Moaddel Ruin, Wainer Irving W
Gerontology Research Center, National Institutes in Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
J Pharm Biomed Anal. 2007 Jan 17;43(2):399-406. doi: 10.1016/j.jpba.2006.08.021. Epub 2006 Nov 13.
Cellular membrane fragments have been immobilized on the surface of a silica-based liquid chromatographic support and on the surface of glass capillaries to create immobilized receptor and drug transporter columns. These columns have included phases containing one subtype of the nicotinic receptor (alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4) and the P-glycoprotein transporter. A key question in the application of these columns to drug discovery and development is the ability of the immobilized receptor or transporter to undergo ligand and/or co-factor induced conformational changes. Using frontal affinity chromatographic techniques and non-linear chromatographic techniques it has been demonstrated that the immobilized nicotinic receptors undergo agonist-induced conformational shifts from the resting to desensitized states with corresponding changes in binding affinities and enantioselectivities. Ligand-induced allosteric interactions and ATP-driven conformational changes have also been demonstrated with the immobilized Pgp stationary phase. The results demonstrate that the immobilized proteins retained their ability to undergo conformational mobility and that this is an attractive alternative to allow for the full characterization of multiple protein conformations.
细胞膜片段已固定在硅胶基液相色谱载体表面和玻璃毛细管表面，以制备固定化受体和药物转运体柱。这些柱包含含有一种烟碱样受体亚型（α3β2、α3β4、α4β2、α4β4）和P-糖蛋白转运体的固定相。将这些柱应用于药物发现和开发的一个关键问题是固定化受体或转运体经历配体和/或辅因子诱导的构象变化的能力。使用前沿亲和色谱技术和非线性色谱技术已证明，固定化烟碱样受体经历激动剂诱导的从静息状态到脱敏状态的构象转变，同时结合亲和力和对映体选择性发生相应变化。固定化Pgp固定相也已证明配体诱导的变构相互作用和ATP驱动的构象变化。结果表明，固定化蛋白保留了其进行构象移动的能力，这是一种有吸引力的替代方法，可用于全面表征多种蛋白构象。
