Zehnpfennig Britta, Urbatsch Ina L, Galla Hans-Joachim
Institute of Biochemistry, University of Munster, 48149 Munster, Germany.
Biochemistry. 2009 May 26;48(20):4423-30. doi: 10.1021/bi9001908.
ABCC3 (MRP3) is a member of the family of multidrug resistance-associated proteins (MRP), which belong to the largest family of membrane transport proteins, namely, the ATP binding cassette (ABC) transporters. Members of this family contribute to the excretion of several organic anions from cells and play a critical role in conferring resistance against drugs used in the treatment of cancer. The overexpression of ABCC3 in the yeast Pichia pastoris and its subsequent purification made possible the study of substrate-dependent ATPase activity [Chloupkova, M., et al. (2007) Biochemistry 46, 7992-8003]. Here we describe the successful reconstitution of purified ABCC3 in proteoliposomes and ABCC3-dependent uptake of the anticancer drug methotrexate (MTX), as well as the physiological substrate leukotriene C(4) (LTC(4)). Our results show specific transport in a cell-free environment and in the absence of other proteins, revealing positive allosteric cooperativity for ABCC3-mediated substrate translocation. The ABCC3-mediated transport of MTX indicates a Hill coefficient of 2.3 +/- 1.7, a maximum transport rate (V(max)) of >2 micromol min(-1) mg(-1), and a K(M) in the millimolar range, whereas the translocation of LTC(4) into proteoliposomes displayed a Hill coefficient of 2.3 +/- 0.5 with a maximum transport rate of 4.7 +/- 0.8 nmol min(-1) mg(-1), and a K(M) in the micromolar range (1.7 +/- 0.3 microM). The transport of both substrates, MTX and LTC(4), was inhibited by etoposide, confirming a higher affinity of ABCC3 for LTC(4) than for MTX. The technical advances described in this report represent the basis for the extended and detailed kinetic characterization of ABCC3 with a wide range of implications for the investigation of other human ABC transporters.
ABCC3（多药耐药相关蛋白3）是多药耐药相关蛋白（MRP）家族的成员，该家族属于最大的膜转运蛋白家族，即ATP结合盒（ABC）转运体。这个家族的成员有助于细胞排出多种有机阴离子，并在赋予对癌症治疗药物的抗性方面发挥关键作用。ABCC3在酵母毕赤酵母中的过表达及其随后的纯化使得研究底物依赖性ATP酶活性成为可能[Chloupkova，M.等人（2007年）《生物化学》46卷，7992 - 8003页]。在此，我们描述了纯化的ABCC³在蛋白脂质体中的成功重构以及ABCC³依赖性的抗癌药物甲氨蝶呤（MTX）和生理底物白三烯C4（LTC4）的摄取。我们的结果显示在无细胞环境且不存在其他蛋白质的情况下存在特异性转运，揭示了ABCC³介导的底物转运存在正构变构协同作用。ABCC³介导的MTX转运表明希尔系数为2.3±1.7，最大转运速率（Vmax）＞2微摩尔·分钟⁻¹·毫克⁻¹，米氏常数（Km）在毫摩尔范围内，而LTC4转运到蛋白脂质体中的希尔系数为2.3±0.5，最大转运速率为4.7±0.8纳摩尔·分钟⁻¹·毫克⁻¹，米氏常数在微摩尔范围内（1.7±0.3微摩尔）。MTX和LTC4这两种底物的转运均被依托泊苷抑制，证实ABCC³对LTC4的亲和力高于对MTX的亲和力。本报告中描述的技术进展为对ABCC³进行扩展和详细的动力学表征奠定了基础，这对其他人类ABC转运体的研究具有广泛的意义。
