Further Characterization of Growth Hormone-Dependent Somatomedin-Binding Proteins in Rat Serum and Demonstration of Somatomedin-Binding Proteins Produced by Rat Liver Cells in Culture*

Abstract

The somatomedin-like peptide multiplication-stimulating activity (MSA) binds specifically to rat serum. The pattern of MSA binding is GH dependent. Specific binding of [125I]iodo-MSA in normal rat serum is primarily in the gamma-globulin region (peak II) on Sephadex G-200, while MSA binding in hypophysectomized (hypox) rat serum is near the albumin region (peak III). This study further characterizes the peak II and peak III somatomedin-binding proteins produced by rat liver cells in culture. [125I]Iodo-MSA binding to normal rat serum is abolished by trypsin pretreatment of rat serum, suggesting that MSA binds to protein components of serum. The only detectable somatomedin activity (measured by [3H]thymidine incorporation into chick embryo fibroblast DNA) in fractions of normal rat serum chromatographed on Sephadex G-200 coincides with peak II binding of [125I]iodo-MSA. In hypox rat serum, the majority of detectable somatomedin activity is in the peak III region. There is complete displacement of the human somatomedins [125I]iodoinsulin-like growth factor I and II and [125I]iodosomatomedin A from the rat serum-binding sites by unlabeled MSA, suggesting that the human somatomedins bind to the same sites as MSA. Treatment of normal rat serum with 1 M acetic acid dissociates somatomedin activity from its binding proteins and converts somatomedin-binding proteins from peak II to peak III. Scatchard analysis of competitive binding data using [125I]iodo-MSA yields a binding affinity that is not appreciably different for either normal or hypox rat sera. The binding capacity of normal or acid-treated normal rat serum for MSA is significantly greater than that for comparably treated hypox rat sera. Although the site of synthesis of somatomedin-binding proteins in vivo is unknown, specific somatomedin-binding proteins are synthesized by two rat liver cell lines in culture. These rat liver cell somatomedin-binding proteins have the same molecular size and the same binding affinity for MSA as the peak III somatomedin-binding protein(s) in rat serum.

Acute metabolic effects and half-lives of intravenously administered insulinlike growth factors I and II in normal and hypophysectomized rats.

“…The finding that most of the endogenous rat somatomedin is associated with the 150,000-200,000-mol wt carrier protein (33) would fit the observation that the half-life of injected IGF is prolonged in the presence of the latter protein. Both observations suggest that at least in vivo the apparent affinity of IGFs towards this protein is greater than that toward the 40,000-50,000-mol wt carrier protein.…”

Modulation of insulinlike growth factor I binding to human fibroblast monolayer cultures by insulinlike growth factor carrier proteins released to the incubation media.

“…Aliquots of media were incubated with 1251I-MSA III-2 and the indicated concentrations of unlabeled peptides in 0.4 ml of PBS containing 2 mg/ml fatty acid-free bovine serum albumin (Sigma Chemical Co.) overnight at 4°C. Tracer MSA not complexed to binding protein was removed by adsorption with activated charcoal as previously described (19,25). Complexes of 125I-MSA III-2 with carrier protein remained in the charcoal supernate and were quantitated.…”

Developmental Pattern of a Serum Binding Protein for Multiplication Stimulating Activity in the Rat

“…In adult rat serum, IGF activity is found associated with larger proteins (12). Most of the IGF activity is associated with a gamma globulin-size protein (peak II); the remainder of the IGF activity is complexed to a protein slightly smaller than albumin (peak III).…”