Doige C A, Yu X, Sharom F J
Guelph-Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Canada.
Biochim Biophys Acta. 1992 Aug 24;1109(2):149-60. doi: 10.1016/0005-2736(92)90078-z.
In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.
对多药耐药细胞系的体外研究表明，一种膜蛋白——P-糖蛋白，对多种结构和功能各异的抗癌药物具有耐药性。P-糖蛋白（Pgp）的氨基酸序列显示出两个ATP结合共有序列，据报道纯化后的该蛋白具有低水平的ATP酶活性。作为我们进一步表征P-糖蛋白ATP酶活性目标的一部分，我们开发了一种程序，可快速部分纯化出高活性形式的该蛋白。来自多药耐药的CHRC5中国仓鼠卵巢细胞的质膜囊泡经过两步程序处理，该程序包括用不同浓度的两性离子去污剂CHAPS进行选择性提取。所得提取物富含P-糖蛋白（纯度约为30%），并显示出一种ATP酶活性（比活性为543 nmol mg-1 min-1），而在来自药物敏感细胞的类似制剂中未发现这种活性。该ATP酶比活性比先前报道的免疫沉淀Pgp高10倍以上，比免疫亲和纯化的Pgp高280倍。这种ATP酶活性可与其他离子驱动ATP酶和膜相关磷酸酶的活性区分开来，因此，有人提出它直接归因于P-糖蛋白。P-糖蛋白ATP酶的最佳活性需要Mg2+，ATP与Mg2+的摩尔比为0.75:1，ATP的表观Km为0.88 mM。P-糖蛋白ATP酶可被钒酸盐以及巯基修饰试剂N-乙基马来酰亚胺、HgCl2和对氯汞苯磺酸盐完全抑制。某些药物和化学增敏剂，包括秋水仙碱、孕酮、硝苯地平、维拉帕米和三氟拉嗪，可使P-糖蛋白ATP酶活性激活高达50%。
