Lin M C, Nicosia S, Lad P M, Rodbell M
J Biol Chem. 1977 Apr 25;252(8):2790-2.
In this study, we report the preparation of [3H]glucagon and its characteristics of binding to receptors in the rat liver plasma membrane. Binding of the labeled hormone is optimal at pH 7.0. In the absence of GTP, [3H]glucagon binding to receptors is slow and the time of equilibration is inversely proportional to the hormone concentration. In the presence of GTP, equilibrium is reached within 30 s regardless of hormone levels, and the kinetics of binding are in accord with the kinetics of activation of adenylate cyclase by native glucagon in the presence of the nucleotide. Equilibrium binding measurements indicate that, in the absence of GTP, the binding isotherm is sigmoidal with an apparent Kd of 2 nM. The addition of GTP results in a complex binding isotherm with about 90% of the binding sites having a considerably lower apparent dissociation constant (greater than 10 nM) and a small population of sites having high affinity for the hormone. The binding properties of [3H]glucagon are compared with those of 125I-glucagon, and the implications of the actions of GTP on glucagon binding are discussed in relation to the overall regulation of adenylate cyclase by hormone and the nucleotide.
在本研究中，我们报告了[3H]胰高血糖素的制备及其与大鼠肝细胞膜受体结合的特性。标记激素的结合在pH 7.0时最佳。在不存在GTP的情况下，[3H]胰高血糖素与受体的结合缓慢，平衡时间与激素浓度成反比。在存在GTP的情况下，无论激素水平如何，30秒内即可达到平衡，且结合动力学与在核苷酸存在下天然胰高血糖素激活腺苷酸环化酶的动力学一致。平衡结合测量表明，在不存在GTP的情况下，结合等温线呈S形，表观解离常数（Kd）为2 nM。加入GTP会导致复杂的结合等温线，约90%的结合位点具有明显较低的表观解离常数（大于10 nM），一小部分位点对激素具有高亲和力。将[3H]胰高血糖素的结合特性与125I-胰高血糖素的结合特性进行了比较，并结合激素和核苷酸对腺苷酸环化酶的整体调节，讨论了GTP对胰高血糖素结合作用的意义。
