Biosensor measurement of the binding of insulin-like growth factors I and II and their analogues to the insulin-like growth factor-binding protein-3

Anders Heding, Raj Gill, Yasushi Ogawa, Pierre De Meyts, Ronald M. Shymko*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF. Affinities (Kd values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available. In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcoreTM instrument (Pharmacia Biosensor AB). IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium Kd of 0.23 nM. IGF-II had similar kinetics with slightly higher affinity. Analogues with mutations in the first 3 amino acids of the B-region (des(1-3) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I. Replacement of residues 28-37 by Gly-Gly-Gly-Gly or deletion of residues 29-41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor. Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate. Insulin had approximately 1000 times lower affinity than IGF-I.

Original languageEnglish
Pages (from-to)13948-52
Number of pages5
JournalThe Journal of Biological Chemistry
Volume271
Issue number24
DOIs
Publication statusPublished - 14 Jun 1996

Keywords

  • Biosensing Techniques
  • Cloning, Molecular
  • Escherichia coli
  • Humans
  • Insulin-Like Growth Factor Binding Protein 3/metabolism
  • Insulin-Like Growth Factor I/metabolism
  • Insulin-Like Growth Factor II/metabolism
  • Kinetics
  • Mathematics
  • Models, Theoretical
  • Protein Binding
  • Recombinant Proteins/metabolism
  • Substrate Specificity

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