Effects of lipids on ATPase activity of purified Chinese hamster P-glycoprotein.
Effects of lipids on ATPase activity of purified Chinese hamster P-glycoprotein.
复制标题
脂质对纯化的中国仓鼠 P-糖蛋白 ATP 酶活性的影响。
DOI：
10.1006/abbi.1995.1020
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发表时间：
1995
影响因子：
3.9
通讯作者：
Senior,AE
中科院分区：
文献类型：
--
作者：
Urbatsch,IL;Senior,AE
中国仓鼠P-糖蛋白(“多药耐药蛋白”)是用I.L.Urbatsch，M.K.Al-Shawl和A.E.High(1994，生物化学33,7069-7076)纯化并重组到蛋白脂质体中的。在辛基糖苷增溶和活性红120层析步骤中，脂质的存在被发现是保留ATPase活性所必需的。羊脑或牛肝脂提取物可以替代以前使用的大肠杆菌脂类，长春花碱、秋水仙碱和柔红霉素对纯化的重组P-糖蛋白的ATPase活性有刺激作用，对羊脑和牛肝脂类有刺激作用，但对大肠杆菌脂类没有影响。基础(即不受药物刺激)ATPase活性在三种脂类中是不同的。对纯化的重组P-糖蛋白中的药物结合部位进行了阿奇多丁标记；长春花碱、秋水仙碱和柔红霉素在所有三种脂类中竞争标记。因此，脂类环境可以显著影响纯化的重组P-糖蛋白ATPase活性的特征以及药物结合和催化部位之间的明显偶联。
Chinese hamster P-glycoprotein ("multidrug-resistance protein") was purified and reconstituted in proteoliposomes by the procedure of I. L. Urbatsch, M. K. Al-Shawl, and A. E. Senior (1994, Biochemistry 33, 7069-7076). The presence of lipid during the octylglucoside solubilization and Reactive Red 120 chromatography steps was found to be mandatory for retention of ATPase activity. Sheep brain or bovine liver lipid extracts could be substituted for the Escherichia coli lipids used previously, Stimulation of ATPase activity of purified, reconstituted P-glycoprotein by vinblastine, colchicine, and daunomycin was seen with sheep brain and bovine liver lipids, but not with E. coli lipids. Basal (i.e., not drug-stimulated) ATPase activity was different in the three lipids. Azidopine labeling of the drug binding sites in purified, reconstituted P-glycoprotein was carried out; vinblastine, colchicine, and daunomycin competed for labeling in all three lipids. It is therefore evident that the lipid environment can significantly influence the characteristics of purified, reconstituted P-glycoprotein ATPase activity and the apparent coupling between drug-binding and catalytic sites.