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Amniotic fluid contains materials other than insulin which react in a somatomedin C radioreceptor assay using human placental membranes. The material in mid-gestational amniotic fluid which reacted with the somatomedin C radioreceptor assay eluted slightly after albumin from a Sephadex G-150 column equilibrated with 0.1M NH4HCO3. Neither boiling nor treatment of this fraction with 1% formic acid yielded small molecular weight somatomedin-like peptides. Separation of the somatomedin reactive material (Sm RM) from albumin was achieved by gel filtration through Ultrogel AcA44 in 3.1M NH4HCO3. The active product had an apparent molecular weight of 33,000 to 35,000 Daltons; its isoelectric point determined by focusing was between 4.1 and 5.1. The purified amniotic fluid protein displaced somatomedin C in the somatomedin C radioreceptor assay but did not compete with insulin in the insulin radioreceptor assay. Sm RM produced only a slight stimulation of thymidine uptake in human fibroblasts but was inactive in stimulating sulfate uptake in hypox rat costal cartilage. In human fibroblast cultures Sm RM inhibited the stimulation of thymidine uptake induced by human serum and by purified rat somatomedin. When Sm RM was added to the 125I somatomedin C, some of the radioactivity eluted from gel filtration at pH 8.6 was converted to a molecular weight complex of about 43,000. We conclude that the material which we have isolated from mid-gestational amniotic fluid is a protein which may bind somatomedin and make it unavailable to the somatomedin receptor of human placenta and human fibroblasts.

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Rat basic somatomedin (Sm) was prepared with an improved purification scheme from the pooled sera of Wistar Furth rats previously inoculated with cells from the pituitary tumor MStT/W15. Amicon hollow fiber diafiltration facilitated the processing of large batches of serum, eliminating the necessity of running smaller portions of material on gel filtration columns to achieve the same qualitative separation. The yield from this technique was excellent. Basic rat Sm was separated from a C3ades Arg component of complement by a narrow pH range isoelectric focusing step. Subsequent chromatography on carboxymethyl-cellulose and Sephadex resins was very efficient, providing a 100-fold purification, with a recovery of Sm activity of approximately 65%. The final product was judged to be pure by a variety of criteria. Structural analysis of this material has demonstrated that the amino-terminal sequences of rat basic Sm and human insulin-like growth factor I are strikingly similar, confirming the homology proposed earlier on the basis of receptor cross-reactivity.

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