Green A, Frank B H, Rubenstein A, Tager H, Olefsky J M
Endocrinology. 1983 Dec;113(6):1963-71. doi: 10.1210/endo-113-6-1963.
[Leu-B25]insulin is a low affinity insulin analog which does not increase the rate of dissociation of 125I-insulin from insulin receptors (i.e. does not display negative cooperativity). We have studied the characteristics of binding of this analog to IM-9 cultured lymphocytes, in order to determine the contribution of negative cooperativity to the curvilinear nature of Scatchard plots typical of insulin binding data. The affinity of [LeuB25]insulin for receptors was approximately 1% that of insulin, as determined by its ability to inhibit 125I-insulin binding. Monoiodinated preparations of insulin and of [LeuB25]insulin were produced, labeled in the tyrosine at position 14 of the A chain. These 125I-TyrA14-labeled species were used in all studies. Both native insulin and a serum containing antiinsulin receptor antibodies were equally potent at inhibiting binding of 125I-[LeuB25]insulin and 125I-native insulin, suggesting that they bind to the same population of receptors. Native insulin (100 ng/ml) increased the rate of dissociation of both 125I-insulin and 125I-[LeuB25]insulin. However, [LeuB25]insulin (2.5 micrograms/ml) did not increase the rates of dissociation of either 125I-insulin or 125I-[LeuB25]insulin (i.e. it did not display negative cooperativity). Competition curves and Scatchard plots were constructed using 125I-[LeuB25]insulin and unlabeled analog. Half-maximal inhibition of 125I-[LeuB25]insulin binding was seen at a [LeuB25]insulin concentration of approximately 500 ng/ml. More importantly, the Scatchard plot of these binding data was markedly curvilinear, as is typical of insulin binding data. In summary, a non-cooperative insulin analog, [LeuB25]insulin, yielded curvilinear Scatchard plots, indicating that negative cooperativity is not the full explanation of the curvilinear nature of Scatchard plots of insulin binding data. Therefore, an alternative explanation, such as the existence of heterogeneous receptor states, with differing affinities for the hormone, must contribute to the nonlinearity of these plots.
[亮氨酸 - B25]胰岛素是一种低亲和力胰岛素类似物，它不会增加125I - 胰岛素从胰岛素受体上的解离速率（即不表现出负协同性）。我们研究了这种类似物与IM - 9培养淋巴细胞结合的特性，以确定负协同性对胰岛素结合数据典型的Scatchard图曲线性质的贡献。通过其抑制125I - 胰岛素结合的能力确定，[亮氨酸B25]胰岛素对受体的亲和力约为胰岛素的1%。制备了胰岛素和[亮氨酸B25]胰岛素的单碘化制剂，标记在A链第14位的酪氨酸上。所有研究均使用这些125I - 酪氨酸A14标记的物质。天然胰岛素和含有抗胰岛素受体抗体的血清在抑制125I - [亮氨酸B25]胰岛素和125I - 天然胰岛素结合方面同样有效，这表明它们与相同群体的受体结合。天然胰岛素（100 ng/ml）增加了125I - 胰岛素和125I - [亮氨酸B25]胰岛素的解离速率。然而，[亮氨酸B25]胰岛素（2.5微克/ml）并未增加125I - 胰岛素或125I - [亮氨酸B25]胰岛素的解离速率（即它不表现出负协同性）。使用125I - [亮氨酸B25]胰岛素和未标记的类似物构建竞争曲线和Scatchard图。在[亮氨酸B25]胰岛素浓度约为500 ng/ml时观察到125I - [亮氨酸B25]胰岛素结合的半数最大抑制。更重要的是，这些结合数据的Scatchard图明显呈曲线状，这是胰岛素结合数据的典型特征。总之，一种非协同性胰岛素类似物[亮氨酸B25]胰岛素产生了曲线状的Scatchard图，表明负协同性并不能完全解释胰岛素结合数据的Scatchard图的曲线性质。因此，一种替代解释，例如存在对激素具有不同亲和力的异质性受体状态，必定导致了这些图的非线性。
