Velaz L, Hemric M E, Benson C E, Chalovich J M
Department of Biochemistry, East Carolina University, Greenville, North Carolina 27858-4354.
J Biol Chem. 1989 Jun 5;264(16):9602-10.
The binding of 125I- and 14C-caldesmon to actin and actin-tropomyosin was studied using a cosedimentation technique and was analyzed by the method of McGhee and von Hippel [1974) J. Mol. Biol. 86, 469-489) for the binding of large ligands to a homogeneous lattice. The binding was adequately described by a single class of binding sites with a stoichiometry between 1:7 and 1:10. The binding exhibited a small degree of positive cooperativity (omega = 5-6) which was the same in the presence and absence of tropomyosin. The association constant for the binding of caldesmon to an isolated binding site was enhanced, from about 6 X 10(5) to about 1.4 X 10(6) M-1, by the presence of smooth muscle tropomyosin. Caldesmon inhibited the actin-activated ATPase activity of skeletal myosin subfragment 1 in both the absence and presence of tropomyosin. Maximum inhibition of ATPase activity occurred when one caldesmon molecule bound to seven actin monomers. A greater degree of inhibition was observed in the presence of tropomyosin than in the absence. This greater inhibition cannot be explained totally by the increased strength of binding of caldesmon to actin in the presence of tropomyosin. Finally, Ca2+-calmodulin completely reversed the binding of caldesmon to actin.
利用共沉降技术研究了¹²⁵I-和¹⁴C-钙调蛋白与肌动蛋白及肌动蛋白-原肌球蛋白的结合，并采用McGhee和von Hippel [1974] J. Mol. Biol. 86, 469 - 489]的方法分析了大配体与均匀晶格的结合。该结合可由一类结合位点充分描述，化学计量比在1:7至1:10之间。结合表现出小程度的正协同性（ω = 5 - 6），在有和没有原肌球蛋白的情况下相同。平滑肌原肌球蛋白的存在使钙调蛋白与单个分离结合位点的结合常数从约6×10⁵ M⁻¹提高到约1.4×10⁶ M⁻¹。在有和没有原肌球蛋白的情况下，钙调蛋白均抑制骨骼肌肌球蛋白亚片段1的肌动蛋白激活的ATP酶活性。当一个钙调蛋白分子与七个肌动蛋白单体结合时，ATP酶活性出现最大抑制。与没有原肌球蛋白时相比，有原肌球蛋白时观察到更大程度的抑制。这种更大程度的抑制不能完全用原肌球蛋白存在时钙调蛋白与肌动蛋白结合强度的增加来解释。最后，Ca²⁺-钙调蛋白完全逆转了钙调蛋白与肌动蛋白的结合。
