Abstract
An insulin-like growth factor II variant (IGF-II variant) was purified from Cohn fraction IV1 of human plasma by ion exchange, gel filtration, and reversed-phase high pressure liquid chromatography. The amino-terminal sequence of the first 35 amino acid residues showed a replacement of Ser-29 of IGF-II with the tetrapeptide Arg-Leu-Pro-Gly of IGF-II variant. Peptides isolated and sequenced after digestion with endoproteinase Asp-N and endoproteinase Glu-C disclosed no differences with the sequence predicted from an IGF-II variant cDNA clone isolated by Jansen, M., van Shaik, F. M. A., van Tol, H., Van den Brande, J. L., and Sussenbach, J. S. (1985) FEBS Lett., 179, 243-246. The molecular ion of intact IGF-II variant was 7809.4 mass units, as measured by plasma desorption mass spectrometry. This is in close agreement with the molecular ion of 7812.8 mass units calculated from the determined sequence and indicates the entire amino acid sequence had been accounted for. Binding of IGF-II variant to purified insulin-like growth factor I (IGF-I) receptors demonstrated a 2-3-fold lower affinity for this receptor compared with IGF-I or IGF-II. The dissociation constants for IGF-I, IGF-II, and IGF-II variant are 0.23, 0.38, and 0.80 nM, respectively. In a growth assay, the concentration of IGF-II and IGF-II variant required to stimulate the half-maximal growth of MCF-7 cells was 4 and 13 nM, respectively. Finally, the amount of IGF-II variant that can be purified by this method constitutes approximately 25% of the total IGF-II isolated from Cohn fraction IV1 of human plasma.
Highlights
$Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11722, and the fhmbardi Cancer ResearchCenter, Georgetown University Medical Center, Washington, D.C. 20007
Insulin-like growth factors were adsorbed from an extract of Cohn fraction IV, with SP Sephadex C-25 using a modification of the procedure previously described [28]
IGF-I1 was separated from IGF-binding proteins on Sephadex G-50 in1.0M acetic acid
Summary
Assay of ZGF-ZZ-IGF-I1 receptor binding activity in column fractions was determined throughout using a soluble rat placental membrane-derived IGF-I1 radioreceptor assay (RRA). Fractions were collected every 13min, and those containing IGF-I1 were pooled and pumped onto a 0.7 X 25-cmAquapore butyl reversed-phase column equilibrated in 95% solvent A (0.1%trifluoroacetic acid in water), 5% solvent B (0.075% trifluoroacetic acid in acetonitrile). The resulting peptides were separated on an Applied Biosystems model 130A microbore HPLC system with a 2.1 X 210-mm AppliedBiosystems PTC-CIScolumn equilibrated in 98% solvent A (0.1% trifluoroacetic acid in water), 2% solvent B (70% acetonitrile, .09% trifluoroacetic acid) using a linear gradient from 2% B to 80% Bover 55 min. Amino Acid Analysis and Sequence Determination-Purified proteins were hydrolyzed with argon-purged, constant boiling 6 N HCl containing 1%(v/v) phenol at 110 "C for 18 h.Amino acids were derivatized with phenylisothiocyanate and separated with an Applied Biosystems model130A PTC analyzer. Following 96 h of growth, the cells were removedfrom the cluster dishes with phosphate-buffered saline containing 0.02% EDTA and thenumber of cells/well wasdetermined using a hemocytometer
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