Characterization of a Protein in Mid-Term Human Amniotic Fluid which Reacts in the Somatomedin-C Radioreceptor Assay*

Abstract

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- Iike peptides. Separation of the somatomedin reactive material (Sm RM) from albumin was achieved by gel filtration through Ultrogel AcA44 in 0.1M 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 thymidine uptake in human fibroblasts but was inactive in stimulating sulfate uptake in hypox costal cartilage. In human fibroblast cultures Sm RM inhibited the stimulation of thymidine uptake induced by human serum and by purified 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.