Purified rat acid-labile subunit and recombinant human insulin-like growth factor (IGF)-binding protein-3 can form a...

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.