Santai C T, Fritz F, Roepe P D
Department of Chemistry, Lombardi Cancer Center Program in Tumor Biology, Georgetown University, Washington, D.C. 20057, USA.
Biochemistry. 1999 Mar 30;38(13):4227-34. doi: 10.1021/bi981930m.
In the previous paper we presented a variety of data consistent with significant perturbations in 9.3 yeast plasma membrane ion transport upon overexpression of the hu MDR 1 protein. Thus, in this paper, we compare formation of DeltapH for inside-out yeast plasma membrane vesicles (ISOV) prepared from control 9.3/pVT versus 9.3/hu MDR 1 yeast. Since MDR 1 ATPase activity has a broader, more alkaline pH profile relative to endogenous yeast H+ ATPase activity, we analyzed H+ pumping at pH >/= 8.0 in detail in order to selectively amplify hu MDR 1 contributions to H+ movement over those of the endogenous yeast H+ ATPase. We observed: (1) imposition of a Cl- gradient oriented outside to in enhances acidification for 9.3/pVT ISOV (as expected), but decreases acidification for 9.3/hu MDR 1 ISOV; (2) imposition of a Cl- gradient oriented inside to out decreases acidification for 9.3/pVT ISOV (as expected) but enhances acidification for 9.3/hu MDR 1 ISOV; (3) a Na+ gradient oriented in the same direction as the Cl- gradient amplifies the effects due to hu MDR 1 when both gradients are oriented inside to out, but not outside to in. The data are most easily explained by interesting Na+, Cl-, and ATP-dependent H+ transport mediated by hu MDR 1 protein as previously suggested [Hoffman and Roepe (1997) Biochemistry 36, 11153-11168]. These data may help to resolve a variety of conflicting reports in the literature regarding ion transport mediated by hu MDR 1 and have implications for the physiology of a number of polarized epithelia in which hu MDR 1 is endogenously expressed.
在之前的论文中，我们展示了多种数据，这些数据与人类多药耐药蛋白1（hu MDR 1）过表达时9.3酵母质膜离子转运的显著扰动相一致。因此，在本文中，我们比较了从对照9.3/pVT酵母与9.3/hu MDR 1酵母制备的内翻式酵母质膜囊泡（ISOV）的ΔpH形成情况。由于MDR 1 ATP酶活性相对于内源性酵母H⁺ ATP酶活性具有更宽、更碱性的pH谱，我们详细分析了pH≥8.0时的H⁺泵浦情况，以便选择性地放大hu MDR 1对H⁺移动的贡献，使其超过内源性酵母H⁺ ATP酶的贡献。我们观察到：（1）施加外向内的Cl⁻梯度会增强9.3/pVT ISOV的酸化（如预期），但会降低9.3/hu MDR 1 ISOV的酸化；（2）施加内向外的Cl⁻梯度会降低9.3/pVT ISOV的酸化（如预期），但会增强9.3/hu MDR 1 ISOV的酸化；（3）当Na⁺梯度和Cl⁻梯度都为内向外时，与Cl⁻梯度方向相同的Na⁺梯度会放大由hu MDR 1引起的效应，但当两者都为外向内时则不会。正如之前所提出的[霍夫曼和罗普（1997年）《生物化学》36卷，11153 - 11168页]，这些数据最容易用由hu MDR 1蛋白介导的有趣的Na⁺、Cl⁻和ATP依赖性H⁺转运来解释。这些数据可能有助于解决文献中关于hu MDR 1介导的离子转运的各种相互矛盾的报道，并对许多内源性表达hu MDR 1的极化上皮细胞的生理学有影响。
