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.
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 BIAcoretrade mark 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.
Heding
A
A
Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark.
Gill
R
R
Ogawa
Y
Y
De Meyts
P
P
Shymko
R M
RM
D015374:Biosensing Techniques; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D018972:Insulin-Like Growth Factor Binding Protein 3; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II