| Polyamine Transport Assay Using Reconstituted Yeast Membranes
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Author:
Date:
2021-01-20
[Abstract] ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.
[摘要] [摘要] ATP13A2 / PARK9是一种晚期内/溶酶体P5B转运ATPase，与多种神经退行性疾病有关。我们最近将ATP13A2表征为溶酶体多胺出口者，这为未知的哺乳动物多胺转运系统的分子身份提供了线索。在这里，我们逐步描述了从过量表达人ATP13A2的酵母细胞中测量重组囊泡中放射性标记的多胺转运的方案。该方案是我们最新出版物的一部分（van Veen等，2020），将有助于表征其他假定的多胺转运蛋白的转运功能，例如P5B转运ATPase的同工型。
[背景] ATP13A2 / PARK9编码一种普遍表达的晚期内-/溶酶体膜蛋白，与一系列神经退行性疾病有关，例如早发性帕金森氏病（Di Fonzo等，2007 ；Lin等，2008）和Kufor -Rakeb综合征（伴痴呆的早期帕金森病）（Ramirez等，2006 ；Park等，2011）。ATP13A2属于P型转运ATPase ，是一类活性转运蛋白，由于ATP水解而暂时形成磷酸中间产物（Kuhlbrandt ，2004年）。ATP13A2是P5亚家族的成员，该家族已在20多年前通过基因组测序鉴定出来（Axelsen和Palmgren ...
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| Measurement of Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Culture Cells for Assessment of the Energy Metabolism
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Author:
Date:
2018-05-20
[Abstract] Mammalian cells generate ATP by mitochondrial (oxidative phosphorylation) and non-mitochondrial (glycolysis) metabolism. Cancer cells are known to reprogram their metabolism using different strategies to meet energetic and anabolic needs (Koppenol et al., 2011; Zheng, 2012). Additionally, each cancer tissue has its own individual metabolic features. Mitochondria not only play a key role in energy metabolism but also in cell cycle regulation of cells. Therefore, mitochondria have emerged as a potential target for anticancer therapy since they are structurally and functionally different from their non-cancerous counterparts (D'Souza et al., 2011). We detail a protocol for measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements ...
[摘要] 哺乳动物细胞通过线粒体（氧化磷酸化）和非线粒体（糖酵解）代谢产生ATP。已知癌细胞使用不同的策略重新编程它们的代谢以满足能量和合成代谢需要（Koppenol等人，2011; Zheng，2012）。此外，每个癌症组织都有其自己的个体代谢特征。线粒体不仅在能量代谢中起关键作用，而且在细胞的细胞周期调控中也起关键作用。因此，线粒体作为抗癌治疗的潜在靶标已经出现，因为它们在结构和功能上与其非癌对应物不同（D'Souza等人，2011）。我们详细介绍了利用海马XF24细胞外通量分析仪（图1）测量活细胞中氧耗率（OCR）和细胞外酸化率（ECAR）测量的方案。 Seahorse XF24细胞外通量分析仪持续测量细胞上清液中的氧浓度和质子流量（Wu等人，2007）。这些测量结果在OCR和ECAR值中转换，并能够直接定量线粒体呼吸和糖酵解。有了这个协议，我们试图评估三种不同癌细胞系的基线粒体功能和线粒体应激反应细胞毒性测试先导化合物甲磺卡西林，以研究其作用机制。将细胞铺在XF24细胞培养板中并保持24小时。在分析之前，将培养基替换为无缓冲的DMEM pH7.4，然后使细胞在非代谢通量分析前使用Seahorse XF在非CO 2孵育器中平衡以允许精确测量Milli-pH单位改变。 ...
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