Lee C Y, Rechler M M
Growth and Development Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Endocrinology. 1995 Nov;136(11):4982-9. doi: 10.1210/endo.136.11.7588232.
Insulin-like growth factors (IGFs) circulate in plasma mainly as part of a 150-kilodalton (kDa) complex with 40- to 45-kDa IGF-binding protein-3 (IGFBP-3) and an approximately 85-kDa acid-labile subunit (ALS) that does not bind IGFs directly. This complex sequesters IGFs in plasma, thereby providing a potential reservoir of the growth factors for tissues while constraining their potential hypoglycemic effects. Although it has been thought that IGFBP-3 must first bind IGF-I or IGF-II before it can complex with ALS to form the 150-kDa complex, we recently showed that unoccupied 150-kDa binary complexes of IGFBP-3 and ALS are abundant in adult rat serum. We now demonstrate that IGFBP-3 and rat (r)ALS can form 150-kDa complexes in the absence of IGFs. ALS was purified from rat serum by anion exchange chromatography and affinity chromatography on an IGF-I-Sepharose column to which human (h) IGFBP-3 had been noncovalently bound. The preparation contained less than 0.1 ng IGF-I/microgram(s) purified ALS. In the absence of IGF, radiolabeled (r)ALS and recombinant hIGFBP-3 formed complexes that could be immunoprecipitated by antiserum to hIGFBP-3; these complexes were identified by direct quantitation of the precipitated radioactivity or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE). Inclusion of IGF-I in the incubation increased complex formation. Complex formation also was demonstrated by incubation of unlabeled rALS with hIGFBP-3, followed by affinity cross-linking. Complexes were fractionated by SDS-PAGE, blotted, and shown to contain unoccupied IGF-binding sites by their ability to bind radioiodinated IGF-II. In addition, when rALS was subjected to SDS-PAGE and blotted, radioiodinated recombinant hIGFBP-3 bound to the 85-kDa protein. In this experiment IGFBP-3 binding was slightly increased by coincubation with IGF-I. Thus, purified rALS can form a 150-kDa complex with hIGFBP-3 in the absence of IGF in vitro. The efficiency of complex formation was increased to variable extents by coincubation with IGF-I depending on the assay method.
胰岛素样生长因子（IGFs）在血浆中主要以150千道尔顿（kDa）复合物的形式循环，该复合物与40至45 kDa的胰岛素样生长因子结合蛋白-3（IGFBP-3）以及一个约85 kDa的酸不稳定亚基（ALS）结合，后者并不直接结合IGFs。这种复合物将IGFs隔离在血浆中，从而为组织提供了生长因子的潜在储存库，同时限制了它们潜在的降血糖作用。尽管一直认为IGFBP-3必须先结合IGF-I或IGF-II，才能与ALS形成150 kDa的复合物，但我们最近发现，在成年大鼠血清中，IGFBP-3和ALS的未占据150 kDa二元复合物很丰富。我们现在证明，在没有IGFs的情况下，IGFBP-3和大鼠（r）ALS可以形成150 kDa的复合物。通过阴离子交换色谱和在已非共价结合人（h）IGFBP-3的IGF-I-琼脂糖柱上进行亲和色谱，从大鼠血清中纯化ALS。该制剂中每微克纯化的ALS中IGF-I含量低于0.1 ng。在没有IGF的情况下，放射性标记的（r）ALS和重组hIGFBP-3形成的复合物可以被抗hIGFBP-3血清免疫沉淀；这些复合物通过对沉淀放射性的直接定量或通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDA-PAGE）进行鉴定。在孵育中加入IGF-I会增加复合物的形成。通过将未标记的rALS与hIGFBP-3孵育，然后进行亲和交联，也证明了复合物的形成。通过SDS-PAGE对复合物进行分级分离、印迹，并通过其结合放射性碘化IGF-II的能力表明其含有未占据的IGF结合位点。此外，当rALS进行SDS-PAGE并印迹时，放射性碘化的重组hIGFBP-3与85 kDa的蛋白质结合。在该实验中，与IGF-I共同孵育会使IGFBP-3的结合略有增加。因此，在体外没有IGF的情况下，纯化的rALS可以与hIGFBP-3形成150 kDa的复合物。根据测定方法的不同，与IGF-I共同孵育会使复合物形成效率在不同程度上提高。
