P-glycoprotein in proteoliposomes with low residual detergent:: The effects of cholesterol
P-glycoprotein in proteoliposomes with low residual detergent:: The effects of cholesterol
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DOI：
10.1007/s11095-007-9326-0
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发表时间：
2007-11-01
影响因子：
3.7
通讯作者：
Kramer, Stefanie D.
中科院分区：
文献类型：
--
作者：
Bucher, Karsten;Belli, Sara;Kramer, Stefanie D.
有证据表明胆固醇影响P-糖蛋白（P-gp）的ATP酶和转运功能。为了研究胆固醇对脂质环境中P-gp的影响，我们用卵磷脂（PhC）和PhC/胆固醇蛋白脂质体（PhC/cholesterol proteoliposomes）重构了P-gp，并通过连续透析法从P-gp、脂质、十二烷基硫酸钠和胆酸盐组成的胶束制备了P-gp蛋白脂质体。在已建立的酶测定中研究了基础和调节剂诱导的ATP酶活性。调节剂的亲和力P-gp和脂质bilayers. In平衡dialysis.In胆固醇的基础ATP酶活性的情况下是6倍低于在存在20或40%的胆固醇，和没有P-gp结合和ATP酶诱导检测到的测试调制器维拉帕米和孕酮。在分别含有20%和40%胆固醇的脂蛋白体中，调节剂显示出显著的P-gp结合和ATP酶激活。胆固醇以三种方式影响P-gp：（a）增强其基础ATP酶活性，（B）使P-gp对调节剂维拉帕米和孕酮敏感，（c）影响ATP酶半数激活时的调节剂浓度。
There is evidence that cholesterol affects the ATPase and transport functions of P-glycoprotein (P-gp). To study the influence of cholesterol on P-gp in a well defined lipid environment, we reconstituted P-gp in egg phosphatidylcholine (PhC) and PhC/cholesterol proteoliposomes with negligible residual amounts of detergents.P-gp proteoliposomes were prepared by continuous dialysis from micelles consisting of P-gp, lipids, sodium dodecyl sulfate and cholate. Basal and modulator-induced ATPase activities were studied in an established enzyme assay. Modulator affinities to P-gp and to the lipid bilayers were determined by equilibrium dialysis.In the absence of cholesterol the basal ATPase activity was six fold lower than in the presence of 20 or 40% cholesterol, and no P-gp binding and ATPase induction was detected for the tested modulators verapamil and progesterone. In proteoliposomes containing 20 and 40% cholesterol, respectively, the modulators showed significant P-gp binding and ATPase activation. The concentration of the modulators for half maximal activation of the ATPase was higher with 40% than with 20% cholesterol.Cholesterol influences P-gp in three ways: (a) it enhances its basal ATPase activity, (b) it renders P-gp sensitive towards the modulators verapamil and progesterone and (c) it affects the modulator concentration at half maximal ATPase activation.