80%; further increases lowered the velocity. When the actin filaments with caldesmon bound at stoichiometric levels were used, filament velocity was unaffected until 50% of the myosin was unphosphorylated, but further increases in the percentage of unphosphorylated myosin induced a decrease in the velocity, and at 95% unphosphorylated myosin, filament movement had ceased. The decreased filament velocity in the presence of caldesmon was also observed when phosphorylated myosin was mixed with myosin rod instead of unphosphorylated myosin, but was not observed when the 38 kDa caldesmon C-terminal fragment, which lacks the myosin-binding domain, was used instead of intact caldesmon. These data indicate that the decreased filament velocity in the presence of caldesmon reflects the mechanical load produced by the tethering of actin to myosin through the interaction of the caldesmon N-terminal domain and the myosin S-2 region. The tethering effect mediated by caldesmon may play a role in smooth muscle contraction when a large number of myosin heads are dephosphorylated, as in force maintenance.
使用体外运动分析方法，研究了不同轻链磷酸化水平的平滑肌肌球蛋白对钙调蛋白介导的肌动蛋白丝运动的影响。通过以单体形式混合不同比例的完全磷酸化和未磷酸化的肌球蛋白，同时保持总肌球蛋白浓度恒定，获得不同磷酸化水平的肌球蛋白。在30℃下，使用完全磷酸化的肌球蛋白时，含有原肌球蛋白的肌动蛋白丝的平均速度为1.20±0.046微米/秒。当涂覆在硝酸纤维素表面的未磷酸化肌球蛋白的百分比增加到80%时，该速度没有改变；进一步增加则会降低速度。当使用化学计量水平结合钙调蛋白的肌动蛋白丝时，直到50%的肌球蛋白未磷酸化时，丝速度不受影响，但未磷酸化肌球蛋白百分比的进一步增加会导致速度降低，当95%的肌球蛋白未磷酸化时，丝运动停止。当磷酸化的肌球蛋白与肌球蛋白杆而不是未磷酸化的肌球蛋白混合时，也观察到了在钙调蛋白存在下丝速度的降低，但当使用缺乏肌球蛋白结合结构域的38 kDa钙调蛋白C末端片段代替完整的钙调蛋白时，未观察到这种情况。这些数据表明，在钙调蛋白存在下丝速度的降低反映了通过钙调蛋白N末端结构域和肌球蛋白S-2区域的相互作用，肌动蛋白与肌球蛋白拴系产生的机械负荷。当大量肌球蛋白头部去磷酸化时，如在维持力的情况下，钙调蛋白介导的拴系效应可能在平滑肌收缩中起作用。
