Abstract
Human amniotic fluid has been shown to contain a protein that binds insulin-like growth factor I and II (IGF-I and IGF-II). Partially purified preparations of this protein have been reported to inhibit the biologic actions of the IGFs. In these studies our laboratory has used a modified purification procedure to obtain a homogeneous preparation of this protein as determined by polyacrylamide gel electrophoresis and amino acid sequence analysis. During purification the ion exchange chromatography step resulted in two peaks of material with IGF binding activity termed peaks B and C. Each peak was purified separately to homogeneity. Both peaks were estimated to be 31,000 daltons by polyacrylamide gel electrophoresis and their amino acid compositions were nearly identical. Amino acid sequence analysis showed that both peaks had identical N-terminal sequences through the first 28 residues. Neither protein had detectable carbohydrate side chains and each had a similar affinity for radiolabeled IGF-I (1.7-2.2 x 10(10) liters/mol). In contrast, these two forms had marked differences in bioactivity. Concentrations of peak C material between 2 and 20 ng/ml inhibited IGF-I stimulation of [3H]thymidine incorporation into smooth muscle cell DNA. In contrast, when peak B (100 ng/ml) was incubated with IGF-I there was a 4.4-fold enhancement of stimulation of DNA synthesis. Additionally, pure peak B was shown to adhere to cell surfaces, whereas peak C was not adherent. The non-adherent peak C inhibited IGF-I binding to its receptor and to adherent peak B. We conclude that human amniotic fluid contains two forms of IGF binding protein that have very similar physiochemical characteristics but markedly different biologic actions. Since both have similar if not identical amino acid compositions, N-terminal sequences, and do not contain carbohydrate, we conclude that they differ in some other as yet undefined post-translational modification.
Highlights
From the Departments of Medicine and Microbiology, Universityof North Carolina School of Medicine, Chapel Hill,North Carolina 27514
Many cell types protein that binds insulin-like growth factor I and I1 and tissues secrete IGF-I [3, 4], it is uncertain whether this (IGF-I and IGF-11)
The non-adherent peak C inhibited IGF-I creased when low concentrations of unlabeled IGF-I areadded binding to its receptor and to adherent peak B.We [18].Since this form of binding protein is present in many concludethat human amniotic fluid contains two formstypes of extracellular fluids it has the potential to alter the of IGF binding protein that have very similarphysio- cellular responses to IGF-I
Summary
From the Departments of Medicine and Microbiology, Universityof North Carolina School of Medicine, Chapel Hill,North Carolina 27514. After 48 h at 4 "Cthe bound and free "'I-IGF-Iwere separated by adding a 1:250 dilution of a rabbit anti-binding proteinantibody which had been prepared using a mixture of peaks B and C binding proteins and the incubation continued for 24 h. The gel was washed sequentially with 1) 0.5% periodic acid, 2)0.5% sodium arsenite/5% acetic acid, 3) 0.1% sodium arsenite/5% acetic acid, 4) 5% acetic acid, 5) Schiffs reagent (overnight), and 6) 0.6% sodium metabisulfite/O.Ol M HCl. To further determine whether either peak B or peak C contained carbohydrate, 1.5 pg of each protein was applied to a concanavalin A-Sepharose column that hadbeen equilibrated in 0.02 Tris, pH7.5, 2 mM CaClz, and 2 mMMgClZ. After 2 h at 4 'C the media was aspirated,the monolayers were washed four times in PBS, and the cell-associated lZ5I-IGF-wI as determined as previously described[18].Nonspecific binding wasdetermined in the presence of 500 ng/ml unlabeled IGF-I andthat value was subtracted for all points.
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