Ambudkar S V, Lelong I H, Zhang J, Cardarelli C O, Gottesman M M, Pastan I
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8472-6. doi: 10.1073/pnas.89.18.8472.
Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl beta-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine greater than daunomycin greater than actinomycin D greater than verapamil greater than colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 microM) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 mumol per min per mg of protein).
多药耐药的人类肿瘤细胞过度表达MDR1基因产物P-糖蛋白，据信该蛋白作为一种依赖ATP的外排泵发挥作用。在本研究中，我们证明部分纯化的P-糖蛋白在人工膜中重构后，可催化药物刺激的ATP水解。用人多药耐药细胞系KB-V1的质膜蛋白在0.4%磷脂和20%（体积/体积）甘油存在的情况下，用1.4%（重量/体积）辛基β-D-吡喃葡萄糖苷进行增溶，粗去污剂提取物在DEAE-琼脂糖CL-6B上进行层析。富含P-糖蛋白但不含钠钾ATP酶的0.1M NaCl级分，通过去污剂稀释法进行重构。P-糖蛋白在蛋白脂质体中占重构蛋白的25%-30%。加入长春碱后，蛋白脂质体的ATP水解受到3.5倍的刺激，但不受疏水性抗肿瘤药物喜树碱的影响，喜树碱不被P-糖蛋白转运。仅当蛋白在蛋白脂质体中重构时才观察到长春碱的刺激作用，这表明要么底物结合位点被去污剂掩盖，要么可溶性P-糖蛋白的构象可能不适合底物诱导的激活。已知其他几种由P-糖蛋白转运的药物以剂量依赖方式增强ATP酶活性，相对效力如下：阿霉素 = 长春碱 > 柔红霉素 > 放线菌素D > 维拉帕米 > 秋水仙碱。钒酸盐抑制基础和长春碱刺激的ATP酶活性（在7-10微摩尔时观察到50%抑制），但不受抑制其他ATP酶和磷酸酶的试剂的影响。这些数据表明，P-糖蛋白与其他离子转运ATP酶类似，表现出高水平的ATP水解（每毫克蛋白每分钟5-12微摩尔）。
