Liu F, Baxter R C, Hintz R L
Department of Pediatrics, Stanford University, California 94305.
J Clin Endocrinol Metab. 1992 Nov;75(5):1261-7. doi: 10.1210/jcem.75.5.1385469.
Insulin-like growth factors (IGFs) circulate in human adult serum predominantly as a high mol wt complex of 150 kilodaltons (kDa), which is made up of three subunits: alpha, the acid-labile subunit, a glycoprotein of around 85-100 kDa; beta, the acid-stable IGF-binding protein subunit, which is the 40-kDa GH-dependent glycoprotein IGF-binding protein-3 (IGFBP-3); and gamma, the 7.5 IGF-I or -II peptide subunit. During human pregnancy, all three subunits are elevated when measured by RIA. However, recent ligand blotting studies have shown that IGFBP-3 is markedly reduced during pregnancy. When pregnancy serum is acidified and neutralized to inactivate endogenous alpha-subunit, ternary complex formation is normal with exogenous radiolabeled alpha-subunit, indicating functional IGFBP-3. We have attempted to clarify the status of IGFBP-3 and the high mol wt (alpha beta gamma) complex in human term pregnancy serum by further analyses. Term pregnancy (TP) or nonpregnancy (NP) pooled sera were fractionated on a S-300 neutral column. The high mol wt (125-150 kDa) and the low mol wt (30-40 kDa) IGF-IGFBP complexes were identified by both RIA for IGFBP-3 and ligand blotting; each was pooled separately. Half of each pool was lyophilized and rechromatographed in acid to separate the IGF-I peptides from their IGFBPs. The IGFBP fractions were recovered for further studies. When the IGFBPs from the high mol wt complex were cross-linked to [125I]IGF-I or -II, bands of 48, 34, 26, and 21 kDa, which were more intense with [125I]IGF-II, were observed, and all were immunoprecipitable by anti-IGFBP-3 antibody. The smaller forms were elevated in TP serum. Affinity cross-linking analysis with [125I]alpha-subunit showed that the IGFBPs from the high mol wt, but not the low mol wt, complex can reform the ternary 150-kDa complex when cold IGF-I or IGF-II is added exogenously. A smaller 130-kDa complex was also present, and it was the predominant form in TP serum. Both bands were immunoprecipitable by anti-IGFBP-3 antibody. When purified alpha-subunit or fractions from neutral Sephacryl S-300 chromatography were cross-linked to covalent [125I]IGF-II:IGFBP-3, a specific band at 150 kDa was observed with pure alpha-subunit as well as with S-300 150- to 100-kDa serum fractions. These results suggest that 1) functional alpha-subunit is present in TP serum; 2) intact as well as smaller fragments of IGFBP-3 are present in the big complex of TP serum and are able to bind IGF and complex with alpha-subunit; and 3) IGFBP-3 in TP serum has reduced binding to [125I]IGF-I, but not to [125I]IGF-II.
胰岛素样生长因子（IGFs）在成人血清中主要以150千道尔顿（kDa）的高分子量复合物形式循环，该复合物由三个亚基组成：α，即酸不稳定亚基，一种分子量约为85 - 100 kDa的糖蛋白；β，即酸稳定的IGF结合蛋白亚基，是40 kDa的生长激素依赖性糖蛋白胰岛素样生长因子结合蛋白-3（IGFBP-3）；γ，即7.5 kDa的IGF-I或-II肽亚基。在人类妊娠期间，通过放射免疫分析（RIA）检测发现所有三个亚基的水平均升高。然而，最近的配体印迹研究表明，妊娠期间IGFBP-3显著减少。当将妊娠血清酸化并中和以灭活内源性α亚基时，外源性放射性标记的α亚基能正常形成三元复合物，表明存在功能性IGFBP-3。我们试图通过进一步分析来阐明足月妊娠血清中IGFBP-3和高分子量（αβγ）复合物的状态。将足月妊娠（TP）或非妊娠（NP）混合血清在S-300中性柱上进行分离。通过针对IGFBP-3的RIA和配体印迹法鉴定高分子量（125 - 150 kDa）和低分子量（30 - 40 kDa）的IGF-IGFBP复合物；分别收集每个复合物。每个收集物的一半进行冻干，然后在酸性条件下重新进行色谱分离，以将IGF-I肽与其IGFBPs分离。回收IGFBP部分用于进一步研究。当将高分子量复合物中的IGFBPs与[125I]IGF-I或-II进行交联时，观察到48、34、26和21 kDa的条带，用[125I]IGF-II时条带更明显，并且所有条带都能被抗IGFBP-3抗体免疫沉淀。较小的形式在TP血清中升高。用[125I]α亚基进行亲和交联分析表明，高分子量复合物中的IGFBPs，而非低分子量复合物中的IGFBPs，在外源添加冷IGF-I或IGF-II时能够重新形成150 kDa的三元复合物。还存在一种较小的130 kDa复合物，它是TP血清中的主要形式。两条带都能被抗IGFBP-3抗体免疫沉淀。当将纯化的α亚基或来自中性Sephacryl S-300色谱的部分与共价[125I]IGF-II:IGFBP-3进行交联时，用纯α亚基以及S-300 150 - 100 kDa血清部分观察到150 kDa的特异性条带。这些结果表明：1）TP血清中存在功能性α亚基；2）TP血清的大复合物中存在完整的以及较小片段的IGFBP-3，它们能够结合IGF并与α亚基形成复合物；3）TP血清中的IGFBP-3与[125I]IGF-I的结合减少，但与[125I]IGF-II的结合未减少。
