Evidence supporting a two-receptor model for insulin binding by cultured embryonic heart cells.
Evidence supporting a two-receptor model for insulin binding by cultured embryonic heart cells.
复制标题
支持培养的胚胎心脏细胞结合胰岛素的双受体模型的证据。
DOI：
10.1210/endo-107-1-195
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发表时间：
1980
期刊：
影响因子：
4.8
通讯作者：
L. J. Elsas
中科院分区：
文献类型：
--
作者：
Frances B. Wheeler;Arthur C. Santora;L. J. Elsas
在15℃下培养的胚胎心脏细胞与胰岛素结合的曲线Scatchard图被解释为一个双受体模型(Santora II，A.C.，F.B.Wheeler，R.L.DeHaan，和L.J.Elsas II，Endocrinology 104：1059,1979)。目前的研究进一步验证了这一假设。125I标记的激素结合和2-甲氨基-[1-14C]异丁酸([14C]meAIB)转运均在24℃相同的缓冲液中进行测定。平衡鸡胰岛素结合实验得到了曲线Scatchard功能，与高亲和力、低容量和低亲和力的高容量受体结合在24℃时一致。胰岛素刺激的模型氨基酸[14C]meAIB(一种特定的丙氨酸偏好系统底物)的转运与低亲和力受体的预期占有率平行。在竞争结合分析中，将增殖刺激活性(MSA)、胰岛素原和胰岛素竞争高亲和力胰岛素结合的能力与它们刺激运输的能力进行了比较。与34 PM鸡[125I]胰岛素结合竞争的相对效价为胰岛素大于或等于MSA(1：0，05：0.03)。相反，刺激[14C]meAIB转运的相对效力是MSA大于胰岛素大于胰岛素原(3：1：0.28)。胰岛素对转运的最大刺激作用与MSA或胰岛素原无相加关系。与[125I]MSA结合竞争的相对效力与促进转运的相对效力相同：MSA大于胰岛素大于胰岛素原(2-3：1：0.2-0.3)。以恒定的摩尔比加入超过胰岛素的胰岛素原，减少低亲和力胰岛素结合的程度大于减少高亲和力胰岛素结合的程度。此外，根据我们的生物效力数据预测并直接支持与每个受体的结合程度，其中胰岛素原在低亲和力受体上的效力几乎是胰岛素的三分之一，但在高亲和力受体上的效力只有胰岛素的5%。不同的相对亲和力表明，这两种受体对胰岛素分子的不同部分具有结合位点特异性。每一项胰岛素原和胰岛素摩尔比恒定的实验都是与胰岛素是唯一未标记的激素的实验平行进行的。对Michaelis-Menten方程的加权非线性最小二乘回归拟合结果的分析表明，以胰岛素为唯一未标记激素的数据需要一个双受体模型来拟合。当汇集每个平行实验中的数据时，也需要低亲和力结合参数。下列动力学实验表明，未标记的胰岛素没有促进结合的[125I]胰岛素的解离。
Curvilinear Scatchard plots for insulin binding by cultured embryonic heart cells at 15 C were interpreted assuming a two-receptor model (Santora II, A. C., F.B. Wheeler, R.L. DeHaan, and L.J. Elsas II, Endocrinology 104:1059, 1979). The present studies test this hypothesis further. Both 125I-labeled hormone binding and 2-methylamino-[1-14C]isobutyric acid ([14C]meAIB) transport were assayed at 24 C in identical buffers. Equilibrium chicken insulin-binding experiments yielded curvilinear Scatchard functions, consistent with binding to both high affinity, low capacity and low affinity, high capacity receptor sites at 24 C. Insulin-stimulated transport of the model amino acid [14C]meAIB (a specific alanine-preferring system substrate) paralleled the expected occupancy of the low affinity receptors. In competitive binding assays, the abilities of multiplication-stimulating activity (MSA), proinsulin, and insulin to compete for primarily high affinity insulin binding were compared with their abilities to stimulate transport. The relative potencies for binding competition with 34 pM chicken [125I]insulin were insulin greater than proinsulin greater than or equal to MSA (1:0,05:0.03). In contrast, the relative potencies for stimulating [14C]meAIB transport were MSA greater than insulin greater than proinsulin (3:1:0.28). Maximal stimulation of transport by insulin was not additive to that by MSA or proinsulin. The relative potency profile for binding competition with [125I]MSA was the same as that for stimulation of transport: MSA greater than insulin greater than proinsulin (2-3:1:0.2-0.3). Proinsulin, added in excess of insulin at a constant molar ratio, reduced low affinity insulin binding to a greater extent than it reduced high affinity insulin binding. Moreover, the extent of binding to each receptor was predicted from, and thus directly supported, our biological potency data, in which proinsulin was nearly one third as potent as insulin at the low affinity receptor yet possessed only 5% of insulin's potency at the high affinity receptor. The different relative affinity profiles suggest that these two receptors had binding site specificities for different portions of the insulin molecule. Each experiment with proinsulin and insulin in a constant molar ratio was carried out in parallel with an experiment in which insulin was the only unlabeled hormone. Analysis of the results from weighted nonlinear least squares regression fits of data to Michaelis-Menten equations showed that a two-receptor model was necessary to fit the data obtained with insulin as the only unlabeled hormone. Low affinity site binding parameters were also needed when data within each parallel experiment were pooled. The following kinetic experiments indicated the absence of enhanced dissociation of bound [125I]insulin by unlabeled insulin...