title: Purification and characterization of the insulin-like growth factor-binding protein-1 phosphoform found in normal plasma.
abstract: Our previous work has shown that, in the normal circulation, insulin-like growth factor-binding protein-1 (IGFBP-1) is present as a single highly phosphorylated species. In this study, we have purified this previously uncharacterized isoform of IGFBP-1 to determine its ligand-binding affinity and the potential significance of highly phosphorylated IGFBP-I. Immunoaffinity chromatography was used to isolate IGFBP-1 from normal human plasma and from human hepatoma (Hep G2) cell medium as an alternative source of the IGFBP-1 phosphoform in the circulation. The affinity of this highly phosphorylated IGFBP-1 was compared with that of nonphosphorylated IGFBP-1 and recombinant human (rh) IGFBP-3 by equilibrium binding to IGF-II and IGF-II. Anion exchange (IEX) HPLC, nondenaturing electrophoresis, alkaline phosphatase treatment, and ligand-binding studies indicated that the highly phosphorylated IGFBP-1 from HepG2 cells was comparable with IGFBP-1 from plasma. In binding to IGF-I, the plasma phosphoform of IGFBP-1 was found to have a higher affinity (2.3 +/- 1.1 x 10(10) M-1) than nonphosphorylated IGFBP-1 (2.5 +/- 1.7 x 10(9) M-1, P < 0.002). However, when binding to IGF-II, phosphorylation had no affect on the affinity of IGFBP-1 (3.6 +/- 2 x 10(9) M-1 vs. 1.8 +/- 3 x 10(9) M-1, P not significant). Therefore, in the circulation, IGF-I has a considerably higher affinity than IGF-II for IGFBP-1 (P < 0.02). The affinity of phosphorylated IGFBP-1 from plasma (2.3 +/- 1.1 x 10(10) M-1) also was significantly higher than the affinity of IGFBP-3 for IGF-I (5.6 +/- 4.2 x 10(9) M-1, P < 0.005). These data suggest that the highly phosphorylated IGFBP-1 in the normal circulation will preferentially bind IGF-I rather than IGF-II, whereas in pregnancy, the affinity of IGFBP-1 for IGF-I will be reduced because of the appearance of non- and lesser-phosphorylated forms. This lends support to the theory that changes in IGFBP-1 phosphorylation may influence the modulatory effects of IGFBP-1 on IGF bioavailability.
authors.affiliation: Department of Medicine, University of Manchester, United Kingdom. mwestwoo@fs2.scg.man.ac.uk
authors.forename: M
authors.initials: M
authors.lastname: Westwood
authors.forename: J M
authors.initials: JM
authors.lastname: Gibson
authors.forename: A
authors.initials: A
authors.lastname: White
mesh_terms: D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000737:chemistry; D005260:Female; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000097:blood* / Q000302:isolation & purification* / Q000378:metabolism; D007536:Isomerism; D008024:Ligands; D010766:Phosphorylation; D011994:Recombinant Proteins; D012016:Reference Values; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D003470:Culture Media; D018970:Insulin-Like Growth Factor Binding Protein 1; D008024:Ligands; D011994:Recombinant Proteins