NAD⁺ content and its role in mitochondria.
NAD⁺ content and its role in mitochondria.
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DOI：
10.1007/978-1-4939-1875-1_4
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发表时间：
2015
影响因子：
--
通讯作者：
Wei Li;A. Sauve
中科院分区：
文献类型：
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作者：
Wei Li;A. Sauve
烟酰胺腺嘌呤二核苷酸 (NAD+) 是参与细胞能量代谢和能量产生的中心代谢辅酶/共底物。它可以很容易地被两个电子当量还原并形成 NADH 形式，这是大多数生理条件下 NAD+ 的少数种类。 NAD+不仅在细胞的氧化还原反应中发挥着重要作用，而且作为信号分子也发挥着重要作用。例如，NAD+ 通过参与丙酮酸脱氢酶、三羧酸循环和氧化磷酸化化学，在线粒体功能中发挥关键作用。它还可作为脱酰基酶 SIRT3、SIRT4 和 SIRT5 的底物，这些酶可改变线粒体内赖氨酸的蛋白质翻译后修饰。最近的工作强调了线粒体 NAD+ 动态变化的生物学意义。这增加了对标准化和有效的方法来测量该细胞器中 NAD+ 含量的需求。为了测定细胞中，特别是线粒体中的 NAD+ 浓度，我们描述了两种测定 NAD+ 的方法：酶循环测定法和同位素稀释法。循环测定包含样品 NAD+、乳酸、乳酸脱氢酶、心肌黄酶和刃天青。同位素稀释测定使用合成的 18 O-NAD+作为内标，并且通过HPLC分级处理的样品，然后通过阳性模式MS中的 16 O-和 18 O-NAD+峰(664/666)比率确定NAD+浓度。
Nicotinamide adenine dinucleotide (NAD+) is a central metabolic coenzyme/cosubstrate involved in cellular energy metabolism and energy production. It can readily be reduced by two electron equivalents and forms the NADH form, which is the minority species to NAD+under most physiologic conditions. NAD+plays an important role in not only oxidation–reduction reactions in cells but also as a signaling molecule. For example, NAD+plays a key role in mitochondrial function via participation in pyruvate dehydrogenase, tricarboxylic acid cycle, and oxidative phosphorylation chemistries. It also serves as a substrate for deacylases SIRT3, SIRT4, and SIRT5, which modify protein posttranslational modifications on lysine within the mitochondrial compartment. Recent work has highlighted the biological significance of dynamic changes to mitochondrial NAD+. This has increased the need for standardized and effective methods to measure NAD+contents in this organelle. To determine NAD+concentrations in cells, and specifically in mitochondria, we describe two assays for NAD+determinations: An Enzymatic Cycling Assay and Isotope Dilution. The cycling assay contains sample NAD+, lactate, lactate dehydrogenase, diaphorase, and resazurin. The isotope dilution assay uses synthetic18O-NAD+as an internal standard, and treated samples are fractionated by HPLC and then NAD+concentration determined by the16O- and18O-NAD+peak (664/666) ratio in positive mode MS.