SYNTHESIS AND PROPERTIES OF FLUORESCENT BETA-ADRENOCEPTOR LIGANDS
SYNTHESIS AND PROPERTIES OF FLUORESCENT BETA-ADRENOCEPTOR LIGANDS
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DOI：
10.1021/bi00197a015
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发表时间：
1994-08-09
期刊：
影响因子：
2.9
通讯作者：
HELMREICH, EJM
中科院分区：
文献类型：
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作者：
HEITHIER, H;HALLMANN, D;HELMREICH, EJM
我们描述了合成bordifluoropyrromethene（BODIPY），荧光素，和相关的β-肾上腺素能配体CGP 12177的荧光衍生物。用这些探针，我们筛选了用人β（2）-肾上腺素受体基因稳定转化的昆虫（Sf 9）细胞，每个细胞表达（2-3.5）× 10（5）人β（2）-肾上腺素受体。在这些衍生物中，只有BODIPY-CGP给出了足够强的受体特异性信号，用于通过荧光分光光度法或光子计数法测量β-肾上腺素受体特异性结合的开和关速率常数和平衡解离常数。通过动力学测量获得了BODIPY-CGP结合的类似K-D值（约100%）。250 pM）和在平衡条件下（400 +/-180 pM），并且这些在与用[H-3]CGP 12177获得的那些（200 +/-32 pM）相同的范围内。非荧光CGP 12177可以特异性地将细胞结合的荧光猝灭至接近背景水平。在4 ℃和30 ℃下，BODIPY-CGP染色的Sf 9细胞中β 2-肾上腺素受体的分布主要局限于细胞表面。因此，β-肾上腺素受体表达细胞可以用BODIPY-CGP特异性染色，并且单个细胞上的β-肾上腺素受体可以通过在荧光显微镜下的光子计数来评估。也可以通过荧光激活流式细胞术扫描细胞。
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 beta(2)-adrenoceptor gene and expressing (2-3.5) x 10(5) human beta(2)-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 K-D 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 [H-3]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 beta(2)-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.