Santora A C, Wheeler F B, DeHaan R L, Elsas L J
Endocrinology. 1979 Apr;104(4):1059-68. doi: 10.1210/endo-104-4-1059.
The characteristics of insulin receptors were studied in cultured embryonic chick heart cells which demonstrated insulin-responsive amino acid transport. Binding of [125I]iodoinsulin was time dependent, reversible, saturable, species specific, and proportional to cell number. Optimum binding occurred at pH 7.8 in the presence of 0.1% bovine serum albumin. Curvilinear Scatchard plots were found for chicken and bovine insulin binding at 15 and 30 C. Equilibrium association constants (Kas) and maximum capacities were calculated based on a two-receptor model. When studied at 15 C, chicken insulin was bound with Kas of 5.0 and 0.026 nM-1 for the high and low affinity receptors, respectively. Bovine insulin bound with Kas of 2.1 and 0.03 nM-1. The binding capacities of 600 and 9000 molecules/cell for the high and low affinity receptors, respectively, were the same for both species of ligand. At 30 C, the Ka of the high affinity chicken insulin receptor interaction decreased to 1.6 nM-1, whereas the low affinity Ka was not changed. In competitive binding assays, chicken insulin was 4 and 250 times more potent than bovine and guinea pig insulin, respectively. Human GH and mouse epidermal growth factor did not compete with chicken insulin. Although mouse epidermal growth factor did not compete with insulin, it did stimulate 2-aminoisobutyric acid accumulation. The maximal stimulation by this hormone was less than and additive to that produced by the maximal stimulation of insulin. These data indicate that insulin and epidermal growth factor acted through different receptors to stimulate amino acid transport. When insulin-stimulated functional response was compared to calculated receptor occupancy in this two-receptor model, stimulation of amino acid transport paralleled occupancy of the low affinity receptor such that at half-maximal transport stimulation, approximately 50% of these receptors were occupied. Half-maximal stimulation of 2-aminoisobutyric acid transport occurred at 18- and 20-nM concentrations of bovine and chicken insulins, respectively. Guinea pig insulin failed to stimulate 2-aminoisobutyric acid uptake even at 350 nM. Both the affinity of binding in the low affinity class and the insulin concentration for half-maximal stimulation of 2-aminoisobutyric acid transport were independent of temperature in the temperature ranges studied. We conclude that 14-day embryonic chick heart cells possess at least two classes of receptors which bind insulin. Occupancy of the lower affinity class of insulin receptors correlates quantitatively with insulin stimulation of 2-aminoisobutyric acid transport.
在已证明具有胰岛素反应性氨基酸转运功能的培养鸡胚心脏细胞中研究了胰岛素受体的特性。[125I]碘胰岛素的结合具有时间依赖性、可逆性、饱和性、物种特异性，且与细胞数量成正比。在0.1%牛血清白蛋白存在的情况下，pH 7.8时发生最佳结合。在15℃和30℃下，鸡和牛胰岛素结合的Scatchard图呈曲线。基于双受体模型计算了平衡缔合常数（Kas）和最大容量。在15℃研究时，鸡胰岛素与高亲和力和低亲和力受体结合的Kas分别为5.0和0.026 nM-1。牛胰岛素结合的Kas为2.1和0.03 nM-1。两种配体的高亲和力和低亲和力受体的结合容量分别为600和9000个分子/细胞。在30℃时，高亲和力鸡胰岛素受体相互作用的Ka降至1.6 nM-1，而低亲和力Ka不变。在竞争性结合试验中，鸡胰岛素的效力分别比牛胰岛素和豚鼠胰岛素高4倍和250倍。人生长激素和小鼠表皮生长因子不与鸡胰岛素竞争。虽然小鼠表皮生长因子不与胰岛素竞争，但它确实刺激了2-氨基异丁酸的积累。该激素的最大刺激作用小于胰岛素最大刺激作用产生的效果，且二者具有加和性。这些数据表明胰岛素和表皮生长因子通过不同的受体发挥作用来刺激氨基酸转运。当在这个双受体模型中将胰岛素刺激的功能反应与计算出的受体占有率进行比较时，氨基酸转运的刺激与低亲和力受体的占有率平行，因此在转运刺激达到半最大值时，大约50%的这些受体被占据。牛胰岛素和鸡胰岛素分别在18 nM和20 nM的浓度下对2-氨基异丁酸转运产生半最大刺激作用。豚鼠胰岛素即使在350 nM时也未能刺激2-氨基异丁酸的摄取。在所研究的温度范围内，低亲和力类别中结合亲和力以及2-氨基异丁酸转运半最大刺激所需的胰岛素浓度均与温度无关。我们得出结论，14日龄鸡胚心脏细胞至少拥有两类结合胰岛素的受体。低亲和力类胰岛素受体的占有率与胰岛素对2-氨基异丁酸转运的刺激在数量上相关。
