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• W2021277909 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 BIAcore™ 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. 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 BIAcore™ 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. INTRODUCTIONInsulin-like growth factor-I and -II (IGF-I and IGF-II) 1The abbreviations used are: IGFinsulin-like growth factorIGFBPIGF-binding proteinNHSN-hydroxysuccinimideECDN-ethyl-N′-(3-diethylaminopropyl)carbodiimide. are small proteins that stimulate a variety of growth-promoting and metabolic effects via an interaction with the IGF-I receptor (1Lowe W.L. LeRoith D. Insulin-like Growth Factors: Molecular and Cellular Aspects. CRC Press Inc., Boca Raton, FL1991: 49Google Scholar). The peptides consist of four regions: A- and B-regions, which are homologous to the A and B chains of insulin; a C-region, which is analogous to but unrelated to the C peptide of insulin that connects the A- and B-regions; and a short carboxyl-terminal D-region, with no counterpart in insulin (2LeRoith D. Clemmons D. Nissley P. Rechler M.M. Ann. Intern. Med. 1992; 116: 854-862Google Scholar, 3Sussenbach J.S. Steenbergh P.H. Holthuizen P. Growth Regul. 1992; 2: 1-9Google Scholar).IGF-I and -II form complexes with six different IGF-binding proteins (IGFBPs) in the circulation and in the extracellular environment (4Martin J.L. Baxter R.C. Curr. Opin. Endocrinol. Diabetes. 1994; 1: 16-21Google Scholar). In the circulation the majority of IGFs are bound in a 140-kDa complex consisting of IGFBP-3 of 40–50 kDa, an approximately 85-kDa so-called acid-labile subunit, and IGF-I or IGF-II (5Baxter R.C. Martin J.L. Beniac V.A. J. Biol. Chem. 1989; 264: 11843-11848Google Scholar, 6Kaufmann U. Zapf J. Torretti B. Froesch E.R. J. Clin. Endocrinol. & Metab. 1977; 44: 160-166Google Scholar, 7Furlanetto R.W. J. Clin. Endocrinol. & Metab. 1980; 51: 12-19Google Scholar, 8White R.M. Nissley S.P. Moses A.C. Rechler M.M. Johnsonbaugh R.E. J. Clin. Endocrinol. & Metab. 1981; 53: 49-57Google Scholar, 9Hintz R.L. Liu F. Rosenfeld R.G. Kemp S.F. J. Clin. Endocrinol. & Metab. 1981; 53: 100-104Google Scholar, 10Baxter R.C. Martin J.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6898-6902Google Scholar). The IGFs bind to IGFBP-3 with high affinity, and the acid-labile subunit is then capable of binding to the formed complex with a somewhat lower affinity (11Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Google Scholar). The major biological function of IGFBP-3 is believed to be to extend the circulating half-lives of IGFs, since IGFs bound to the 140-kDa complex are cleared from the circulation much slower than free IGFs (12Guler H. Zapf J. Schmid C. Froesch E.R. Acta Endocrinol. 1989; 121: 753-758Google Scholar).Wild type IGFBP-3 is heavily glycosylated, which results in an apparent molecular mass of 40–50 kDa on denaturing SDS-polyacrylamide gel electrophoresis, while the non-glycosylated molecule has a molecular mass of approximately 29 kDa. The non-glycosylated protein's binding characteristics are reported to be identical to those of the wild type molecule (13Tressel T.J. Tatsuno G.P. Spratt K. Sommer A. Biochem. Biophys. Res. Commun. 1991; 178: 625-633Google Scholar).Attempts have been made to identify the regions of IGF-I involved in the high affinity binding to IGFBP-3 by constructing insulin/IGF-I hybrids and by using site-directed mutagenesis of the IGF-I gene. From the studies involving insulin/IGF-I hybrids, it was established that the B-region but not the D-region of IGF-I is important for binding to IGFBP-3 (14Joshi S. Burke G.T. Katsoyannis P.G. Biochemistry. 1985; 24: 4208-4214Google Scholar, 15Joshi S. Ogawa H. Burke G.T. Tseng L.Y.-H. Rechler M.M. Katsoyannis P.G. Biochem. Biophys. Res. Commun. 1985; 133: 423-429Google Scholar, 16De Vroede M.A. Rechler M.M. Nissley S.P. Joshi S. Burke G.T. Katsoyannis P.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3010-3014Google Scholar). From the studies using site-directed mutagenesis, it was found that the C-region is of less importance than the B-region for high affinity binding to IGFBP-3 (17Bayne M.L. Applebaum J. Underwood D. Chicchi G.G. Green B.G. Hayes N.S. Cascieri M.A. J. Biol. Chem. 1989; 264: 11004-11008Google Scholar), and that residues 3 and 4, the region between residues 8 and 18, and residues 49–51 appear to be most important for IGFBP-3 binding (11Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Google Scholar). In this study we have investigated the detailed association and dissociation binding kinetics between IGFBP-3 and IGF-I, IGF-II, insulin, and seven synthetic IGF-I analogues with mutations in the B- or C-regions and with normal and swapped disulfide bridges. In order to generate detailed kinetics, we have employed the BIAcore™ instrument developed by Pharmacia, and analyzed the data by using computer fitting programs developed at the Hagedorn Research Institute.The BIAcore™ technology makes it possible to visualize macromolecular interactions directly and in “real time” (18Fägerstam L.G. Frostell-Karlsson A. Karlsson R. Persson B. Ronnberg I. J. Chromatogr. 1992; 597: 397-410Google Scholar). The BIAcore is a biosensor-based instrument that uses surface plasmon resonance as the detection principle. One molecule of the interaction to be studied is immobilized covalently to a sensor chip, and the other interactant is then passed over the chip in solution. The detection system measures and displays on a computer screen a signal proportional to the mass of protein bound to the surface. In this way, the association phase can be directly visualized as the ligand-containing solution flows over the surface, and the subsequent dissociation is similarly displayed after the flow switches to buffer containing no ligand (18Fägerstam L.G. Frostell-Karlsson A. Karlsson R. Persson B. Ronnberg I. J. Chromatogr. 1992; 597: 397-410Google Scholar).The determination of the binding kinetics of IGF-I analogues toward IGFBP-3 is important for the development of IGF-I analogues with reduced affinity for IGFBP-3 and normal affinity for the IGF-I receptor, which could be of clinical interest.RESULTSTo study the binding of IGF-I/analogue to IGFBP-3, the binding protein was immobilized to the BIAcore sensor chip as described above. Kinetic binding assays were carried out for IGF-I, IGF-II, human insulin, and seven IGF-I analogues with the following code names and mutations: des(1–3) IGF-I (deletion of amino acids 1–3), long IGF-I (possessing a 13-amino acid extension at the NH2 terminus, and amino acid at position 3 changed from Glu to Arg), 4-Gly IGF-I (IGF-I with residues 28–37 replaced by a 4-glycine bridge), mini-IGF-I (deletion of the C-region residues 28–41). There are three proteins with swapped disulfide bridges: IGF-I swap, mini-IGF-I swap, and 4-Gly IGF-I swap, where swap indicates that the disulfide bridge normally connecting residue 52 to 47 now connects residue 52 to 48, and the disulfide bridge normally connecting residue 6 to 48 now connects residue 6 to 47. The analogues can be divided into four groups according to their mutations; the wild type hormones IGF-I, IGF-II and insulin, the C-region mutants 4-Gly IGF-I and mini-IGF-I, the B-region mutants des(1–3) IGF-I and long IGF-I, and finally the analogues with swapped disulfide bridges IGF-I swap, 4-Gly IGF-I swap, and mini-IGF-I swap. Fig. 3 shows examples of association and dissociation curves for the analogues grouped according to their mutations. Sensorgrams are shown prior to correction for refractive index shift, and the figures show the curve for IGF-I for comparison. In each experiment, the assays were carried out at a ligand concentration of 50 nM on a surface with the same amount of immobilized IGFBP-3. Fig. 3a shows that IGF-I and IGF-II have very similar curves with fast association and slow dissociation, whereas insulin has a very low binding affinity for the binding protein. In Fig. 3b, curves for the two analogues with the C-region mutations are shown. Both analogues have almost identical kinetics, which resemble the kinetics of IGF-I, although they do display a slower association and a faster dissociation rate.In Fig. 3c are shown kinetic curves for the two analogues with B-region mutations. These mutations reduce the analogue's affinity for IGFBP-3 when compared to wild type IGF-I, by decreasing the association rate and increasing the dissociation rate. This finding is in agreement with previously published work carried out with crude acid-stable serum-binding proteins (21Bayne M.L. Applebaum J. Chicchi G.G. Hayes N.S. Green B.G. Cascieri M.A. J. Biol. Chem. 1988; 263: 6233-6239Google Scholar) and also with work carried out with a 4-kDa binding protein secreted from bovine kidney cells (22Szabo L. Mottershead D.G. Ballard F.J. Wallace J.C. Biochem. Biophys. Res. Commun. 1988; 151: 207-214Google Scholar). Fig. 3d shows binding curves for the three IGF-analogues with swapped disulfide bridges. It is clear from this picture that swapping of the disulfide bridges in IGF-I greatly reduces its affinity for IGFBP-3.Data points obtained from the above-mentioned curves were used to calculate kinetic constants. This was done by fitting the binding model described earlier to the data points after correction for the bulk refractive index shift. Fig. 4 shows an example of a curve fit done on corrected data from IGF-I, IGF-II, and insulin experiments. The association, dissociation, and equilibrium dissociation constants calculated from these data are presented in Table I. The analogues in the table have been grouped according to their mutations. IGF-I and IGF-II have the fastest association rate and the slowest dissociation rate of all the analogues, resulting in the highest affinities, with IGF-II having about twice the affinity of IGF-I for IGFBP-3. The calculated affinities of the other analogues are in good agreement with the degree of binding observed in the kinetic binding curves in Fig. 3.Fig. 4Association and dissociation curves for IGF-I (○), IGF-II (□), and insulin (⋄) corrected for bulk refractive index shift. A two-site binding model is fitted to the data points, as described under “Data Analysis.” The ordinate gives the measured signal in RU, representing the mass of protein bound.View Large Image Figure ViewerDownload (PPT)Table IBinding parameters derived from the BIAcore experimentsPeptideka× 104± S.E.kd× 104± S.E.Kd± S.E.s−1 nM−1s−1nMIGF-I3.5 ± 0.370.78 ± 0.110.23 ± 0.04IGF-II5.2 ± 0.740.62 ± 0.0050.12 ± 0.02Insulin0.04 ± 0.0089.5 ± 2.8251 ± 914-Gly IGF-I1.8 ± 0.111.4 ± 0.150.8 ± 0.096Mini-IGF-I1.5 ± 0.22.3 ± 0.21.5 ± 0.24Des (1Lowe W.L. LeRoith D. Insulin-like Growth Factors: Molecular and Cellular Aspects. CRC Press Inc., Boca Raton, FL1991: 49Google Scholar, 2LeRoith D. Clemmons D. Nissley P. Rechler M.M. Ann. Intern. Med. 1992; 116: 854-862Google Scholar, 3Sussenbach J.S. Steenbergh P.H. Holthuizen P. Growth Regul. 1992; 2: 1-9Google Scholar)IGF-I1.6 ± 0.219 ± 0.665.6 ± 0.85Long IGF-I0.53 ± 0.066.3 ± 0.00311.9 ± 1.34-Gly Swap0.32 ± 0.192.1 ± 0.446.6 ± 1.4Mini-Swap0.36 ± 0.142.3 ± 0.296.4 ± 2.6IGF-I Swap0.11 ± 0.051.7 ± 1.015.5 ± 11.8 Open table in a new tab DISCUSSIONIn this study non-glycosylated IGFBP-3 was found to have comparable affinity for IGF-I and IGF-II with a slight preference for IGF-II, which is in agreement with what has been reported previously (23Martin J.L. Baxter R.C. J. Biol. Chem. 1986; 261: 8754-8760Google Scholar). We found the equilibrium dissociation constants for IGF-I and IGF-II to be 0.23 nM and 0.12 nM, respectively, which is approximately 5 times higher than reported by Martin et al. and Sommer et al. (23Martin J.L. Baxter R.C. J. Biol. Chem. 1986; 261: 8754-8760Google Scholar, 24Sommer A. Spratt S.K. Tatsumo G.P. Tressel T. Lee R. Maack C.A. Growth Regul. 1993; 3: 46-49Google Scholar). The discrepancy might be explained by differences in the assays used. Martin et al. (23Martin J.L. Baxter R.C. J. Biol. Chem. 1986; 261: 8754-8760Google Scholar) used competition assays with 125I-labeled IGF, and we have found that the binding kinetics tend to be faster on the BIAcore than in solution, 3A. Heding, L. Schäffer, I. Søndergaard, P. De Meyts, and R. M. Shymko, manuscript in preparation. which might account for the difference. Sommer et al. (24Sommer A. Spratt S.K. Tatsumo G.P. Tressel T. Lee R. Maack C.A. Growth Regul. 1993; 3: 46-49Google Scholar) used the BIAcore instrument, but do not describe experimental conditions such as temperature, pH, buffer composition, immobilization level, and method of data analysis for determining kinetic constants.Human insulin was found to have a measurable, but extremely low, affinity for the binding protein-3. It has been reported that insulin is unable to bind to serum-binding proteins (16De Vroede M.A. Rechler M.M. Nissley S.P. Joshi S. Burke G.T. Katsoyannis P.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3010-3014Google Scholar, 22Szabo L. Mottershead D.G. Ballard F.J. Wallace J.C. Biochem. Biophys. Res. Commun. 1988; 151: 207-214Google Scholar); the BIAcore assay used in this study therefore appears to be more sensitive for the detection of low affinity interactions than the competition assays used previously.The C-region mutants 4-Gly IGF-I and mini-IGF-I displayed a similar affinity for IGFBP-3, with Kd values 3 and 6 times higher than the Kd value of wild type IGF-I. Bayne et al. (17Bayne M.L. Applebaum J. Underwood D. Chicchi G.G. Green B.G. Hayes N.S. Cascieri M.A. J. Biol. Chem. 1989; 264: 11004-11008Google Scholar) have reported that 4-Gly IGF-I has a 2–3-fold higher affinity than IGF-I for acid-stable human serum-binding proteins and purified IGFBP-3. This slight discrepancy might be due to the different natures of the binding assays used: competition assays with 125I-labeled IGF-I as opposed to the direct binding of unlabeled ligands in the BIAcore assay. The 2-fold lower affinity of mini-IGF-I for IGFBP-3 compared with that of 4-Gly IGF-I might be explained by the complete absence of the C-region in mini-IGF-I, since this presumably causes a greater conformational change in the molecule than the replacement of the C-region residues 28–37 with a 4-glycine bridge. The relatively high affinity of these analogues for IGFBP-3 is in contrast to their affinity for the IGF-I receptor, which has been found by Bayne et al. (17Bayne M.L. Applebaum J. Underwood D. Chicchi G.G. Green B.G. Hayes N.S. Cascieri M.A. J. Biol. Chem. 1989; 264: 11004-11008Google Scholar) and Cascieri et al. (25Cascieri M.A. Bayne M.L. LeRoith D. Raizada M.K. Molecular and Cellular Biology of Insulin-like Growth factors and Their Receptors. Plenum Publishing Corp., New York1989: 285Google Scholar) to be approximately 30 times lower than that of the wild type molecule. Gill et al.2 found that 4-Gly IGF-I has 100 times lower affinity for the IGF-I receptor than wild type IGF-I and that mini-IGF-I has an immeasurably low receptor affinity. These results indicate that the C-region is crucial for receptor binding but is of less importance for the binding to IGFBP-3.It has been reported by several groups that amino acid 3 in the B-region of IGF-I is important for its binding to binding proteins (11Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Google Scholar, 16De Vroede M.A. Rechler M.M. Nissley S.P. Joshi S. Burke G.T. Katsoyannis P.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3010-3014Google Scholar, 21Bayne M.L. Applebaum J. Chicchi G.G. Hayes N.S. Green B.G. Cascieri M.A. J. Biol. Chem. 1988; 263: 6233-6239Google Scholar, 22Szabo L. Mottershead D.G. Ballard F.J. Wallace J.C. Biochem. Biophys. Res. Commun. 1988; 151: 207-214Google Scholar, 26Forbes B. Szabo L. Baxter R.C. Ballard F.J. Wallace J.C. Biochem. Biophys. Res. Commun. 1988; 157: 196-202Google Scholar). It is therefore not surprising that des(1–3) IGF-I and long IGF-I have a greatly reduced affinity for IGFBP-3, since both analogues have a mutation at amino acid 3. Both analogues are known to be more mitogenic than IGF-I in L6 myoblasts (27Ballard F.J. Francis G.L. Ross M. Bagley C.J. May B. Wallace J.C. Biochem. Biophys. Res. Commun. 1987; 149: 398-404Google Scholar). Since des(1–3) IGF-I and a related analogue with amino acids 3 and 4 mutated to glutamine and alanine respectively have a normal affinity for the IGF-I receptor (21Bayne M.L. Applebaum J. Chicchi G.G. Hayes N.S. Green B.G. Cascieri M.A. J. Biol. Chem. 1988; 263: 6233-6239Google Scholar, 27Ballard F.J. Francis G.L. Ross M. Bagley C.J. May B. Wallace J.C. Biochem. Biophys. Res. Commun. 1987; 149: 398-404Google Scholar), the increased mitogenic potency of these analogues appears to result from their low affinity for IGFBP-3 and possibly other binding proteins. Long IGF-I has half the affinity of des(1–3) IGF-I for IGFBP-3, which indicates that the 13 amino acid amino-terminal extension of long IGF-I has a disturbing effect on the binding to IGFBP-3.It is clear from the low affinities of IGF-I swap, 4-Gly IGF-I swap and mini-IGF-I swap that swapping of the disulfide bridges of IGF-I greatly impairs the hormone's ability to bind to IGFBP-3. This is primarily due to a decreased association rate. The swapped IGF-I analogues also have extremely low IGF-I receptor affinity,2 which indicates that swapping of the disulfide bridges causes a major conformational change of the entire hormone as found by Miller et al. (28Miller J.A. Narhi L.O. Hua Q.X. Rosenfeld R. Arakawa T. Rohde M. Prestrelski S. Lauren S. Stoney K.S. Tsai L. Weiss M.A. Biochemistry. 1993; 32: 5203-5213Google Scholar). These findings are important as IGF-I swap is secreted from recombinant organisms (Saccharomyces cerevisiae and E. coli) and arises from the refolding of denatured IGF-I in quantities similar to those of the normally folded product (29Elliott S. Fagin K.D. Narhi L.O. Miller J.A. Jones M. Koski R. Peters M. Hsieh P. Sachdev R. Rosenfeld R.D. Rohde M.F. Arakawa T. J. Protein Chem. 1990; 9: 95-104Google Scholar, 30Meng H. Burleigh B.D. Kelly G.M. J. Chromatogr. 1988; 443: 183-192Google Scholar, 31Hejnæs K.R. Bayne S. Nørskov L. Sørensen H.H. Thomsen J. Schäffer L. Wollmer A. Skriver L. Protein Eng. 1992; 5: 797-806Google Scholar).In this study we found IGFBP-3 to have a high and a low affinity binding site for the IGFs, where the high affinity binding site is responsible for approximately 90% of the binding. However, it is unclear whether the low affinity binding site has any biological relevance, since in these experiments IGFBP-3 is immobilized to the sensor chip via amine groups in a random way, and some IGFBP-3 molecules might be bound to the sensor chip close to the ligand binding site, thus impairing their affinity for the ligand.We have found that the BIAcore assay used in this study has several advantages over the classic 125I-based tracer assays. Most importantly, the BIAcore system is label-free and shows the binding of IGF-I analogues to IGFBP-3 in real time. The BIAcore system is also time saving since the system's robotic unit allows for large series of samples to be assayed automatically. Since the other five IGF-I-binding proteins are structurally related to IGFBP-3 (4Martin J.L. Baxter R.C. Curr. Opin. Endocrinol. Diabetes. 1994; 1: 16-21Google Scholar), we believe that it should also be possible to use the assay described here for the study of interactions between these proteins and IGF-I analogues. INTRODUCTIONInsulin-like growth factor-I and -II (IGF-I and IGF-II) 1The abbreviations used are: IGFinsulin-like growth factorIGFBPIGF-binding proteinNHSN-hydroxysuccinimideECDN-ethyl-N′-(3-diethylaminopropyl)carbodiimide. are small proteins that stimulate a variety of growth-promoting and metabolic effects via an interaction with the IGF-I receptor (1Lowe W.L. LeRoith D. Insulin-like Growth Factors: Molecular and Cellular Aspects. CRC Press Inc., Boca Raton, FL1991: 49Google Scholar). The peptides consist of four regions: A- and B-regions, which are homologous to the A and B chains of insulin; a C-region, which is analogous to but unrelated to the C peptide of insulin that connects the A- and B-regions; and a short carboxyl-terminal D-region, with no counterpart in insulin (2LeRoith D. Clemmons D. Nissley P. Rechler M.M. Ann. Intern. Med. 1992; 116: 854-862Google Scholar, 3Sussenbach J.S. Steenbergh P.H. Holthuizen P. Growth Regul. 1992; 2: 1-9Google Scholar).IGF-I and -II form complexes with six different IGF-binding proteins (IGFBPs) in the circulation and in the extracellular environment (4Martin J.L. Baxter R.C. Curr. Opin. Endocrinol. Diabetes. 1994; 1: 16-21Google Scholar). In the circulation the majority of IGFs are bound in a 140-kDa complex consisting of IGFBP-3 of 40–50 kDa, an approximately 85-kDa so-called acid-labile subunit, and IGF-I or IGF-II (5Baxter R.C. Martin J.L. Beniac V.A. J. Biol. Chem. 1989; 264: 11843-11848Google Scholar, 6Kaufmann U. Zapf J. Torretti B. Froesch E.R. J. Clin. Endocrinol. & Metab. 1977; 44: 160-166Google Scholar, 7Furlanetto R.W. J. Clin. Endocrinol. & Metab. 1980; 51: 12-19Google Scholar, 8White R.M. Nissley S.P. Moses A.C. Rechler M.M. Johnsonbaugh R.E. J. Clin. Endocrinol. & Metab. 1981; 53: 49-57Google Scholar, 9Hintz R.L. Liu F. Rosenfeld R.G. Kemp S.F. J. Clin. Endocrinol. & Metab. 1981; 53: 100-104Google Scholar, 10Baxter R.C. Martin J.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6898-6902Google Scholar). The IGFs bind to IGFBP-3 with high affinity, and the acid-labile subunit is then capable of binding to the formed complex with a somewhat lower affinity (11Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Google Scholar). The major biological function of IGFBP-3 is believed to be to extend the circulating half-lives of IGFs, since IGFs bound to the 140-kDa complex are cleared from the circulation much slower than free IGFs (12Guler H. Zapf J. Schmid C. Froesch E.R. Acta Endocrinol. 1989; 121: 753-758Google Scholar).Wild type IGFBP-3 is heavily glycosylated, which results in an apparent molecular mass of 40–50 kDa on denaturing SDS-polyacrylamide gel electrophoresis, while the non-glycosylated molecule has a molecular mass of approximately 29 kDa. The non-glycosylated protein's binding characteristics are reported to be identical to those of the wild type molecule (13Tressel T.J. Tatsuno G.P. Spratt K. Sommer A. Biochem. Biophys. Res. Commun. 1991; 178: 625-633Google Scholar).Attempts have been made to identify the regions of IGF-I involved in the high affinity binding to IGFBP-3 by constructing insulin/IGF-I hybrids and by using site-directed mutagenesis of the IGF-I gene. From the studies involving insulin/IGF-I hybrids, it was established that the B-region but not the D-region of IGF-I is important for binding to IGFBP-3 (14Joshi S. Burke G.T. Katsoyannis P.G. Biochemistry. 1985; 24: 4208-4214Google Scholar, 15Joshi S. Ogawa H. Burke G.T. Tseng L.Y.-H. Rechler M.M. Katsoyannis P.G. Biochem. Biophys. Res. Commun. 1985; 133: 423-429Google Scholar, 16De Vroede M.A. Rechler M.M. Nissley S.P. Joshi S. Burke G.T. Katsoyannis P.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3010-3014Google Scholar). From the studies using site-directed mutagenesis, it was found that the C-region is of less importance than the B-region for high affinity binding to IGFBP-3 (17Bayne M.L. Applebaum J. Underwood D. Chicchi G.G. Green B.G. Hayes N.S. Cascieri M.A. J. Biol. Chem. 1989; 264: 11004-11008Google Scholar), and that residues 3 and 4, the region between residues 8 and 18, and residues 49–51 appear to be most important for IGFBP-3 binding (11Baxter R.C. Bayne M.L. Cascieri M.A. J. Biol. Chem. 1992; 267: 60-65Google Scholar). In this study we have investigated the detailed association and dissociation binding kinetics between IGFBP-3 and IGF-I, IGF-II, insulin, and seven synthetic IGF-I analogues with mutations in the B- or C-regions and with normal and swapped disulfide bridges. In order to generate detailed kinetics, we have employed the BIAcore™ instrument developed by Pharmacia, and analyzed the data by using computer fitting programs developed at the Hagedorn Research Institute.The BIAcore™ technology makes it possible to visualize macromolecular interactions directly and in “real time” (18Fägerstam L.G. Frostell-Karlsson A. Karlsson R. Persson B. Ronnberg I. J. Chromatogr. 1992; 597: 397-410Google Scholar). The BIAcore is a biosensor-based instrument that uses surface plasmon resonance as the detection principle. One molecule of the interaction to be studied is immobilized covalently to a sensor chip, and the other interactant is then passed over the chip in solution. The detection system measures and displays on a computer screen a signal proportional to the mass of protein bound to the surface. In this way, the association phase can be directly visualized as the ligand-containing solution flows over the surface, and the subsequent dissociation is similarly displayed after the flow switches to buffer containing no ligand (18Fägerstam L.G. Frostell-Karlsson A. Karlsson R. Persson B. Ronnberg I. J. Chromatogr. 1992; 597: 397-410Google Scholar).The determination of the binding kinetics of IGF-I analogues toward IGFBP-3 is important for the development of IGF-I analogues with reduced affinity for IGFBP-3 and normal affinity for the IGF-I receptor, which could be of clinical interest." @default.
• W2021277909 created "2016-06-24" @default.
• W2021277909 creator A5015494879 @default.
• W2021277909 creator A5030660467 @default.
• W2021277909 creator A5052331621 @default.
• W2021277909 creator A5071526017 @default.
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• W2021277909 date "1996-06-01" @default.
• W2021277909 modified "2023-10-14" @default.
• W2021277909 title "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" @default.
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