Binding properties of insulin-like growth factor binding protein-3 (IGFBP-3), IGFBP-3 N- and C-terminal fragments, and structurally related proteins mac25 and connective tissue growth factor measured using a biosensor.
We measured the binding of IGF-I and IGF-II to recombinant human N-terminal [residues 1-97; recombinant human IGF-binding protein-3(1-97) (rhIGFBP-3(1-97))] and C-terminal (residues 98-264; rhIGFBP-3(98-264)) IGFBP-3 fragments and compared it with IGF binding to intact IGFBP-3 using biosensor analysis. Experiments were carried out in different configurations, either with binding protein or fragment immobilized or with IGF immobilized. These experiments showed that IGF-I and IGF-II bind to IGFBP-3 with affinities of 4-5 x 10(9) M(-1) and similar binding kinetics. The affinities of both rhIGFBP-3(1-97) and rhIGFBP-3(98-264) for IGF proteins were approximately 3 orders of magnitude less than that of full-length IGFBP-3. These results further support the concept that high affinity binding of IGF to IGF-binding proteins results from a two-site interaction of IGF with both the N- and C-terminal regions of the binding protein. Binding of insulin to IGFBP-3 and its N- and C-terminal fragments and of IGF-I and IGF-II to the structurally related proteins mac25 and connective tissue growth factor was also investigated. Weak insulin binding to full-length IGFBP-3 could be demonstrated in a few experiments, but we found that binding of IGF-I, IGF-II, and insulin to mac25 or connective tissue growth factor was below the detection limit of the biosensor instrument.
Vorwerk
Peter
P
Department of Pediatric Oncology, Otto von Guericke University, Emanuel Larisch Weg 17-19, D-39112 Magdeburg, Germany. peter.vorwerk@medizin.uni-magdeburg.de
Hohmann
Bianka
B
Oh
Youngman
Y
Rosenfeld
Ron G
RG
Shymko
Ronald M
RM
D015374:Biosensing Techniques; D001710:Biotin / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D055513:Connective Tissue Growth Factor; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008838:Microcomputers; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011786:Quality Control; D011994:Recombinant Proteins / Q000494:pharmacology; D020349:Surface Plasmon Resonance
C526317:CCN2 protein, human; D002352:Carrier Proteins; D006133:Growth Substances; D017874:Immediate-Early Proteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C102529:insulin-like growth factor binding protein-related protein 1; D055513:Connective Tissue Growth Factor; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D001710:Biotin