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Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader

Ray, Manisha ; Nagai, Kazufumi ; Kihara, Yasuyuki ; Kussrow, Amanda ; Kammer, Michael N. ; Frantz, Aaron ; Bornhop, Darryl J. ; Chun, Jerold

Journal of lipid research, 2020-08, Vol.61 (8), p.1244-1251 [Periódico revisado por pares]

United States: Elsevier Inc

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  • Título:
    Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader
  • Autor: Ray, Manisha ; Nagai, Kazufumi ; Kihara, Yasuyuki ; Kussrow, Amanda ; Kammer, Michael N. ; Frantz, Aaron ; Bornhop, Darryl J. ; Chun, Jerold
  • Assuntos: free-solution assay-compensated interferometric reader ; G protein-coupled receptor ; interferometry ; lipid signaling ; lysophospholipids ; Methods ; molecular interaction ; receptor binding assay
  • É parte de: Journal of lipid research, 2020-08, Vol.61 (8), p.1244-1251
  • Notas: ObjectType-Article-1
    SourceType-Scholarly Journals-1
    ObjectType-Feature-2
    content type line 23
    Present address of K. Nagai: Ono Pharmaceutical Company, Ltd., Osaka 618-8585, Japan.
  • Descrição: Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.
  • Editor: United States: Elsevier Inc
  • Idioma: Inglês
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