Institute of Physiology, Pathophysiology and Biophysics, Department of Biomedical Sciences, University of Veterinary Medicine Vienna, 1210 Vienna, Austria.
Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany.
Biomolecules. 2020 Apr 29;10(5):685. doi: 10.3390/biom10050685.
Several mitochondrial proteins, such as adenine nucleotide translocase (ANT), aspartate/glutamate carrier, dicarboxylate carrier, and uncoupling proteins 2 and 3, are suggested to have dual transport functions. While the transport of charge (protons and anions) is characterized by an alteration in membrane conductance, investigating substrate transport is challenging. Currently, mainly radioactively labeled substrates are used, which are very expensive and require stringent precautions during their preparation and use. We present and evaluate a fluorescence-based method using Magnesium Green (MgGr), a Mg-sensitive dye suitable for measurement in liposomes. Given the different binding affinities of Mg for ATP and ADP, changes in their concentrations can be detected. We obtained an ADP/ATP exchange rate of 3.49 ± 0.41 mmol/min/g of recombinant ANT1 reconstituted into unilamellar liposomes, which is comparable to values measured in mitochondria and proteoliposomes using a radioactivity assay. ADP/ATP exchange calculated from MgGr fluorescence solely depends on the ANT1 content in liposomes and is inhibited by the ANT-specific inhibitors, bongkrekic acid and carboxyatractyloside. The application of MgGr to investigate ADP/ATP exchange rates contributes to our understanding of ANT function in mitochondria and paves the way for the design of other substrate transport assays.
几种线粒体蛋白，如腺嘌呤核苷酸转运体（ANT）、天冬氨酸/谷氨酸载体、二羧酸载体和解偶联蛋白 2 和 3，被认为具有双重运输功能。虽然电荷（质子和阴离子）的转运以膜电导率的变化为特征，但研究底物转运具有挑战性。目前，主要使用放射性标记的底物，这些底物非常昂贵，并且在制备和使用过程中需要严格的预防措施。我们提出并评估了一种使用镁绿（MgGr）的荧光法，这是一种适用于脂质体测量的镁敏感染料。鉴于镁对 ATP 和 ADP 的不同结合亲和力，可以检测到它们浓度的变化。我们获得了 3.49±0.41mmol/min/g 重组 ANT1 重新构建到单层脂质体中的 ADP/ATP 交换率，这与使用放射性测定法在线粒体和蛋白脂质体中测量的值相当。仅从 MgGr 荧光计算得出的 ADP/ATP 交换率取决于脂质体中 ANT1 的含量，并被 ANT 特异性抑制剂绷酸和羧基甜菜碱抑制。将 MgGr 应用于研究 ADP/ATP 交换率有助于我们理解 ANT 在线粒体中的功能，并为设计其他底物转运测定方法铺平了道路。
