Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor
Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor
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DOI：
10.1074/jbc.m110.155119
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发表时间：
2010-11-26
影响因子：
4.8
通讯作者：
Olsen, Lars F.
中科院分区：
文献类型：
--
作者：
Ozalp, Veli C.;Pedersen, Tina R.;Olsen, Lars F.
腺苷5 '-三磷酸是所有活细胞中的通用分子，在其中它在生物能量学和细胞信号传导中起作用。为了了解ATP的浓度是如何通过细胞代谢调节的，进而了解它是如何调节细胞中酶的活性的，如果我们能够在真实的时间内测量完整细胞中的ATP浓度将是有益的。使用一种新的基于适配体的ATP纳米传感器，它可以很容易地监测细胞内ATP在真核细胞中的时间分辨率为秒，我们已经进行了第一次在线测量的细胞内浓度的ATP在酵母酿酒酵母。这些ATP测量结果表明，酵母细胞中的ATP浓度不是稳定的。除了振荡的ATP浓度之外，我们还观察到在饥饿的细胞中浓度高，并且当诱导糖酵解时开始降低。ATP浓度的降低被证明是由膜结合ATP酶的活性引起的，例如线粒体F0 F1 ATP酶水解ATP和质膜ATP酶（PMA 1）。这两种ATP酶的活性受到细胞中葡萄糖浓度的严格控制。最后，细胞内ATP的测量表明，2-脱氧葡萄糖（2-DG）可能有更复杂的功能，而不仅仅是一个分解代谢块。令人惊讶的是，添加2-DG仅诱导ATP的中度下降。此外，我们的研究结果表明，2-DG可能会抑制PMA 1激活后，加入葡萄糖。
Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F0F1 ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.