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.