Senior A E, al-Shawi M K, Urbatsch I L
Department of Biochemistry and Biophysics, University of Rochester Medical Center, New York 14642, USA.
Methods Enzymol. 1998;292:514-23. doi: 10.1016/s0076-6879(98)92040-7.
We have developed two defined experimental systems for biochemical investigation of P-glycoprotein, namely, plasma membranes highly enriched in Pgp, obtained from the CR1R12 Chinese hamster ovary cell line, and pure, reconstituted Pgp, obtained by solubilization of Pgp from CR1R12 plasma membranes, Reactive Red 120 chromatography, and reconstitution in liposomes. Studies of the ATPase catalytic mechanism by kinetic methods and covalent inactivation have been greatly facilitated by the availability of these experimental systems. The technique of vanadate trapping of nucleotide has been particularly useful. As a result of these studies, we now have explicit, testable, proposals for (1) the normal catalytic pathway of ATP hydrolysis, (2) a postulated alternating catalytic site cycle, and (3) coupling of ATP hydrolysis to drug transport. The experimental methods described here should prove valuable for future studies of Pgp and of ABC transporters in general.
我们开发了两种用于P-糖蛋白生化研究的特定实验系统，即从CR1R12中国仓鼠卵巢细胞系获得的高度富含Pgp的质膜，以及通过从CR1R12质膜溶解Pgp、活性红120色谱法和在脂质体中重构而获得的纯化、重构的Pgp。这些实验系统的可用性极大地促进了通过动力学方法和共价失活对ATP酶催化机制的研究。钒酸盐捕获核苷酸的技术特别有用。这些研究的结果使我们现在对以下方面有了明确的、可检验的提议：（1）ATP水解的正常催化途径，（2）假定的交替催化位点循环，以及（3）ATP水解与药物转运的偶联。这里描述的实验方法对于未来Pgp和一般ABC转运蛋白的研究应该是有价值的。
