Structure, specificity, and regulation of the insulin-like growth factor-binding proteins in adult rat serum.

Insulin-like growth factor-I (IGF-I), the principal IGF in adult rat serum, occurs complexed to specific binding proteins. After fractionation of serum on Sephadex G-200 at neutral pH, 62% of the immunoreactive IGF-I is recovered in the 150K region, 38% in the 40K region, and none is present as free 7.5K IGF-I. Adult rat serum also contains unoccupied binding sites for IGFs that also are predominantly (77%) located in the 150K region and have preferential binding affinity for IGF-II. IGF-binding protein components in the 150K and 40K regions were evaluated by affinity cross-linking to 125I-labeled IGFs and by ligand blotting (i.e. incubation of nitrocellulose blots of sodium dodecyl sulfate (SDS)-gels with [125I]IGFs). Affinity cross-linking of the 150K region revealed a major 43K binding protein complex and several minor covalent complexes of 97-210K that are formed during the cross-linking reaction. The 40K region of the gel filtration column contains a predominant 32K complex and smaller amounts of the 43K complex. Ligand blotting of the 150K region identifies a predominant cluster of binding components of about 40K and a smaller 29K protein. The apparent molecular masses of the 40K and 29K proteins are decreased by incubation with N-glycanase, indicating that they contain N-linked oligosaccharides. These glycoprotein components, designated gp40 and gp29, presumably combine with an acid-labile nonbinding subunit of about 100K to generate the 150K complex. The gp40 cluster represents glycosylation variants of a 34K protein; gp29 has been shown to correspond to an amino-terminal fragment of gp40. Ligand blotting of the 40K region indicates that it contains smaller amounts of gp40 and gp29, possibly representing free subunits not combined with the nonbinding subunit, as well as two proteins of apparent molecular mass 24K and 30K (p24 and p30) that are not glycosylated. Although p30 is similar in size to the binding protein from BRL-3A cells (BP-3A) that is present in fetal rat serum, immunoprecipitation and immunoblotting of whole and fractionated adult serum with an antiserum to BP-3A indicate that p30 in adult rat serum is an antigenically distinct protein. Serum levels of gp40 and gp29 are decreased by hypophysectomy and are restored by GH treatment; p24 and p30 show similar but smaller changes.