Characterization of insulin-like growth factor-binding protein in ovine amniotic fluid.
We have characterized an insulin-like growth factor (IGF)-binding protein present in ovine amniotic fluid. Using an activated charcoal-binding assay, whole amniotic fluid specifically bound approximately 20-30% of 125I-labelled human (h) IGF-II added, while the binding of 125I-labelled hIGF-I was minimal. Radioimmunoassay for IGF-I or -II in ovine biological fluids showed that values in amniotic fluid were 9- to 13-fold less than in fetal plasma, while gel filtration of amniotic fluid on Sephadex G-50 eluted with 1 mol acetic acid/l revealed no additional binding activity which had been complexed to IGFs at neutral pH. Together, these observations suggest that the binding activity in amniotic fluid is largely unsaturated. Competition studies for the displacement of 125I-labelled IGF-II binding to amniotic fluid by increasing amounts of unlabelled IGF-I or -II, using the charcoal assay, showed that IGF-II was 30-fold more potent than IGF-I. Scatchard analysis revealed a single class of binding site for IGF-II, with a binding affinity of 0.68 +/- 0.18 litres/nmol (mean +/- S.D., n = 3). Ligand blot analysis of amniotic fluid by separation on 8% SDS-PAGE, transfer to nitrocellulose membranes, incubation with 125I-labelled IGF-II and autoradiography revealed a single band of IGF-binding protein with approximate molecular size of 38 kDa. Additional IGF-binding species of 20, 28, 48 and greater than 180 kDa were present in ovine fetal plasma. Separation of amniotic fluid on Concanavalin A-Sepharose revealed that it had little carbohydrate content. These results show that ovine amniotic fluid contains an unsaturated, non-glycosylated IGF-binding protein with high affinity for IGF-II.(ABSTRACT TRUNCATED AT 250 WORDS)
Lawson Research Institute, St Joseph's Health Centre, London, Ontario, Canada.
J F
JF
Wang
L J
LJ
Fraher
D J
DJ
Hill
D000653:Amniotic Fluid / Q000737:chemistry*; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D005591:Chemical Fractionation / Q000379:methods; D002845:Chromatography / Q000379:methods; D005260:Female; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000032:analysis; D007335:Insulin-Like Growth Factor II / Q000032:analysis; D011247:Pregnancy; D011863:Radioimmunoassay; D011869:Radioligand Assay / Q000379:methods; D012756:Sheep / Q000378:metabolism*; D013002:Somatomedins / Q000032:analysis*
D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II