Fujii T, Oomatsuzawa A, Kuzumaki N, Kondo Y
Department of Functional Polymer Science, Faculty of Textile Science and Technology, Shinshu University, Nagano.
J Biochem. 1994 Jul;116(1):121-7. doi: 10.1093/oxfordjournals.jbchem.a124484.
Calponin, a major calmodulin-, actin-, and tropomyosin-binding protein in smooth muscle, interacted with brain S100 and the properties of the interaction were investigated in detail. From fluorescence labeling and chemical cross-linking experiments, the apparent Kd value was calculated to be 7 x 10(7) M-1 in the presence of Ca2+ with 1 mol of S100 bound per mol of calponin. The addition of S100 to the mixture of calponin and F-actin caused the removal of calponin from actin filaments in the presence of Ca2+ but not in the presence of EGTA or Zn2+. Ca2+ and S100 could relieve calponin-induced actomyosin Mg(2+)-ATPase inhibition. Both the removal of calponin from F-actin and the restoration of ATPase inhibition by S100 were more effective than those by calmodulin. At low ionic strength, the binding was observed irrespective of Ca2+ concentration and it was greatly weakened with increasing salt concentration. The formation of the complex in the presence of Ca2+ was less sensitive, with only 45% inhibition at 100 mM NaCl, where the complex in the absence of Ca2+ had almost disappeared. This was confirmed by S-100 Sepharose 4B chromatography. Addition of Ca2+ and S100 also led to a decrease in the affinity of calponin for tropomyosin. Domain mapping with chymotryptic digestion revealed that the S100 binding site resided within the N-terminal 22 kDa fragment of calponin, where the bindings of calmodulin and actin also occur.
钙调蛋白是平滑肌中一种主要的钙调素、肌动蛋白和原肌球蛋白结合蛋白，它与脑S100相互作用，并对这种相互作用的特性进行了详细研究。通过荧光标记和化学交联实验，在存在Ca2+且每摩尔钙调蛋白结合1摩尔S100的情况下，表观解离常数（Kd）值计算为7×10⁻⁷ M⁻¹。在Ca2+存在下，将S100添加到钙调蛋白和F-肌动蛋白的混合物中会导致钙调蛋白从肌动蛋白丝上脱离，但在EGTA或Zn2+存在下则不会。Ca2+和S100可以缓解钙调蛋白诱导的肌动球蛋白Mg²⁺-ATP酶抑制。S100使钙调蛋白从F-肌动蛋白上脱离以及恢复ATP酶抑制的效果都比钙调素更有效。在低离子强度下，无论Ca2+浓度如何都能观察到结合，并且随着盐浓度的增加结合会大大减弱。在Ca2+存在下复合物的形成对盐浓度不太敏感，在100 mM NaCl时只有45%的抑制，而在没有Ca2+时复合物几乎消失。这通过S-100琼脂糖4B层析得到了证实。添加Ca2+和S100还导致钙调蛋白对原肌球蛋白的亲和力降低。用胰凝乳蛋白酶消化进行结构域定位表明，S100结合位点位于钙调蛋白N端22 kDa片段内，钙调素和肌动蛋白的结合也发生在此处。
