Olwin B B, Storm D R
Biochemistry. 1985 Dec 31;24(27):8081-6. doi: 10.1021/bi00348a037.
The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.
测定了钙离子（Ca2+）与钙调蛋白敏感磷酸二酯酶结合到钙调蛋白上的偶联自由能，并与另外两种钙调蛋白结合蛋白——肌钙蛋白I和肌球蛋白轻链激酶的偶联能进行了比较。通过用喹啉2（Quin 2）定量测定Ca2+与与钙调蛋白结合蛋白复合的钙调蛋白的结合情况来监测游离Ca2+浓度，从而确定偶联自由能。在等摩尔兔骨骼肌肌钙蛋白I、兔骨骼肌肌球蛋白轻链激酶和牛心钙调蛋白敏感磷酸二酯酶存在的情况下，Ca2+与钙调蛋白结合的解离常数（-Kd）的几何平均值分别为2.1、1.1和0.55微摩尔。四个Ca2+以及这些蛋白质与钙调蛋白结合的自由能偶联分别为-4.48、-6.00和-7.64千卡。磷酸二酯酶与钙调蛋白结合的不依赖Ca2+的Kd估计为80毫摩尔，这表明在低Ca2+条件下，细胞内钙调蛋白与这种酶之间不会形成复合物。大的自由能偶联值反映了钙调蛋白与钙调蛋白结合蛋白复合时Ca2+亲和力的增加，并定义了每个系统预期的Ca2+结合的明显正协同性。这些数据表明，在体外，各种钙调蛋白调节酶的自由能偶联差异可能导致酶对Ca2+的敏感性不同。
