Purification and Characterization of Multiplication‐Stimulating Activity

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Abstract
Multiplication-stimulating activity (MSA) refers to a family of insulin-like growth factors that have been purified from serum-free medium conditioned by a Buffalo rat liver cell line (BRL-3A). Using Dowex ion-exchange chromatography, gel chromatography on Sephadex G-75, and preparative disc acrylamide gel electrophoresis, several polypeptides with the full biological multiplication-stimulating activity were isolated. One of these polypeptides, designated MSA II-1, was used previously to study the relationship of the activity to the insulin-like growth factors (somatomedins) purified from human plasma. Polypeptide II-1 is a single chain polypeptide of MW 8700. Glycine is the COOH-terminal amino acid. Edman degradation of carboxymethylated MSA II-1 did not reveal a free NH2-terminus. A polypeptide of lower MW than MSA II-1 was also purified. This polypeptide (MSA III-2) was more potent than MSA II-1 in the rat-liver-membrane radioreceptor assay and in a competitive binding assay utilizing the rat-serum somatomedin-binding protein(s). The relationship of these various polypeptides was investigated by gel filtration in guanidine hydrochloride and by acrylamide gel electrophoresis of the reduced and native polypeptides.

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