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Endocrinology Vol. 138, No. 3 1130-1136
Copyright © 1997 by The Endocrine Society

Articles

Purification and Characterization of the Insulin-Like Growth Factor-Binding Protein-1 Phosphoform Found in Normal Plasma1

Melissa Westwood, J Martin Gibson and Anne White

Endocrine Sciences Group, Department of Medicine, University of Manchester, Manchester, M13 9PT, United Kingdom

Address all correspondence and requests for reprints to: Melissa Westwood, Endocrine Sciences Research Group, Department of Medicine, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, United Kingdom. E-mail: mwestwoo{at}fs2.scg.man.ac.uk

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-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 1010 M-1) than nonphosphorylated IGFBP-1 (2.5 ± 1.7 x 109 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 109 M-1vs. 1.8 ± 3 x 109 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 1010 M-1) also was significantly higher than the affinity of IGFBP-3 for IGF-I (5.6 ± 4.2 x 109 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.




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