Quantitative analysis of EGFR affinity to immobilized glycolipids by surface plasmon resonance |
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Authors: | Haga Yoshimi Hakomori Sen-itiroh Hatanaka Kenichi |
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Institution: | a Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan b Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA c Department of Microbiology, University of Washington, Seattle, WA 98195, USA |
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Abstract: | EGF-induced activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, its dimer, and other mimetics. However, details of the interaction, such as kinetic properties, have not yet been clarified. The direct interaction is now defined by the surface plasmon resonance (SPR) technique. To determine the affinity of EGFR for lyso-GM3 or lyso-GM3 mimetic, these glycolipid ligands were covalently immobilized onto a sensor chip, and binding affinities were investigated. Results of these studies confirmed the direct interaction of lyso-GM3 or its mimetic with EGFR. A strong interaction between EGFR and lyso-GM3 or its mimetic was indicated by increased binding of EGFR to glycolipid-immobilized surface, in an EGFR dose-dependent manner. |
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Keywords: | BSA bovine serum albumin DMEM Dulbecco&rsquo s modified Eagle&rsquo s medium EGFR epidermal growth factor receptor ESIMS electrospray ionization mass spectrometry FBS fetal bovine serum GM3 NeuAcα3Galβ4Glcβ1Cer MAM Maackia amurensis lectin PBS phosphate-buffered saline PNGase peptide-N-glycosidase SPR surface plasmon resonance RU resonance unit Kd dissociation rate constants Ka association rate constants |
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