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Synthesis and properties of fluorescent ß-adrenoceptor ligands

MPS-Authors
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Hallmann,  Dieter
Department of Membrane Biophysics, MPI for biophysical chemistry, Max Planck Society;

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Reiländer,  Helmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Arndt-Jovin,  Donna J.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Jovin,  Thomas M.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Citation

Heithier, H., Hallmann, D., Boege, F., Reiländer, H., Dees, C., Jaeggi, K. D., et al. (1994). Synthesis and properties of fluorescent ß-adrenoceptor ligands. Biochemistry, 33(31), 9126-9134. doi:10.1021/bi00197a015.


Cite as: http://hdl.handle.net/21.11116/0000-0007-B39A-1
Abstract
We describe the synthesis of bordifluoropyrromethene (BODIPY), fluorescein, and related fluorescent derivatives of the beta-adrenergic ligand CGP 12177. With these probes we screened insect (Sf9) cells stably transformed with the human ß2-adrenoceptor gene and expressing (2-3.5) x 105 human ß2-adrenoceptors per cell. Among these derivatives only BODIPY-CGP gave a receptor-specific signal sufficiently strong for measuring the on- and off-rate constants and the equilibrium dissociation constant of beta-adrenoceptor-specific binding by spectrofluorometry or photon counting. Similar KD values for BODIPY-CGP binding were obtained by kinetic measurements (approx. 250 pM) and under equilibrium conditions (400 +/- 180 pM), and these were in the same range as those obtained with [3H]CGP 12177 (200 +/- 32 pM). The cell-bound fluorescence could be quenched specifically with nonfluorescent CGP 12177 to near background levels. The disposition of the ß2-adrenoceptors in BODIPY-CGP-stained Sf9 cells was mainly restricted to the cell surface at 4 and 30 degrees C. Hence, beta-adrenoceptor-expressing cells can be stained specifically with BODIPY-CGP, and beta-adrenoceptors on a single cell can be assessed by photon counting under the fluorescence microscope. Cells can also be scanned by fluorescence-activated flow cytometry.