期刊
ANALYTICAL BIOCHEMISTRY
卷 402, 期 1, 页码 32-39出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.03.022
关键词
Fluorescence anisotropy; G protein-coupled receptors; Melanocortin 4 receptor; Cy3B-NDP-alpha-MSH; TAMRA-NDP-alpha-MSH; Ligand binding kinetics
资金
- Estonian Science Foundation [7569]
- Estonian Ministry of Education and Science [0182734s06]
Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC4) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligand, Cy3B-NDP-alpha-MSH and TAMRA-NDP-alpha-MSH. were used and compared in assays that monitored their binding to MC4 receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC4 receptors was saturable and with high affinity. All studied MC4 receptor-specific nonlabeled ligands displaced fluoroligands' binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3 h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters. (C) 2010 Elsevier Inc. All rights reserved.
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