Urbatsch I L, al-Shawi M K, Senior A E
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Biochemistry. 1994 Jun 14;33(23):7069-76. doi: 10.1021/bi00189a008.
A simple and rapid procedure is described for purification of P-glycoprotein (Pgp) from a multidrug-resistant Chinese hamster ovary cell line (CR1R12) in which the plasma membranes are highly enriched in Pgp (Al-Shawi, M.K., Senior A.E. (1993) J. Biol. Chem, 268, 4197-4206). The procedure consisted of octylglucoside solubilization of Pgp from plasma membranes and chromatography on Reactive Red 120 agarose. The purified Pgp displayed substantial verapamil-stimulated MgATPase activity (kcat = 9.2 s-1, KM(MgATP) = 0.8 mM). A range of other compounds known to interact with Pgp in whole cells also stimulated the MgATPase activity. Catalytic activity in presence of verapamil was characterized in terms of pH dependence, magnesium versus calcium specificity, kinetic parameters, nucleotide specificity, and inhibitors. There was potent inactivation of MgATPase activity by NEM and NBD-Cl, which was diminished greatly by MgATP protection. Vanadate was also an effective inhibitor. Predominantly, the catalytic features seen resembled those reported previously for the plasma membrane-bound form of Pgp. The catalytic nucleotide-binding sites are therefore preserved in their native folded conformation in the purified Pgp preparation.
本文描述了一种简单快速的方法，用于从多药耐药的中国仓鼠卵巢细胞系（CR1R12）中纯化P-糖蛋白（Pgp），该细胞系的质膜中富含Pgp（Al-Shawi, M.K., Senior A.E.（1993）J. Biol. Chem, 268, 4197 - 4206）。该方法包括用辛基葡糖苷从质膜中溶解Pgp，并在活性红120琼脂糖上进行色谱分离。纯化后的Pgp表现出显著的维拉帕米刺激的MgATP酶活性（kcat = 9.2 s-1，KM(MgATP) = 0.8 mM）。一系列已知在完整细胞中与Pgp相互作用的其他化合物也刺激了MgATP酶活性。在维拉帕米存在下的催化活性通过pH依赖性、镁与钙的特异性、动力学参数、核苷酸特异性和抑制剂进行了表征。NEM和NBD-Cl对MgATP酶活性有强烈的失活作用，而MgATP保护可大大减弱这种作用。钒酸盐也是一种有效的抑制剂。主要地，观察到的催化特征与先前报道的质膜结合形式的Pgp相似。因此，在纯化的Pgp制剂中，催化核苷酸结合位点以其天然折叠构象得以保留。
