Abstract
A G-protein-coupled receptor-cell-membrane stationary phase (CMSP) has been prepared by immobilizing cell membranes on the surface of silica, as carrier. The resulting HEK293 alpha1A adrenoceptor cell-membrane stationary phase can be used for rapid on-line chromatographic determination of potential subtype-selective alpha1-adrenoceptor ligand-binding affinities for alpha 1-adrenoceptor subtypes. The objective of the research was to study whether cell lines stably overexpressing subtype receptors could improve the sensitivity and specificity of cell-membrane chromatography (CMC) compared with use of homogenized tissue and cells in primary culture. Effects of mobile-phase ionic strength, sample concentration, and the presence of competitive agents on ligand-receptor interaction in CMSP were also evaluated. We found that cell lines stably overexpressing subtype receptors led to improved sensitivity and specificity in CMC. The technique leads to useful procedures-cell-membrane stationary phases may, for example, facilitate exploration of ligand-receptor interaction and determination of ligand-receptor binding affinity in initial screening and separation of lead compounds or active components in Chinese traditional natural medicine and herbs. This might eventually be an important contribution and an addition to our collection of techniques.
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