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• W2036763753 abstract "Elevated insulin-like growth factor (IGF)-1 levels are prognostic for the development of prostate and breast cancers and exacerbate the complications of diabetes. In each case, perturbation of the balance between IGF-1/2, the IGF-1 receptor, and the IGF-binding proteins (IGFBPs) leads to elevated IGF-1 sensitivity. Blockade of IGF action in these diseases would be clinically significant. Unfortunately, effective IGF antagonists are currently unavailable. The IGFBPs exhibit high affinity and specificity for the IGFs and serve as natural IGF antagonists, limiting their mitogenic/anti-apoptotic effects. As an initial step in designing IGFBP-based agents that antagonize IGF action, we have begun to analyze the structure of the IGF-binding site on IGFBP-2. To this end, two IGF-1 photoprobes,N αGly1-(4-azidobenzoyl)-IGF-1 (abG1IGF-1) andN αGly1-([2–6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionoyl)-IGF-1 (bedG1IGF-1), selective for the IGFBPs were synthesized by derivatization of the α-amino group of Gly1, known to be part of the IGFBP-binding domain. Mass spectrometric analysis of the reduced, alkylated, and trypsin-digested abG1IGF-1·recombinant human IGFBP-2 (rhIGFBP-2) complex indicated photoincorporation near the carboxyl terminus of rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of avidin-purified tryptic peptides of the bedG1IGF-1·rhIGFBP-2 complex revealed photoincorporation within residues 212–227. Taken together, these data indicate that the IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2, providing evidence that the IGF-1-binding domain is located within the C terminus of IGFBP-2. Elevated insulin-like growth factor (IGF)-1 levels are prognostic for the development of prostate and breast cancers and exacerbate the complications of diabetes. In each case, perturbation of the balance between IGF-1/2, the IGF-1 receptor, and the IGF-binding proteins (IGFBPs) leads to elevated IGF-1 sensitivity. Blockade of IGF action in these diseases would be clinically significant. Unfortunately, effective IGF antagonists are currently unavailable. The IGFBPs exhibit high affinity and specificity for the IGFs and serve as natural IGF antagonists, limiting their mitogenic/anti-apoptotic effects. As an initial step in designing IGFBP-based agents that antagonize IGF action, we have begun to analyze the structure of the IGF-binding site on IGFBP-2. To this end, two IGF-1 photoprobes,N αGly1-(4-azidobenzoyl)-IGF-1 (abG1IGF-1) andN αGly1-([2–6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionoyl)-IGF-1 (bedG1IGF-1), selective for the IGFBPs were synthesized by derivatization of the α-amino group of Gly1, known to be part of the IGFBP-binding domain. Mass spectrometric analysis of the reduced, alkylated, and trypsin-digested abG1IGF-1·recombinant human IGFBP-2 (rhIGFBP-2) complex indicated photoincorporation near the carboxyl terminus of rhIGFBP-2, between residues 266 and 287. Mass spectrometric analysis of avidin-purified tryptic peptides of the bedG1IGF-1·rhIGFBP-2 complex revealed photoincorporation within residues 212–227. Taken together, these data indicate that the IGFBP-binding domain on IGF-1 contacts the distal third of IGFBP-2, providing evidence that the IGF-1-binding domain is located within the C terminus of IGFBP-2. insulin-like growth factor insulin-like growth factor-1 receptor insulin-like growth factor-binding protein recombinant human insulin-like growth factor-binding protein N-hydroxysuccinimidyl 4-azidobenzoate sulfosuccinimidyl [2–6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionate N αGly1(4-azidobenzoyl)-IGF-1 N αGly1-([2–6-(biotinamido)2-(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionoyl)-IGF-1 high performance liquid chromatography matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Insulin-like growth factor (IGF)1-1 and IGF-2 play central roles in a number of cellular processes, including growth, proliferation, differentiation, survival, transformation, and metastasis (1Daughaday W.H. Rotwein P. Endocr. Rev. 1989; 10: 68-91Crossref PubMed Scopus (1619) Google Scholar, 2Baserga R. Cancer Res. 1995; 55: 249-252PubMed Google Scholar). Enhanced activity of the IGFs has been implicated in diabetic complications and cancer. These effects are mediated by the IGF-1 receptor (IGF-1R), a member of the receptor tyrosine kinase family of cell-surface receptors. The IGF-2 receptor, which lacks signaling activity, plays a role in clearing IGF-2 from the cell surface (3Louvi A. Accili D. Efstratiadis A. Dev. Biol. 1997; 189: 33-48Crossref PubMed Scopus (324) Google Scholar, 4Ludwig T. Le Borgne R. Hoflack B. Trends Cell Biol. 1995; 5: 202-205Abstract Full Text PDF PubMed Scopus (79) Google Scholar). The IGFs are regulated at the extracellular level by a family of six IGF-binding proteins (IGFBPs), designated IGFBP-1–6 (5Holly J.M.P. Flyvjberg A. Orskov H. Alberti G. Growth Hormone and Insulin-like Growth Factor I in Human and Experimental Diabetes. John Wiley & Sons Ltd., Chichester, United Kingdom1993: 47-76Google Scholar, 6Clemmons D.R. Jones J.I. Busby W.H. Wright G. Ann. N. Y. Acad. Sci. U. S. A. 1993; 692: 10-21Crossref PubMed Scopus (85) Google Scholar, 7Jones J.I. Clemmons D.R. Endocr. Rev. 1995; 16: 13-34Google Scholar). These six proteins exhibit higher affinities for the IGFs than the IGF-1R, while having negligible affinity for insulin. Renewed interest in the function of the IGF system stems from the observations that IGF-1 and IGF-2, acting through the IGF-1R, increase the tumorigenic potential of breast and prostate cancer cells (8Long L. Rubin R. Baserga R. Brodt P. Cancer Res. 1995; 55: 1006-1009PubMed Google Scholar). Accordingly, increased serum IGF-1 levels have been shown to be prognostic for the development of prostate and breast cancers (9Chan J.M. Stampfer M.J. Giovannucci E. Gann P.H. Ma J. Wilkinson P. Hennekens C.H. Pollak M. Science. 1998; 279: 563-566Crossref PubMed Scopus (1792) Google Scholar, 10Hankinson S.E. Willett W.C. Colditz G.A. Hunter D.J. Michaud D.S. Deroo B. Rosner B. Spitzer F.E. Pollak M. Lancet. 1998; 351: 1393-1396Abstract Full Text Full Text PDF PubMed Scopus (1600) Google Scholar). Alterations in IGFBP expression may also contribute to disease states. For example, IGFBP-3 is a target of the p53 tumor suppressor, and a common p53 mutation results in decreased IGFBP-3 secretion (11Buckbinder L. Talbott R. Velasco-Miguel S. Takenaka I. Faha B. Seizinger B.R. Kley N. Nature. 1995; 377: 646-649Crossref PubMed Scopus (806) Google Scholar, 12Burns T.F. El-Deiry W.S. J. Cell. Physiol. 1999; 181: 231-239Crossref PubMed Scopus (200) Google Scholar), which is likely to cause an increased proliferative response to IGF-1. Also, reduced IGFBP-2 expression resulting from the hyperglycemia of diabetes was recently shown to enhance the sensitivity of renal mesangial cells to the growth and secretory effects of IGF-1, pushing the cells toward a glomerulosclerotic phenotype (13Horney M.J. Shirley D.W. Kurtz D.T. Rosenzweig S.A. Am. J. Physiol. 1998; 274: F1045-F1053PubMed Google Scholar). Because IGF-1 can suppress apoptosis, cells lacking IGF-1 receptors, cells with compromised IGF-1R signaling pathways, or cells treated with the IGFBPs may selectively die by apoptosis (8Long L. Rubin R. Baserga R. Brodt P. Cancer Res. 1995; 55: 1006-1009PubMed Google Scholar). Taken together, these findings suggest that the IGFBPs serve a role as natural IGF antagonists. IGF-1 and IGF-2 are homologous protein hormones of 70 and 67 amino acids in length, respectively (14Humbel R.E. Eur. J. Biochem. 1990; 190: 445-462Crossref PubMed Scopus (679) Google Scholar). Based on studies of chemically modified and mutated IGF-1, a number of residues have been identified as being part of the IGF-1R contact site, in particular the aromatic residues at positions 23–25 (15Blundell T.L. Bedarkar S. Rinderknecht E. Humbel R.E. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 180-184Crossref PubMed Scopus (230) Google Scholar). Cooke et al. (16Cooke R.M. Harvey T.S. Campbell I.D. Biochemistry. 1991; 30: 5484-5491Crossref PubMed Scopus (165) Google Scholar) used NMR and restrained molecular dynamics to elicit the solution structure of IGF-1; this model clearly illustrates an IGFBP-interacting domain on the surface of IGF-1 and its lack of overlap with the receptor-docking site (see Fig. 1). Specifically, this site consists of the N-terminal tripeptide Gly-Pro-Glu (17Szabo L. Mottershead D.G. Ballard F.J. Wallace J.C. Biochem. Biophys. Res. Commun. 1988; 151: 207-214Crossref PubMed Scopus (102) Google Scholar) and residues 49–51 (18Cascieri M.A. Chicchi G.G. Applebaum J. Green B.G. Hayes N.S. Bayne M.L. J. Biol. Chem. 1989; 264: 2199-2202Abstract Full Text PDF PubMed Google Scholar). These two regions come together to form an independent binding domain (see Fig. 1) (16Cooke R.M. Harvey T.S. Campbell I.D. Biochemistry. 1991; 30: 5484-5491Crossref PubMed Scopus (165) Google Scholar). The analog des-1–3-IGF-1 binds to the IGF-1R with high affinity, but has dramatically reduced affinity for the IGFBPs, underscoring the importance of the N-terminal contact site on IGF-1 for binding protein specificity (17Szabo L. Mottershead D.G. Ballard F.J. Wallace J.C. Biochem. Biophys. Res. Commun. 1988; 151: 207-214Crossref PubMed Scopus (102) Google Scholar). In addition, mutations of Glu3 or residues 49–51 result in a ligand with severely reduced binding activity (19Clemmons D.R. Dehoff M.L. Busby W.H. Bayne M.L. Cascieri M.A. Endocrinology. 1992; 131: 890-895Crossref PubMed Google Scholar). In good agreement with these findings, insulin lacks these residues common in the IGFBP-binding region and thus does not bind to the IGFBPs with high affinity. The IGFBPs are globular proteins containing 18 spatially conserved cysteine residues participating in the formation of nine disulfide bonds. They range in size from 200 to 300 amino acids and, based on their high degree of homology, can be divided into three distinct domains, each constituting about one-third of the protein (20Kiefer M.C. Ioh R.S. Bauer D.M. Zapf J. Biochem. Biophys. Res. Commun. 1991; 176: 219-225Crossref PubMed Scopus (91) Google Scholar). The N- and C-terminal regions, designated domains 1 and 3, respectively, share the highest homology (20Kiefer M.C. Ioh R.S. Bauer D.M. Zapf J. Biochem. Biophys. Res. Commun. 1991; 176: 219-225Crossref PubMed Scopus (91) Google Scholar, 21Kiefer M.C. Masiarz F.R. Bauer D.M. Zapf J. J. Biol. Chem. 1991; 266: 9043-9049Abstract Full Text PDF PubMed Google Scholar), whereas the intervening region (domain 2) is highly variable (<30% homology) (6Clemmons D.R. Jones J.I. Busby W.H. Wright G. Ann. N. Y. Acad. Sci. U. S. A. 1993; 692: 10-21Crossref PubMed Scopus (85) Google Scholar). Domains 1 and 3 contain 12 and 6 spatially conserved cysteine residues, respectively, except in the case of IGFBP-6, which is missing 2 cysteine residues in domain 1 (5Holly J.M.P. Flyvjberg A. Orskov H. Alberti G. Growth Hormone and Insulin-like Growth Factor I in Human and Experimental Diabetes. John Wiley & Sons Ltd., Chichester, United Kingdom1993: 47-76Google Scholar); IGFBP-4 has 2 additional cysteine residues in domain 2. Because of the high homology of these proteins in domains 1 and 3 (∼70%) (6Clemmons D.R. Jones J.I. Busby W.H. Wright G. Ann. N. Y. Acad. Sci. U. S. A. 1993; 692: 10-21Crossref PubMed Scopus (85) Google Scholar), the IGF-binding domain has been proposed to reside within one of these regions. Support for this notion is based on studies in which N- or C-terminal IGFBP fragments were found to retain high affinity binding activity for IGF-1 and/or IGF-2 (19Clemmons D.R. Dehoff M.L. Busby W.H. Bayne M.L. Cascieri M.A. Endocrinology. 1992; 131: 890-895Crossref PubMed Google Scholar). IGFBP-related protein-1 and -2, also known as IGFBP-7/Mac25 and IGFBP-8/connective tissue growth factor, respectively, have homologies within their N termini to IGFBP-1–6 (23Hwa V. Oh Y. Rosenfeld R.G. Endocr. Rev. 1999; 20: 761-787Crossref PubMed Scopus (908) Google Scholar). These proteins have lower affinities for the IGFs compared with the IGFBPs and have been reported to interact with insulin (24Yamanaka Y. Wilson E.M. Rosenfeld R.G. Oh Y. J. Biol. Chem. 1997; 272: 30729-30734Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The precise mechanism by which the IGFBPs inhibit IGF-1 and IGF-2 action is presently unknown. It is thought to involve high affinity binding of the IGFs by the IGFBPs, thereby limiting their access to the IGF-1R. A complete understanding of this inhibitory action will come with the solution of the three-dimensional structure of the IGFBPs. As of yet, the IGF-binding domain on the IGFBPs has not been defined. To date, extensive use of molecular techniques has been applied to assess the binding domain on the IGFBPs. However, only sparse structural information has been obtained. Currently, a debate exists as to which domain(s) of the IGFBPs are most crucial for IGF binding. This is further confounded by the paucity of precise structural information about the IGFBPs as recently reviewed by Baxter (25Baxter R.C. Am. J. Physiol. 2000; 278: E967-E976Crossref PubMed Google Scholar). To more precisely identify the points of contact between IGF-1 and IGFBP-2, we chose to pursue a photoaffinity labeling approach, which has been used to identify sites of interaction between a number of interacting proteins. On this basis, we derivatized the α-amino group of the N-terminal glycine of IGF-1 with two different photoaffinity reagents,N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and sulfosuccinimidyl [2–6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionate (SBED), to generate IGF-1 photoprobes capable of selectively labeling the IGF-binding domain on IGFBP-2. The advantage of this approach is that the photoreactive group is inserted within the region shown to be essential for high affinity binding of IGF-1 to the IGFBPs. Since IGF-2, which has a 3-residue N-terminal extension, exhibits a higher affinity for IGFBP-2 than IGF-1, reduced binding affinity of IGF-1 for IGFBP-2 resulting from N-terminal substitutions was not anticipated. In this study, we describe the synthesis and characterization ofN αGly1-(4-azidobenzoyl)-IGF-1 (abG1IGF-1) andN αGly1-([2–6-(biotinamido)-2(p-azidobenzamido)hexanoamido]ethyl-1,3′-dithiopropionoyl)IGF-1 (bedG1IGF-1) and their successful application to photoaffinity label rhIGFBP-2. Based on these direct photoaffinity labeling analyses, our results indicate that the C terminus of IGFBP-2 contains the IGF-binding domain. Recombinant human IGF-1 was provided by Genentech, Inc. (South San Francisco, CA). HSAB was synthesized fromp-aminobenzoic acid, sodium azide (Sigma),and dicyclohexylcarbodiimide (Pierce) and purified according to the method of Galardy et al. (26Galardy R.E. Craig L.C. Jamieson J.D. Printz M.P. J. Biol. Chem. 1974; 249: 3510-3518Abstract Full Text PDF PubMed Google Scholar). HPLC columns were from Vydac Instruments (Hesperia, CA). SBED, UltraLinkTM monomeric avidin-agarose, NeutrAvidinTM-peroxidase, and tris(2-carboxyethylyl)phosphine hydrochloride were obtained from Pierce. Methotrexate was from Immunex Corp. (Seattle, WA), and fetal bovine serum was from Summit Biotechnology (Fort Collins, CO). cDNA encoding human IGFBP-2 (27Binkert C. Landwehr J. Mary J.-L. Schwander J. Heinrich G. EMBO J. 1989; 8: 2497-2502Crossref PubMed Scopus (220) Google Scholar) was obtained from Dr. Jörg Landwehr (Hoffmann-La Roche, Basel, Switzerland). All other materials were of reagent grade or higher. All derivatizations and handling of photoprobes were carried out under subdued lighting or under a red safety light. abG1IGF-1 was synthesized by reaction of recombinant human IGF-1 (1 mg, 130 nmol) with a 10-fold molar excess of HSAB (0.34 mg, 1.3 μmol) for 1 h at 23 °C. Unreacted ester was quenched by the addition of 30 μl of ethanolamine for 30 min, and the entire reaction mixture was lyophilized. The lyophilized reaction mixture was dissolved in 0.5 m acetic acid and 10 μl of trifluoroacetic acid and injected onto a C18 column equilibrated in 0.1% trifluoroacetic acid and 24% acetonitrile at a flow rate of 1 ml/min. After 20 min, a linear gradient of 24–60% acetonitrile was developed over 60 min to elute IGF-1 and the reaction products. Peak 4 was collected, dried in vacuo in a SpeedVac concentrator (Savant Instruments, Inc., Farmingdale, NY), reinjected onto the C18 column equilibrated in 50 mmtriethanolamine phosphate (pH 3.0) containing 27.5% acetonitrile, and eluted with a gradient of 27.5–38% acetonitrile over 60 min. The major peak was collected, dried, and further analyzed. Synthesis and purification of bedG1IGF-1 was carried out essentially as described for abG1IGF-1 using a 1:1 ratio of SBED to protein. IGF-1 and the photoprobes were digested with pepsin at an enzyme/substrate ratio of 1:20 in 0.01 mHCl for 5 h at 23 °C (28Forsberg G. Gunnar P. Ekebacke A. Josephson S. Hartmanis M. Biochem. J. 1990; 271: 357-363Crossref PubMed Scopus (44) Google Scholar). Digests were then injected onto a C18 column equilibrated in 0.1% trifluoroacetic acid, and the fragments were eluted using a linear gradient of 0–60% acetonitrile over 90 min. For each photoprobe, the HPLC fractions containing fragments with potentially modified residues (AE, CG, and D fragments) (see Fig. 3) (28Forsberg G. Gunnar P. Ekebacke A. Josephson S. Hartmanis M. Biochem. J. 1990; 271: 357-363Crossref PubMed Scopus (44) Google Scholar) were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Dried HPLC fractions were dissolved in 0.1% trifluoroacetic acid containing 70% acetonitrile. 0.5-μl aliquots of each fraction were mixed with 1 μl of 50 mmα-cyano-4-hydroxycinnamic acid (Sigma) in 0.1% trifluoroacetic acid and 70% acetonitrile. The mixture was spotted onto a gold-coated, stainless steel sample plate and air-dried. The samples were analyzed using a PerSeptive Biosystems Voyager-DE MALDI-TOF mass spectrometer equipped with a 337-nm nitrogen laser. A delayed extraction source was operated in linear mode (1.2-m ion flight path, 20-kV accelerating voltage), yielding an instrumental resolution of ∼700 (full width at half-maximum) at m/z 1297.5. One mass spectrum was based on 256 averaged mass scans. External mass calibration was performed using angiotensin I (1297.5 Da) and bovine insulin (5734.54 Da) as standards. Mass accuracy was ±0.1%. rhIGFBP-2 was purified from DHFR− Chinese hamster ovary cells (29Urlaub G. Kas E. Carothers A.M. Chasin L.A. Cell. 1983; 33: 405-412Abstract Full Text PDF PubMed Scopus (206) Google Scholar) stably transfected with 30 μg of pCMV-hIGFBP-2 and 2 μg of pMT2 containing murine dihydrofolate reductase (30Kaufman R.J. Davies M.V. Pathak V.K. Hershey J.W. Mol. Cell. Biol. 1989; 9: 946-958Crossref PubMed Scopus (333) Google Scholar) using calcium phosphate. Cells were grown to confluence in roller bottles and subjected to a weekly cycle of 3 days growth in serum-containing medium, followed by 4 days in serum-free medium. Pooled conditioned medium (400–600 ml) was acidified to pH 3–4 with glacial acetic acid and dialyzed against 10 mm acetic acid in Spectrapor 4 membranes (12,000–14,000M r cutoff; Spectrum, Laguna Hills, CA) to remove salt and IGFs. After lyophilization, IGFBP-2 was purified by sequential IGF-1-agarose affinity chromatography and reversed-phase HPLC on a C4 column equilibrated in 0.1% trifluoroacetic acid. IGFBP-2 eluted between 30 and 40% acetonitrile. 2 ml of Affi-Gel 10-activated immunoaffinity support (Bio-Rad) were washed four times with 3 volumes of cold water, followed by the addition of 2 mg of IGF-1 in 100 mm HEPES (pH 7.4). The mixture was agitated overnight at 4 °C, followed by two washes with 100 mmHEPES (pH 8.0). Unreacted sites were then quenched with 2 ml of 1m Tris-HCl (pH 7.9) for 1 h at ambient temperature. The column was then washed with three cycles of alternating low pH/high pH salt washes (0.5 m NaCl and 0.1 m sodium acetate (pH 4.0) and 0.5 m NaCl and 0.1 m Tris (pH 8.0), respectively). Finally, the column was washed and stored in 10 ml of 150 mm NaCl and 50 mm HEPES (pH 7.4) with 0.05% sodium azide. Dialyzed and lyophilized conditioned medium was dissolved in 40–45 ml of 50 mm HEPES (pH 7.4) containing 150 mm NaCl (Buffer A). Insoluble material was removed by centrifugation at 3000 × g for 20 min. 2 ml of IGF-1-agarose in Buffer A were then added, and the slurry was incubated overnight at 4 °C with gentle agitation. The column was subsequently washed with 100 ml of Buffer A, followed by 50 ml of 10% Buffer A. Proteins bound to the column were then eluted with 15 ml of 0.5 m acetic acid, and the column was washed with 20 ml of Buffer A plus 1 ml of 1m HEPES (pH 7.4), followed by 40 ml of Buffer A. The eluate was dried in vacuo using a SpeedVac concentrator. The dried eluate was stored at −20 °C until further purification as described above. Soluble IGFBP-2 binding assays were carried out using polyethylene glycol precipitation and centrifugation (31Bourner M.J. Busby Jr., W.H. Siegel N.R. Krivi G.G. McCusker R.H. Clemmons D.R. J. Cell. Biochem. 1992; 48: 215-226Crossref PubMed Scopus (120) Google Scholar). 1 ng of rhIGFBP-2 was combined with various concentrations of IGF-1, abG1IGF-1, or bedG1IGF-1 ranging from 30 fm to 100 nm in binding assay buffer (100 mm HEPES (pH 7.4), 44 mm NaHCO3, 0.01% bovine serum albumin, 0.01% Triton X-100, and 0.02% NaN3), followed by the addition of 10 nCi of125I-IGF-1 (Amersham Pharmacia Biotech). After a 4-h incubation at room temperature, 250 μl of 0.5% bovine γ-globulin were added, followed by 500 μl of 25% polyethylene glycol (averageM r of 8000; Sigma). The samples were incubated for 10 min at room temperature and centrifuged for 3 min at 15,000 × g. The pellets were washed with 1 ml of 6.25% polyethylene glycol, and bound radioactivity was quantified in a Compugamma spectrometer (LKB-Wallac, Turku, Finland). Counts bound in the presence of 1 μm or 100 nm IGF-1 (nonspecific binding) were subtracted to obtain specific binding. IC50 values were calculated using the equationB = B max/(1 + [ligand]/IC50), where B is the concentration of bound ligand and B max is the maximal binding observed. The Microsoft Excel 97 Solver was used to minimize the sum of the squares of the differences from the mean IC50 values for each IGF-1 concentration by optimizing B restrained by the above equation. The calculated IC50 values were used to generate smooth curves. Equimolar quantities of abG1IGF-1 or bedG1IGF-1 and rhIGFBP-2 were allowed to attain equilibrium binding by co-incubation for 4 h at 23 °C in 100 mm HEPES (pH 7.4) containing 44 mm NaHCO3 and 0.01% Triton X-100. The sample was then placed in ice water and irradiated for 2 h with a prewarmed Fotodyne hand-held, single-wavelength UV lamp (2 × 4-watt 300-nm bulbs) at a distance of 2 cm. The mixture was dried in vacuo, and the proteins were reduced and alkylated using tris(2-carboxyethylyl)phosphine (32Han J.C. Han G.Y. Anal. Biochem. 1994; 220: 5-10Crossref PubMed Scopus (287) Google Scholar) and 4-vinylpyridine (33Liu C. Bowers L.D. J. Mass Spectrom. 1997; 32: 33-42Crossref PubMed Scopus (45) Google Scholar). abG1IGF-1- and bedG1IGF-1-photolabeled IGFBP-2 proteins were separated from unreacted IGFBP-2 by reversed-phase HPLC and trypsinized as described below. Trypsinization of reduced and alkylated proteins was performed according to Honegger and Humbel (34Honegger A. Humbel R.E. J. Biol. Chem. 1986; 261: 569-575Abstract Full Text PDF PubMed Google Scholar). Proteins (20 μm) were dissolved in 100 mm N-ethylmorpholine acetate (pH 8.5) to which was added sufficient trypsin (1 μg/μl) to achieve a 1:50 enzyme/substrate ratio. Mixtures were then incubated for 2 h at 37 °C, followed by a second addition of trypsin. After 2 additional h at 37 °C, the reaction was stopped by the addition of an equal volume of 0.1% trifluoroacetic acid. The mixture was dried in vacuo or applied directly to a C18 reversed-phase HPLC column. Tryptic peptides generated from the bedG1IGF-1·IGFBP-2 complex were applied to an UltraLinkTM monomeric avidin column. Flow-through fractions were collected and pooled. The column was eluted with low pH buffer, and the eluate fractions were pooled. The flow-through and eluate fractions were dried and further analyzed by reversed-phase HPLC on a C18 column equilibrated in 0.1% trifluoroacetic acid and eluted with a linear gradient of acetonitrile. Eluted peaks were analyzed by MALDI-TOF-MS. Samples were dissolved in SDS sample buffer with or without dithiothreitol and resolved on a 10 or 12.5% SDS-polyacrylamide gel according to the procedure of Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) using a Hoefer Scientific Instruments apparatus. The proteins were transferred to nitrocellulose and immunoblotted using a commercial antiserum against intact bovine IGFBP-2 (Upstate Biotechnology, Inc., Lake Placid, NY). Blots were developed with horseradish peroxidase-labeled secondary antibody (Chemicon International, Inc., Temecula, CA) and an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). For sequential anti-IGF-1/anti-IGFBP-2 antibody analysis of the same blot, the membrane was stripped for 20 min at 60 °C in 1m Tris (pH 6.7) containing 10% SDS and 0.1 mβ-mercaptoethanol. The stripped membrane was washed twice with Tris-buffered saline containing Tween for 10 min at 23 °C, followed by blocking for 1 h with 5% nonfat dry milk and reprobed. As shown in Fig.1, the three-dimensional structure of IGF-1 reveals the presence of distinct binding domains for the IGFBPs and the IGF-1R, which do not overlap. IGF-1 contains four primary amines (the ε-amino groups of 3 lysyl residues and the α-amino group of Gly1), all of which are reactive with HSAB and SBED. Fig. 2 shows the structures of these reagents and highlights the relative lengths of the spacer arms from the site of covalent linkage to IGF-1 to the photoreactive azide group. In addition to a significantly longer spacer arm, SBED also contains a reduction-sensitive disulfide linkage and a biotin moiety. As a result, although we anticipated that use of HSAB as a photocross-linking agent would yield a reduction-stable covalent IGF-1·IGFBP-2 complex, use of SBED was expected to produce a reduction-sensitive IGF-1·IGFBP-2 complex that, after reduction, would result in biotinylation of IGFBP-2 at the site of photoincorporation. As shown in Fig. 2, reaction of HSAB and SBED with IGF-1 as described under “Experimental Procedures” resulted in production of three major products (peaks 2–4) in addition to unreacted IGF-1 (peak 1). Analysis of the HSAB reaction products by acidic polyacrylamide gel electrophoresis in 8 m urea and by MALDI-TOF-MS revealed that these three peaks were monoderivatized forms of IGF-1 (data not shown). Peak 2 had previously been shown to represent the elution position of abK27IGF-1 (where K27 is Lys27) (36Yip C.C. Hsu H. Olefsky J. Seely L. Peptides (Elmsford). 1993; 14: 325-330Crossref PubMed Scopus (4) Google Scholar). Peaks 3 and 4 had been tentatively identified as abK65IGF-1 and abG1IGF-1, respectively, based on amino acid sequencing analysis (36Yip C.C. Hsu H. Olefsky J. Seely L. Peptides (Elmsford). 1993; 14: 325-330Crossref PubMed Scopus (4) Google Scholar). No abK68IGF-1 was detected. The three product peaks were further purified by chromatography on a C18 column equilibrated in triethanolamine phosphate and acetonitrile. Structural assignments were confirmed using pepsin digestion as described below. After derivatization with SBED, peaks 2–4 were shown to similarly represent bedK27IGF-1, bedK65IGF-1, and bedG1IGF-1 (Fig. 2 B). To verify that the “ab” and “bed” moieties were covalently bound to the α-amino group of Gly1 for each photoprobe, it was necessary to isolate Gly1 from the 3 Lys residues. Since reduction and alkylation might disrupt the azide moiety, we utilized pepsin digestion of nonreduced abG1IGF-1 and bedG1IGF-1. As reported by Forsberg et al. (28Forsberg G. Gunnar P. Ekebacke A. Josephson S. Hartmanis M. Biochem. J. 1990; 271: 357-363Crossref PubMed Scopus (44) Google Scholar) and shown in Fig.3, pepsin releases a disulfide-linked AE fragment containing Gly1 and no Lys residues. Pepsin digestion of abG1IGF-1 and bedG1IGF-1 was carried out in the dark to avoid photoactivation of the probe, and recombinant human IGF-1 was also digested in parallel to serve as a control for subsequent HPLC and mass spectrometric analyses. For each photoprobe, HPLC purification of the pepsin digestion products revealed that the retention times for the derivatized AE fragments were significantly increased compared with the underivatized AE fragment (data not shown). This was predicted based on the added hydrophobicity of the additional functional groups. Fig.4 A shows the MALDI-TOF-MS of abG1AE. This fragment had the correct mass (predicted average mass of 2419.7 Da; observed mass of 2419.8 Da) and exhibited loss of nitrogen as a result of photoactivation by the 337-nm laser, to yield a second peak at 2394.3 Da. The identities of the CG and D fragments, containing Lys65/Lys68 and Lys27, respectively, were also confirmed by MALDI-TOF-MS and were in good agreement with those of Forsberg et al.(28Forsberg G. Gunnar P. Ekebacke A. Josephson S. Hartmanis M. Biochem. J. 1990; 271: 357-363Crossref PubMed Scopus (44) Google Scholar). Similar observations were made for bedG1IGF-1. As shown in Fig. 5, a peak at 2956.2 Da (predicted average mass of 2955.9 Da) was observed along with a second peak at 2929.4 Da, reflecting the loss of nitrogen resulting from photolysis. Again," @default.
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• W2036763753 title "Synthesis and Characterization of Insulin-like Growth Factor (IGF)-1 Photoprobes Selective for the IGF-binding Proteins (IGFBPs)" @default.
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