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*

The somatomedin-like peptide multiplication-stimulating activity (MSA) binds specifically to rat serum. The pattern of MSA binding is GH dependent. Specific binding of 125Iiodo-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. 125IIodo-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 3Hthymidine incorporation into chick embryo fibroblast DNA) in fractions of normal rat serum chromatographed on Sephadex G-200 coincides with peak II binding of 125Iiodo-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 125Iiodoinsulin-like growth factor I and II and 125Iiodosomatomedin 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 125Iiodo-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.