Pascaud C, Garrigos M, Orlowski S
Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, CEA and URA2096 CNRS, C.E. Saclay, F-91991 Gif/Yvette Cedex, France.
Biochem J. 1998 Jul 15;333 ( Pt 2)(Pt 2):351-8. doi: 10.1042/bj3330351.
P-Glycoprotein, the plasma membrane protein responsible for the multidrug resistance of some tumour cells, is an active transporter of a number of structurally unrelated hydrophobic drugs. We have characterized the modulation of its ATPase activity by a multidrug-resistance-related cytotoxic drug, vinblastine, and different multidrug-resistance-reversing agents, verapamil and the dihydropyridines nicardipine, nimodipine, nitrendipine, nifedipine and azidopine. P-Glycoprotein ATPase activity was measured by using native membrane vesicles containing large amounts of P-glycoprotein, prepared from the highly multidrug-resistant lung fibroblasts DC-3F/ADX. P-Glycoprotein ATPase is activated by verapamil and by nicardipine but not by vinblastine. Among the five dihydropyridines tested, the higher the hydrophobicity, the higher was the activation factor with respect to the basal activity and the lower was the half-maximal activating concentration. The vinblastine-specific binding on P-glycoprotein is reported by the inhibitions of the verapamil- and the nicardipine-stimulated ATPase. These inhibitions are purely competitive, which means that the bindings of vinblastine and verapamil, or vinblastine and nicardipine, on P-glycoprotein are mutually exclusive. In contrast, verapamil and nicardipine display mutually non-competitive interactions. This demonstrates the existence of two distinct specific sites for these two P-glycoprotein modulators on which they can bind simultaneously and separately to the vinblastine site. The nicardipine-stimulated ATPase activity in the presence of the other dihydropyridines shows mixed-type inhibitions. These dihydropyridines have thus different binding sites that interact mutually to decrease their respective, separately determined affinities. This could be due to steric constraints between sites close to each other. This is supported by the observation that vinblastine binding is not mutually exclusive with nifedipine or nitrendipine binding, whereas it is mutually exclusive with nicardipine. Moreover, verapamil binding also interacts with the five dihydropyridines by mixed inhibitions, with different destabilization factors. On the whole our enzymic data show that P-glycoprotein has distinct but interacting binding sites for various modulators of its ATPase function.
P-糖蛋白是一种质膜蛋白，负责某些肿瘤细胞的多药耐药性，是多种结构不相关的疏水性药物的主动转运体。我们已经研究了一种与多药耐药相关的细胞毒性药物长春碱，以及不同的多药耐药逆转剂维拉帕米和二氢吡啶类药物尼卡地平、尼莫地平、尼群地平、硝苯地平和叠氮平对其ATP酶活性的调节作用。通过使用从高度多药耐药的肺成纤维细胞DC-3F/ADX制备的含有大量P-糖蛋白的天然膜囊泡来测量P-糖蛋白ATP酶活性。P-糖蛋白ATP酶被维拉帕米和尼卡地平激活，但不被长春碱激活。在所测试的五种二氢吡啶类药物中，疏水性越高，相对于基础活性的激活因子越高，半数最大激活浓度越低。维拉帕米和尼卡地平刺激的ATP酶活性的抑制作用表明长春碱在P-糖蛋白上有特异性结合。这些抑制作用纯粹是竞争性的，这意味着长春碱与维拉帕米或长春碱与尼卡地平在P-糖蛋白上的结合是相互排斥的。相反，维拉帕米和尼卡地平表现出相互非竞争性相互作用。这证明了这两种P-糖蛋白调节剂存在两个不同的特异性结合位点，它们可以同时并分别与长春碱位点结合。在其他二氢吡啶类药物存在的情况下，尼卡地平刺激的ATP酶活性表现出混合型抑制作用。因此，这些二氢吡啶类药物具有不同的结合位点，它们相互作用以降低各自单独测定的亲和力。这可能是由于彼此靠近的位点之间的空间限制。这一点得到了以下观察结果的支持：长春碱与硝苯地平或尼群地平的结合不是相互排斥的，而与尼卡地平是相互排斥的。此外，维拉帕米的结合也通过混合型抑制作用与五种二氢吡啶类药物相互作用，具有不同的失稳因子。总体而言，我们的酶学数据表明，P-糖蛋白对于其ATP酶功能的各种调节剂具有不同但相互作用的结合位点。
