Barenton B, Patel B A, Khan M N, Guyda H J, Posner B I
Institut National de la Recherche Agronomique, Station de Physiologie Animale, Montpellier, France.
Endocrinology. 1988 Jun;122(6):2499-507. doi: 10.1210/endo-122-6-2499.
We have characterized binding proteins for insulin-like growth factors (IGFs) in hepatic subcellular fractions and in the washed supernatants of these fractions in normal and hypophysectomized (hypox) rats. In the course of assessing IGF-II-binding sites on rat liver microsomes, we observed that [125I] IGF-II binding to the microsomal membranes of hypox rats was much lower than that in normal rats. Paradoxically, binding increased in hypox animals at low concentrations (0.5-5 ng/ml) of unlabeled IGF-II. After resuspension and centrifugation (washing) of the microsomes, no difference was found in [125I]IGF-II binding to hypox vs. normal microsomes. However, the binding of [125I]IGF-II to the washing supernatant (SN) from hypox rat microsomes was greater than binding to that from normal animals. Binding to SN was inhibited by unlabeled IGF-II in a dose-dependent manner. Scatchard analyses indicated that the affinity constant for binding by hypox SN was higher than that of normal SN and the microsomal fractions of both hypox and normal rats. After further subfractionation of the liver, no binding activity was found in SN from plasmalemma, whereas it was about 20% of input counts per min of [125I]IGF-II in SN from combined Golgi-endosome fractions of hypox rat liver. We next compared IGF-binding moieties in microsomal SN with those in plasma using cross-linking of [125I]IGF-II followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal rat plasma, we observed the presence of 42K, 39K, 31K, and 27K binding complexes. In hypox rat plasma only a 42-39K doublet was found. In the SN of normal rat microsomes, the predominant complex migrated at 39K and was distinguishable only after acidification. In the SN of hypox rat microsomes, the 42K complex was predominant, with a minor 34K complex. These studies have identified IGF-binding moieties in hepatic tissues, particularly in hepatic vesicular elements, which interfere in the binding of IGF-II to membrane receptors. Their presence in these receptor-rich elements may influence IGF binding to intracellular receptors and, hence, the biological activity of the peptide.
我们已经对正常和垂体切除（hypox）大鼠肝脏亚细胞组分及其洗涤后的上清液中的胰岛素样生长因子（IGF）结合蛋白进行了表征。在评估大鼠肝脏微粒体上的IGF-II结合位点的过程中，我们观察到，[125I]IGF-II与hypox大鼠微粒体膜的结合远低于正常大鼠。矛盾的是，在未标记的IGF-II浓度较低（0.5 - 5 ng/ml）时，hypox动物的结合增加。微粒体重悬并离心（洗涤）后，[125I]IGF-II与hypox和正常微粒体的结合未发现差异。然而，[125I]IGF-II与hypox大鼠微粒体洗涤上清液（SN）的结合大于与正常动物洗涤上清液的结合。未标记的IGF-II以剂量依赖性方式抑制与SN的结合。Scatchard分析表明，hypox SN结合的亲和常数高于正常SN以及hypox和正常大鼠的微粒体组分。肝脏进一步亚分级后，质膜SN中未发现结合活性，而在hypox大鼠肝脏高尔基体 - 内体联合组分的SN中，其结合活性约为[125I]IGF-II每分钟输入计数的20%。接下来，我们使用[125I]IGF-II交联后进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，比较了微粒体SN和血浆中的IGF结合部分。在正常大鼠血浆中，我们观察到存在42K、39K、31K和27K结合复合物。在hypox大鼠血浆中仅发现42 - 39K双峰。在正常大鼠微粒体的SN中，主要复合物在39K处迁移，且仅在酸化后才可区分。在hypox大鼠微粒体的SN中，42K复合物为主，还有少量34K复合物。这些研究已经确定了肝脏组织中，特别是肝泡状成分中的IGF结合部分，它们会干扰IGF-II与膜受体的结合。它们在这些富含受体的成分中的存在可能会影响IGF与细胞内受体的结合，从而影响该肽的生物活性。
