Abstract
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.
Highlights
The insulin-like growth factors (IGF-I and insulin-like growth factor II (IGF-II))1 are polypeptide mitogens which play diverse roles in development and metabolism across a wide range of vertebrate species
Optimization of Iodination Conditions—When the concentration of NaI was held at 40-fold excess over bIGFBP-2, an increase in the molar ratio of chloramine-T/NaI in the iodination reaction resulted in an increase in the amount of iodine which became incorporated into bIGFBP-2 (Fig. 2a)
These results suggest that the number of reactive tyrosine residues of bIGFBP-2 was reduced when IGF-II was bound
Summary
Materials—Recombinant bIGFBP-2 was transiently expressed in the COS-1 (ATCC: CRL 1650) monkey kidney cell line and purified from medium conditioned by the transfected cells by a method adapted from Szabo et al [24]. Following the addition of 40 nmol of chloramine-T in 10 l of water (corresponding to a chloramine-T/NaI molar ratio of 0.75) to each tube, the iodination reactions proceeded as described above. All of the reaction mixtures were acidified by the addition of 100 l of 4 M acetic acid and the bIGFBP-2 species were purified by HPLC gel filtration as described above and the bIGFBP-2 peaks were lyophilized (Speed-Vac, Savant, Farmingdale, NY). The digested bIGFBP-2 peptides were separated by reverse-phase HPLC (using the same cartridge as described above) at 40 °C with a linear gradient of acetonitrile from 0 to 50% (v/v) in 0.1% (v/v) trifluoroacetic acid over 50 min at 0.5 ml/min. The iodinated bIGFBP-2 species were purified by HPLC gel filtration as described above and the incorporation of iodine was confirmed by the loss of tyrosine-specific fluorescence during the chromatography. The relative affinities of the respective iodinated bIGFBP-2 species for both 125I-labeled IGF-I and IGF-II were determined by charcoal binding assay, as described previously [24]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
