The insulin-like growth factor (IGF) binding site of bovine insulin-like growth factor binding protein-2 (bIGFBP-2) probed by iodination.
The insulin-like growth factor (IGF) binding site of bovine insulin-like growth factor binding protein 2 (bIGFBP-2) has been probed by chemical iodination. Tyrosyl residues of bIGFBP-2 were reacted by chloramine T-mediated iodination. The modification patterns of free bIGFBP-2 and bIGFBP-2 associated with insulin-like growth factor II (IGF-II) were compared by tryptic mapping using electrospray mass spectrometry and N-terminal sequencing. The presence of bound IGF-II resulted in protection of tyrosine at position 60 from iodination measured by the relative loss of tyrosine specific fluorescence and the incorporation of the radioisotope 125I. In addition, the pattern of iodine incorporation of bIGFBP-2 was not different whether IGF-I or IGF-II was the protective ligand. bIGFBP-2, when iodinated alone sustained a 8-fold loss of binding affinity for IGF-I and a 4-fold loss in binding affinity for IGF-II. In contrast, bIGFBP-2 iodinated while complexed with either IGF-I or IGF-II retained the same binding affinity for IGF-I or IGF-II as non-iodinated bIGFBP-2. We conclude that tyrosine 60 lies either in a region of bIGFBP-2 which directly interacts with both IGF-I and IGF-II or lies in a region of bIGFBP-2 which undergoes a conformational change that is important for IGF binding. Furthermore, iodination of tyrosine residues at positions 71, 98, 213, 226, and 269 has no detectable impact on binding of bIGFBP-2 to the IGFs.
Hobba
G D
GD
Cooperative Research Centre for Tissue Growth and Repair, P. O. Box 10065, Gouger Street, Adelaide, South Australia 5005, Australia.
Forbes
B E
BE
Parkinson
E J
EJ
Francis
G L
GL
Wallace
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000737:chemistry*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007455:Iodine / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D013050:Spectrometry, Fluorescence; D014357:Trypsin; D014443:Tyrosine / Q000737:chemistry
D018971:Insulin-Like Growth Factor Binding Protein 2; D014443:Tyrosine; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D007455:Iodine; D014357:Trypsin