Endocrinology, Vol 136, 4982-4989, Copyright © 1995 by Endocrine Society

ARTICLES

Purified rat acid-labile subunit and recombinant human insulin-like growth factor (IGF)-binding protein-3 can form a 150-kilodalton binary complex in vitro in the absence of IGFs

CY Lee and MM Rechler
Growth and Development Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

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