P-glycoprotein senses its substrates and the lateral membrane packing density:: Consequences for the catalytic cycle
P-glycoprotein senses its substrates and the lateral membrane packing density:: Consequences for the catalytic cycle
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DOI：
10.1021/bi800209h
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发表时间：
2008-09-23
期刊：
影响因子：
2.9
通讯作者：
Seelig, Anna
中科院分区：
文献类型：
--
作者：
Aanismaa, Paivi;Gatlik-Landwojtowicz, Ewa;Seelig, Anna
P-糖蛋白（ABCB 1）阻止吸收（例如，血脑屏障）或增强排泄（例如，肾）通过以ATP水解为代价将底物从胞质移动到细胞外膜小叶。它在表现出不同横向堆积密度的膜中转运各种药物和功能。为了获得更多的功能洞察力，我们测量了温度依赖性的P-糖蛋白ATP酶活性在NIH-MDRI-G185细胞膜的存在和不存在三种药物（丙嗪，维拉帕米，和PSC 833），表现出显着不同的转运亲和力。活化熵（Δ H（double dagger））和熵（T Δ S（double dagger））来自Eyring图。在没有药物的情况下，P-糖蛋白ATP酶活性的活化焓和活化自由能分别测定为Δ H（双匕首）= 92.6 +/-4.2 kJ/mol和Δ G（双匕首）= 73.1 +/-7.2 kJ/mol。增加药物浓度降低了活化焓，由此具有最高转运蛋白亲和力的药物具有最强的作用（Δ Δ H（双匕首）=-21%）。活化的自由能对于活化降低（Δ Δ G（双匕首）=类似于-3.8%）并且对于抑制性化合物增加（Δ Δ G（双匕首）=类似于+0.7%）。因此，活化自由能的药物特异性变化仅略高于热能。与文献数据的比较表明，侧膜堆积密度的降低降低了活化自由能的熵和熵贡献。虽然P-糖蛋白ATP酶活性随着侧膜堆积密度的降低仅略有增加，但作用模式从高堆积密度下的强烈熵驱动变为低堆积密度下的基本上熵驱动。这表明转运蛋白和膜形成了一个功能实体。
P-glycoprotein (ABCB1) prevents absorption (e.g., blood-brain barrier) or enhances excretion (e.g., kidney) by moving substrates from the cytosolic to the extracellular membrane leaflet at the expense of ATP hydrolysis. It translocates various drugs and functions in membranes exhibiting different lateral packing densities. To gain more functional insight, we measured the temperature dependence of the P-glycoprotein ATPase activity in NIH-MDRI-G185 cell membranes in the absence and presence of three drugs (promazine, verapamil, and PSC833), exhibiting significantly different transporter affinities. Activation enthalpies (Delta H(double dagger)) and entropies (T Delta S(double dagger)) were derived from Eyring plots. In the absence of drugs, the activation enthalpy and the free energy of activation for P-glycoprotein ATPase activity was determined as Delta H(double dagger) = 92.6 +/- 4.2 kJ/mol and Delta G(double dagger) =73.1 +/- 7.2 kJ/mol, respectively. Increasing the drug concentration reduced the activation enthalpy, whereby the drug with the highest transporter affinity had the strongest effect (Delta Delta H(double dagger) = -21 %). The free energy of activation decreased for activating (Delta Delta G(double dagger)=similar to-3.8%) and increased for inhibitory compounds (Delta Delta G(double dagger)=similar to+0.7%). The drug-specific changes of the free energy of activation are thus barely above thermal energy. A comparison with literature data revealed that a decrease of the lateral membrane packing density reduces the enthalpic and the entropic contribution to the free energy of activation. Although the P-glycoprotein ATPase activity increases only slightly with decreasing lateral membrane packing density, the mode of action changes from strongly entropy-driven at high, to essentially enthalpy-driven at low packing densities. This Suggests that the transporter and the membrane form a functional entity.