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Purification and characterization of the insulin-like growth factor-binding protein-1 phosphoform found in normal plasma

Westwood, M; Gibson, J M; White, A

Endocrinology. 1997;138(3):1130-1136.

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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 lGFBP-1 to determine its ligand-binding affinity and the potential significance of highly phosphorylated IGFBP-1. 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-I 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 has 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 than changes in IGFBP-1 phosphorylation may influence the modulatory effects of IGFBP-1 on IGF bioavailability.

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Created by:
Gibson, Martin
15th January, 2012, 15:04:08
Last modified by:
Gibson, Martin
Last modified:
1st February, 2013, 19:46:47