Multiplication-stimulating activity (MSA): a somatomedin-like polypeptide from cultured rat liver cells

Multiplication-stimulating activity (MSA) has been purified to homogeneity with the use of Dowex chromatography. Sephadex gel filtration, and preparative disc gel electrophoresis. A molecular weight of 8,700 was determined for both native and reduced and alkylated MSA by chromatography on a 6% agarose column in 6 M guanidine HCl. Amino acid analysis of performic acid-oxidized MSA revealed 2--3 cysteic acid residues, and reduction and alkylation resulted in loss of biologic residues, and reduction and alkylation resulted in loss of biologic activity. These results suggested the presence on one intrachain disulfide bond, which is required for biologic activity. Specific receptors for MSA have been identified on chick embryo fibroblasts, human skin fibroblasts, a rat liver cell line, and purified rat liver plasma membranes. The closely related peptides, acid ethanol-soluble nonsuppressible insulin-like activity (NSILA-S) and somatomedin-A, competed for MSA tracer binding to these MSA receptors, whereas unrelated peptides did not compete. MSA receptors were divided into two types based on their reactivity with insulin and proinsulin. Insulin and proinsulin competed potently for MSA binding to chick embryo and human skin fibroblasts (type II) but did not compete for MSA tracer binding to rat liver cells and purified rat liver membranes (type I). In chick embryo fibroblasts, the concentrations of MSA, insulin, proinsulin, and somatomedin-A, which inhibited [125I]MSA binding by 50%, also gave half-maximal stimulation of DNA synthesis, consistent with the MSA receptor being a mediator of DNA synthesis. MSA tracer has also been shown to bind specifically to rat serum proteins. The MSA binding profile on Sephadex G-200 was shown to be growth hormone dependent. Since the serum half-life of somatomedin activity in the rat was also growth hormone dependent, these results suggest that growth hormone induces the binding protein for somatomedin and thereby governs the half-life of somatomedin. Finally, the ability of two rat liver cells lines to multiply in serum-free medium did not depend upon the level of MSA in the conditioned medium.