Vorwerk Peter, Hohmann Bianka, Oh Youngman, Rosenfeld Ron G, Shymko Ronald M
Department of Pediatric Oncology, Otto von Guericke University, Emanuel Larisch Weg 17-19, D-39112 Magdeburg, Germany.
Endocrinology. 2002 May;143(5):1677-85. doi: 10.1210/endo.143.5.8760.
We measured the binding of IGF-I and IGF-II to recombinant human N-terminal [residues 1-97; recombinant human IGF-binding protein-3(1-97) (rhIGFBP-3(1-97))] and C-terminal (residues 98-264; rhIGFBP-3(98-264)) IGFBP-3 fragments and compared it with IGF binding to intact IGFBP-3 using biosensor analysis. Experiments were carried out in different configurations, either with binding protein or fragment immobilized or with IGF immobilized. These experiments showed that IGF-I and IGF-II bind to IGFBP-3 with affinities of 4-5 x 10(9) M(-1) and similar binding kinetics. The affinities of both rhIGFBP-3(1-97) and rhIGFBP-3(98-264) for IGF proteins were approximately 3 orders of magnitude less than that of full-length IGFBP-3. These results further support the concept that high affinity binding of IGF to IGF-binding proteins results from a two-site interaction of IGF with both the N- and C-terminal regions of the binding protein. Binding of insulin to IGFBP-3 and its N- and C-terminal fragments and of IGF-I and IGF-II to the structurally related proteins mac25 and connective tissue growth factor was also investigated. Weak insulin binding to full-length IGFBP-3 could be demonstrated in a few experiments, but we found that binding of IGF-I, IGF-II, and insulin to mac25 or connective tissue growth factor was below the detection limit of the biosensor instrument.
我们使用生物传感器分析方法，测定了胰岛素样生长因子-I（IGF-I）和胰岛素样生长因子-II（IGF-II）与重组人N端[1 - 97位氨基酸残基；重组人胰岛素样生长因子结合蛋白-3（1 - 97）（rhIGFBP-3(1 - 97)）]及C端（98 - 264位氨基酸残基；rhIGFBP-3(98 - 264)）胰岛素样生长因子结合蛋白-3（IGFBP-3）片段的结合情况，并将其与IGF与完整IGFBP-3的结合进行比较。实验采用了不同的配置方式，包括固定结合蛋白或片段，以及固定IGF。这些实验表明，IGF-I和IGF-II以4 - 5×10⁹ M⁻¹的亲和力与IGFBP-3结合，且结合动力学相似。rhIGFBP-3(1 - 97)和rhIGFBP-3(98 - 264)对IGF蛋白的亲和力比全长IGFBP-3低约3个数量级。这些结果进一步支持了以下概念：IGF与胰岛素样生长因子结合蛋白的高亲和力结合是由IGF与结合蛋白的N端和C端区域的双位点相互作用导致的。我们还研究了胰岛素与IGFBP-3及其N端和C端片段的结合，以及IGF-I和IGF-II与结构相关蛋白mac25和结缔组织生长因子的结合。在少数实验中可以证明胰岛素与全长IGFBP-3存在弱结合，但我们发现IGF-I、IGF-II和胰岛素与mac25或结缔组织生长因子的结合低于生物传感器仪器的检测限。
