Booth B A, Bar R S, Boes M, Dake B L, Bayne M, Cascieri M
Veterans Administration Hospital, Department of Internal Medicine, Iowa City, Iowa.
Endocrinology. 1990 Dec;127(6):2630-8. doi: 10.1210/endo-127-6-2630.
Conditioned medium from cultured vascular endothelial cells contains material capable of stimulating acute metabolic processes in endothelial cells. The bioactivity of the conditioned medium is not caused by the copurification of known growth factors produced by the cells, in particular platelet-derived growth factor, basic fibroblast growth factor, or insulin-like growth factor (IGF)-I/II. We now demonstrate that the bioactivity is directly due to an IGF-binding protein(s) (ECBP) and, further, that the bioactive domain of the binding protein differs from the IGF-binding domain. Binding proteins (BPs) from cultured pulmonary artery endothelial cells were purified by sequential passage over sizing, multiplication-stimulating activity affinity, and hydrophobic columns. BP fractions were separated into those with and those without biological activity. The bioactive binding protein(s) was cross-linked with disuccinimidyl suberate to IGF-I or the recombinant IGF analog [1-27,Gly4,38-70]IGF-I (Analog). The IGF-I Analog, by itself, had minimal interaction with the type I IGF receptor in cultured microvessel endothelial cells and no intrinsic bioactivity, but did bind with high affinity to ECBP. All free BP and free IGF-I/Analog were removed from the cross-linked mixture by passage over gel filtration and IGF affinity columns. The cross-linked BP-IGF-I complex did not bind to the type I receptor of cultured endothelial cells, but did stimulate glucose and alpha-aminoisobutyric acid uptake in endothelial cells (approximately 2-fold increase); the magnitude of the response was nearly equal to the effect of ECBP or IGF-I alone. The BP-Analog complex also stimulated glucose and alpha-aminoisobutyric acid uptake, with the magnitude of the response approaching the effect of ECBP alone. The BP-Analog complex also did not react with type I IGF receptors on the cultured endothelial cells. We conclude 1) IGF-BP produced by endothelial cells possess intrinsic biological activity; 2) bioactivity of the BP(s) is retained when the IGF-binding domain of the BP is occupied by IGF-I or an inactive IGF-I analog; and 3) IGF-I bound to the bioactive BP does not react with its receptor and possesses minimal, if any, bioactivity in vitro.
来自培养的血管内皮细胞的条件培养基含有能够刺激内皮细胞急性代谢过程的物质。条件培养基的生物活性并非由细胞产生的已知生长因子（特别是血小板衍生生长因子、碱性成纤维细胞生长因子或胰岛素样生长因子（IGF）-I/II）的共纯化所致。我们现在证明，这种生物活性直接归因于一种或多种IGF结合蛋白（ECBP），并且进一步证明，结合蛋白的生物活性结构域不同于IGF结合结构域。通过依次经过尺寸排阻柱、增殖刺激活性亲和柱和疏水柱，对培养的肺动脉内皮细胞中的结合蛋白（BP）进行纯化。BP组分被分离为具有生物活性和不具有生物活性的组分。将具有生物活性的结合蛋白与辛二酸二琥珀酰亚胺酯交联到IGF-I或重组IGF类似物[1-27，Gly4，38-70]IGF-I（类似物）上。IGF-I类似物本身与培养的微血管内皮细胞中的I型IGF受体相互作用极小，且无内在生物活性，但确实以高亲和力与ECBP结合。通过凝胶过滤柱和IGF亲和柱，从交联混合物中去除所有游离的BP和游离的IGF-I/类似物。交联的BP-IGF-I复合物不与培养的内皮细胞的I型受体结合，但确实能刺激内皮细胞对葡萄糖和α-氨基异丁酸的摄取（增加约2倍）；反应强度几乎与单独的ECBP或IGF-I的作用相当。BP-类似物复合物也能刺激葡萄糖和α-氨基异丁酸的摄取，反应强度接近单独的ECBP的作用。BP-类似物复合物也不与培养的内皮细胞上的I型IGF受体反应。我们得出结论：1）内皮细胞产生的IGF-BP具有内在生物活性；2）当BP的IGF结合结构域被IGF-I或无活性的IGF-I类似物占据时，BP的生物活性得以保留；3）与生物活性BP结合的IGF-I不与其受体反应，并且在体外具有极小的（如果有的话）生物活性。
