G-Protein-Coupled Receptor Chromatographic Stationary Phases. 2. Ligand-Induced Conformational Mobility in an Immobilized 
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- 作者：Farideh Beigi ; Khalid Chakir ; Rui-Ping Xiao ; and Irving W. Wainer
- 刊名：Analytical Chemistry
- 出版年：2004
- 出版时间：December 15, 2004
- 年：2004
- 卷：76
- 期：24
- 页码：7187 - 7193
- 全文大小：255K
- 年卷期：v.76,no.24(December 15, 2004)
- ISSN：1520-6882
文摘
Membranes from a HEK-293 cell line expressing the 
2-adrenergic receptor (2-AR) have been immobilized on anartificial membrane liquid chromatographic stationaryphase. The resulting phase was packed into a glasscolumn (1.8 × 0.5 (i.d.) cm) and used in on-line chromatographic system. Frontal displacement affinity chromatography was used to determine the dissociationconstants (Kd) of CGP 12177A (552.6 nM) and (S)-propranolol (84.3 nM). Zonal displacement chromatography using CGP 12177A as the marker and racemicmixtures of the antagonists nadolol and propranololdemonstrated that the immobilized 2-AR retained itsability to specifically bind these compounds. Similarexperiments with (R)- and (S)-propranolol demonstratedthat the immobilized receptor retained its enantioselectivity as (S)-propranolol displaced the CGP 12177 markerto a great extent that the (R)-enantiomer. The addition ofthe agonist butoxamine to the mobile phase increased theretention of the CGP-12177A as did the addition of theagonist fenoterol. These results indicate that the immobilized 2-AR retained its ability to undergo ligand-induced conformational changes. The data from this studysuggest that the immobilized 2-AR can be used to screenfor ligand binding interactions in both the resting andactive states of the receptor.
2-adrenergic receptor (2-AR) have been immobilized on anartificial membrane liquid chromatographic stationaryphase. The resulting phase was packed into a glasscolumn (1.8 × 0.5 (i.d.) cm) and used in on-line chromatographic system. Frontal displacement affinity chromatography was used to determine the dissociationconstants (Kd) of CGP 12177A (552.6 nM) and (S)-propranolol (84.3 nM). Zonal displacement chromatography using CGP 12177A as the marker and racemicmixtures of the antagonists nadolol and propranololdemonstrated that the immobilized 2-AR retained itsability to specifically bind these compounds. Similarexperiments with (R)- and (S)-propranolol demonstratedthat the immobilized receptor retained its enantioselectivity as (S)-propranolol displaced the CGP 12177 markerto a great extent that the (R)-enantiomer. The addition ofthe agonist butoxamine to the mobile phase increased theretention of the CGP-12177A as did the addition of theagonist fenoterol. These results indicate that the immobilized 2-AR retained its ability to undergo ligand-induced conformational changes. The data from this studysuggest that the immobilized 2-AR can be used to screenfor ligand binding interactions in both the resting andactive states of the receptor.