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.

Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [3H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [3H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10-fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [3H] thymiding incorporation into DNA. MSA causes a 2--10-fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [3H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [3H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.