这份手稿中产生的人物是用 BioRender.com 创作的。我们感谢 Amy Walker 对本文提供反馈。J.B.S.得到了伍斯特生物医学研究基金会资助。

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

<ol>
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作者没有利益冲突需要报告。

随着新兴研究表明哺乳动物线粒体可以在不消耗分子氧的情况下发挥作用，研究人员在OCR测量之外采用正交测定来准确量化线粒体功能至关重要。在这里，我们编制了一系列可用于通过测量线粒体NAD + / NADH平衡，自适应末端电子受体的利用，ATP的产生，从 头 嘧啶生物合成和线粒体衍生的ROS来直接评估复合物I，复合物II，复合物V和DHODH的活性。值得注意的是，这些测定比OCR测量更直接地测量线粒体功能。此外，这些测定为研究人员提供了在缺氧期间量化线粒体功能的易处理方法，由于富马酸盐被用作首选的末端电子受体，因此 OCR 测量在很大程度上无关紧要。最后，这里描述的基于增殖的方法比经典的呼吸测量实验更具成本效益，从而提供了一种广泛可访问的方法来研究哺乳动物系统中的线粒体功能。
利用这些检测来测量培养细胞中的线粒体功能时，有一些关键的考虑因素。关于增殖测定，重要的是调整接种的细胞数量以增加每个细胞系的倍增率。应将细胞接种至至少10%的汇合度，并有足够的空间允许三到四倍，以便可以量化增殖差异。每种测定的另一个考虑因素是用作每个ETC复合物活性对照的小分子的浓度。由于不同的细胞系可能对这些抑制剂表现出不同的敏感性，因此测试这些小分子的剂量以确定最佳浓度至关重要。
体外研究线粒体功能的测定（包括OCR测量和此处描述的所有测定）的普遍局限性是培养基的代谢组成。标准细胞培养基倾向于使系统偏向于表面高水平的线粒体功能。例如，超生理谷氨酰胺水平增加了其TCA循环25的无激素，这促进了线粒体NADH的合成，从而增加了氧化磷酸化。同样，哺乳动物组织中的氧分压范围在3mmHg和100mmHg（约0.1%-13%O2）之间，但在体外是大气压（140mmHg，约21%）26，27。这种过量的O2使线粒体呼吸能力和超氧化物产生最大化28。最近，人们努力设计更生理的培养基29，30。值得注意的是，在人血浆样培养基中培养细胞会降低某些癌细胞系的线粒体呼吸30，T细胞31中的线粒体ROS以及线粒体对癌症治疗的适应32。因此，注意所用培养基的组成并了解其如何影响线粒体功能至关重要。
解释线粒体功能的另一个重要和普遍的限制是线粒体数量差异的可能性。因此，通过定量mtDNA33、用膜电位不敏感染料34测量线粒体质量或线粒体标志物的蛋白质印迹来测量线粒体含量至关重要。这是一个关键的控制，因此线粒体数量的减少不会被误认为线粒体功能的下降。
还有一些适用于此处描述的测定的特定限制和故障排除。首先，鉴于分化的细胞不会增殖，在这种情况下，基于增殖的测定对于评估线粒体功能没有用。用于测量DHODH活性的 13C4-天冬氨酸示踪方案的一个关键限制是细胞中的天冬氨酸摄取效率极低35。为了克服这一潜在的限制，研究人员可以过表达天冬氨酸转运蛋白SLC1A3，以促进 13C4-天冬氨酸摄取35。
使用13C5-谷氨酰胺示踪测量SDH活性的方案的局限性在于，该测定要求细胞利用还原羧化途径来富集M + 3同位素异构体以测量反向活性。由于ATP柠檬酸裂解酶表达低36，HIF稳定性不足37或α-KG：柠檬酸盐比值太低38，一些细胞系无法进行还原羧化通量。为了克服这一限制，可以利用13C4-天冬氨酸示踪来测量SDH正向和反向活动7。在该测定中，SDH正向活性可以通过富马酸盐M + 2：琥珀酸M + 2的比率和琥珀酸盐M + 4：富马酸盐M + 4的反向反应来测量。值得注意的是，这种示踪绕过了还原羧化途径中的大多数酶。
使用DCPIP还原作为读数的复合物I活性测定的局限性是线粒体在结构上不是完整的。冻融线粒体以使NADH吸收用于测定的过程肯定会损害线粒体膜的结构完整性39。该测定应与诸如复合物I增殖测定之类的测定同时进行，以确保观察到的复合物I活性的变化对于完整细胞也是真实的。
在未来的研究中，其中一些技术可以适用于使用模型生物（如小鼠和秀丽隐杆线虫）测量体内线粒体功能。目前用于测量体内线粒体功能的方法集中在生物体水平的OCR，特别是使用小鼠模型时的呼吸交换率。这种方法的一个明显局限性是，氧气除了在线粒体ETC中作为无处不在的末端电子受体的作用外，还具有许多生化和信号功能。例如，氧气被双加氧酶家族中酶的催化活性“消耗”。虽然这些酶有助于细胞耗氧率，但它们不参与、调节或反映线粒体功能。体外经典呼吸测量实验通常控制“非线粒体OCR”，而有机呼吸交换比（RER）实验无法控制这一点，限制了RER作为体内线粒体功能的指标的解释。然而，调整方案以通过13C4-天冬氨酸示踪测量DHODH活性，通过13C5-谷氨酰胺示踪测量复合物II活性，从组织中纯化的线粒体上的复合物I活性，以及使用LC-MS友好化合物（如MitoB）测量线粒体ROS以测量体内线粒体功能.这些询问线粒体功能的直接测定，结合经典的呼吸测量实验，为研究人员提供了对哺乳动物细胞和组织中线粒体功能的更全面和准确的评估。

线粒体功能是细胞健康的关键指标，因为它维持哺乳动物细胞中关键的生物合成、生物能量和信号功能1。绝大多数线粒体功能需要电子流过电子传递链（ETC），ETC中电子流的中断会导致严重的线粒体疾病2。ETC由嵌入线粒体内膜中的一系列还原和氧化（氧化还原）反应组成，这些电子转移反应释放的自由能可用于支持ATP合成，生理过程（例如产热），生物合成途径（例如从头嘧啶生物合成）以及辅助因子（例如NADH）的氧化还原状态的平衡。ETC复合物I和III产生活性氧（ROS）3，4，5，进而调节信号传导关键途径，如HIF，PI3K，NRF2，NFκB和MAPK6。因此，ETC中的电子流指标通常用作哺乳动物细胞中线粒体功能的代理。
呼吸测量实验经常用于测量哺乳动物细胞中的线粒体功能。由于O2是哺乳动物ETC最普遍的末端电子受体，因此其还原被用作线粒体功能的代表。然而，新出现的证据表明，哺乳动物线粒体可以使用富马酸盐作为电子受体来维持依赖于ETC的线粒体功能，包括从头嘧啶生物合成7，NADH氧化7和硫化氢的解毒8。因此，在某些情况下，特别是在缺氧环境中，O2消耗率（OCR）的测量不能提供线粒体功能的精确或准确的指示。
在这里，我们概述了一系列可用于独立于OCR测量线粒体功能的测定。我们提供直接测量复合物 I 介导的 NADH 氧化、二氢乳清酸脱氢酶介导的 从头 嘧啶生物合成、复合物 V 依赖性 ATP 合成、琥珀酸脱氢酶 （SDH） 复合物的净方向性和线粒体衍生的 ROS 的检测方法。这些测定旨在对培养的哺乳动物细胞进行，尽管许多测定可以适应 研究体内 线粒体功能。值得注意的是，该协议中描述的测定是比OCR更直接的线粒体功能测量。此外，它们能够测量缺氧时的线粒体功能，在这种情况下，OCR 不是指示性测量。总之，这些测定与经典的呼吸测量实验相结合，将为研究人员提供对哺乳动物细胞线粒体功能的更全面的评估。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;1.5 mL tube</td>
			<td>Cell Treat</td>
			<td>667443</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL tube</td>
			<td>Cell Treat</td>
			<td>229446</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Cell Treat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Cell Treat</td>
			<td>229112</td>
			<td></td>
		</tr>
		<tr>
			<td>13C4-aspartate</td>
			<td>Sigma-Aldrich</td>
			<td>604852</td>
			<td></td>
		</tr>
		<tr>
			<td>13C5-Glutamine</td>
			<td>Cambridge Isotope Laboratories</td>
			<td>285978-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tube</td>
			<td>Cell Treat</td>
			<td>667411</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Cell Treat</td>
			<td>667421</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm tissue culture dish</td>
			<td>Cell Treat</td>
			<td>229651</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate-buffered saline</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-dichlorophenolindophenol</td>
			<td>Honeywell</td>
			<td>33125</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>37999</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycin</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascentis Express C18</td>
			<td>Sigma-Aldrich</td>
			<td>53825-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle top filter 500 mL, 0.22 &#181;m, PES 9 9 mm membrane diameter</td>
			<td>Cell Treat</td>
			<td>229717</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td></td>
		</tr>
		<tr>
			<td>Brequinar</td>
			<td>Sigma-Aldrich</td>
			<td>SML0113</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Lifter, Double End Flat and Narrow Blade</td>
			<td>Cell Treat</td>
			<td>229305</td>
			<td></td>
		</tr>
		<tr>
			<td>CentriVap -105 Cold Trap</td>
			<td>Labconco</td>
			<td>7385020</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor Tablets</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coulter Counter Cups</td>
			<td>Fisher Scientific</td>
			<td>07-000-694</td>
			<td></td>
		</tr>
		<tr>
			<td>Decylubiquinone</td>
			<td>Sigma-Aldrich</td>
			<td>D7911</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Invitrogen</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5425R</td>
			<td>Eppendorf</td>
			<td>2231000908</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5910 Ri</td>
			<td>Eppendorf</td>
			<td>5943000343</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G5388</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-free DMEM</td>
			<td>Gibco</td>
			<td>11966025</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine-free DMEM</td>
			<td>Thermo Fisher</td>
			<td>11960044</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-Inactivated Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade 35% Ammonium hydroxide</td>
			<td>Thermo Scientific</td>
			<td>460801000</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>900667</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>366927</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade formic acid</td>
			<td>Thermo Scientific</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>563935</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade MeOH</td>
			<td>Sigma-Aldrich</td>
			<td>900688</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC-grade Water</td>
			<td>Sigma-Aldrich</td>
			<td>270733</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Osteosarcome Cell Line 143B</td>
			<td>ATCC</td>
			<td>CRL-8303</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>320331-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotone buffer</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>P2222</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoSox Red</td>
			<td>Invitrogen</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>75351-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Potter-Elvehjem Tissue Grinder, Size 21</td>
			<td>Kimble</td>
			<td>885502-0021</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate-free DMEM media</td>
			<td>Gibco</td>
			<td>11965175</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive Plus Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>726030</td>
			<td></td>
		</tr>
		<tr>
			<td>ReCO2ver Incubator</td>
			<td>Baker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrivap Benchtop Vacuum Concentrator</td>
			<td>Labconco</td>
			<td>7310020</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Millipore Sigma</td>
			<td>20188</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>SeQuant ZIC-pHILIC 5&#956;m 150 x 2.1 mm analytical column</td>
			<td>Sigma-Aldrich</td>
			<td>1.50460.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>SeQuant ZIC-pHILIC guard kit</td>
			<td>Millipore Sigma</td>
			<td>1.50438.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide, Pellets</td>
			<td>Millipore Sigma</td>
			<td>567530-250GM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>SW, TRACEFINDER 5.1 SP3</td>
			<td>Thermo Scientific</td>
			<td>OPTON-31001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tert-butyl hydroperoxide solution</td>
			<td>Sigma-Aldrich</td>
			<td>458139</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>93352</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25-200-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003</td>
			<td></td>
		</tr>
		<tr>
			<td>VANQUISH HORIZON / FLEX HPLC</td>
			<td>Thermo Scientific</td>
			<td>VF-S01-A-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Z2 Coulter Particle count and size analyzer</td>
			<td>Beckman Coulter</td>
			<td>BZ10131270</td>
			<td></td>
		</tr>
	</tbody>
</table>

oxygen-independent assays to measure mitochondrial function in mammals

线粒体电子传递链（ETC）中的电子流动支持哺乳动物细胞中的多方面生物合成，生物能量和信号传导功能。由于氧（O 2）是哺乳动物ETC最普遍的末端电子受体，因此O2消耗率经常被用作线粒体功能的代表。然而，新兴研究表明，该参数并不总是指示线粒体功能，因为富马酸盐可以用作替代电子受体以维持缺氧时的线粒体功能。本文编制了一系列协议，允许研究人员独立于O2消耗率测量线粒体功能。这些测定在研究缺氧环境中的线粒体功能时特别有用。具体来说，我们描述了测量线粒体ATP产生，从头嘧啶生物合成，复合物I氧化NADH和超氧化物产生的方法。结合经典的呼吸测量实验，这些正交和经济的测定将为研究人员提供更全面的线粒体功能评估他们感兴趣的系统。

DHODH，复合物I和复合物V的活性都可以使用增殖测定进行评估。从培养基中剥夺尿苷后，细胞变得更加依赖于嘧啶生物合成的 从头 途径。因此，当细胞在无尿苷培养基中被激发增殖时，它们比在含有尿苷的培养基中培养的细胞对brequinar抑制DHODH活性更敏感（图6A）。类似地，从培养基中剥夺丙酮酸使细胞更依赖于复合物I活性进行增殖。因此，当细胞在无丙酮酸培养基中被激发增殖时，它们比在含丙酮的培养基中培养的细胞对鱼藤酮对复合物I活性的抑制更敏感（图6B）。复合V活性可以通过挑战细胞在含有半乳糖而不是葡萄糖的培养基中增殖来评估。由于半乳糖在糖酵解中产生净零ATP，因此在这种燃料中生长的细胞更依赖于通过复合V活性合成线粒体ATP。因此，在含半乳糖培养基中增殖的细胞比在含葡萄糖培养基中增殖的细胞对寡霉素的复合物V抑制更敏感（图6C）。
SDH活性可以使用13C5-谷氨酰胺示踪和监测其掺入富马酸盐和琥珀酸盐同位素异构体来测量。在载体处理条件下，SDH复合物有利于前向活性，将13C 4-琥珀酸掺入13 C4-富马酸盐高于将13 C 3-富马酸盐掺入13 C3-琥珀酸盐（图7）。在抗霉素处理的条件下，SDH复合物有利于反向活性，并且将13C 3-富马酸盐掺入13 C3-琥珀酸盐大于将13 C 4-琥珀酸掺入13 C4-富马酸盐（图7）。
线粒体内的超氧化物产生可以使用荧光报告基因MitoSox进行测量，其在与超氧化物反应时产生2-羟基-丝分裂乙铓。在这项研究中，在叔丁基过氧化氢存在下用MitoSox处理的细胞具有更高水平的2-羟基-丝分裂乙锭，其方式通过添加NAC（一种淬灭细胞ROS的抗氧化剂）来抑制（图8）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65184/65184fig01.jpg"> 图 1：复杂 V 增殖测定的机理基础。 通过糖酵解氧化葡萄糖和半乳糖。葡萄糖从糖酵解中产生净两个ATP，而半乳糖产生净零ATP，因为UDP-半乳糖需要UTP合成。因此，在半乳糖中生长的细胞由于缺乏糖酵解产生的ATP，更依赖于线粒体ATP的合成。缩写：GALK = 半乳糖激酶;GALT = 半乳糖-1-磷酸尿苷基转移酶;PGM1 = 磷酸葡萄糖变位酶 1;GPI = 葡萄糖-6-磷酸异构酶，UGP = UDP-葡萄糖焦磷酸化酶;GALE = UDP-半乳糖-4-差向异构酶;NDK = 核苷酸二磷酸激酶;UMPK = 尿苷单磷酸激酶;HK = 己糖激酶;PFK = 磷酸果糖激酶;醛缩酶=醛缩酶;TPI = 磷酸三糖异构酶;GAPDH = 甘油醛-3-磷酸脱氢酶;PGK =磷酸甘油酸激酶;PGM = 磷酸葡萄糖变位酶;ENO = 烯醇化酶;PK = 丙酮酸激酶。 <a href="https://www.jove.com/files/ftp_upload/65184/65184fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65184/65184fig02.jpg"> 图2：复合物I增殖测定的机理基础。 在抑制复合物I活性时改变的代谢途径示意图。在高丙酮酸培养基中，通过LDH介导的NADH氧化绕过复合物I抑制。在低丙酮酸培养基中，这种适应不太可行，使细胞更依赖复合物I活性来再氧化NADH。缩写：LDH = 乳酸脱氢酶;TCA循环=三羧酸循环。 <a href="https://www.jove.com/files/ftp_upload/65184/65184fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65184/65184fig03.jpg"> 图 3： 13C4-天冬氨酸示踪时的 DHODH 反应示意图。 Brequinar抑制二氢乳清酸氧化为乳清酸，从而阻止UMP的下游合成。 13C4-天冬氨酸通过DHODH活性掺入 13C3-UMP中。缩写：OMM = 线粒体外膜;IMM = 线粒体内膜;DHODH = 二氢乳清酸脱氢酶。 <a href="https://www.jove.com/files/ftp_upload/65184/65184fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65184/65184fig04.jpg"> 图 4：通过 13C5-谷氨酰胺示踪测量正向和反向复合物 II 活性。为了测量正向复合物II活性（左），监测13C5-谷氨酰胺掺入13 C4-琥珀酸和13C4-富马酸盐。为了测量反向复合物II活性（右），监测13C5-谷氨酰胺掺入13 C 3-琥珀酸和13C3-富马酸盐。缩写：SDH = 琥珀酸脱氢酶;CytC = 细胞色素 C.<a href="https://www.jove.com/files/ftp_upload/65184/65184fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65184/65184fig05.jpg"> 图5：MitoSox Red与超氧化物的反应。 MitoSox与线粒体超氧化物反应形成2-OH-MitoE2+。 <a href="https://www.jove.com/files/ftp_upload/65184/65184fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65184/65184fig06.jpg"> 图 6：用于测量线粒体功能的 基于增殖的测定 （A） 用 5 uM brequinar（一种 DHODH 抑制剂）处理的 143B 骨肉瘤细胞在含有 ±100 ug/mL 尿苷的培养基中的增殖。数据是平均± SEM;N = 3 每个条件。（B）用2uM鱼藤酮（一种复合物I抑制剂）处理的143B骨肉瘤细胞在含有±5mM丙酮酸的培养基中增殖。数据是平均± SEM;N = 3 每个条件。（C）用5 uM寡霉素（一种复合V抑制剂）处理的143B骨肉瘤细胞在以10 mM葡萄糖或10 mM半乳糖为唯一中心碳源的培养基中增殖。数据是平均± SEM;N = 3 每个条件。* 表示 p < 0.05，使用在 Graphpad Prism 中进行单因素方差分析检验。 <a href="https://www.jove.com/files/ftp_upload/65184/65184fig06large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65184/65184fig07.jpg"> 图 7： 13C5-谷氨酰胺示踪以测量复合物 II 活性。 DMSO和500nM抗霉素A处理的Caki1和DLD1细胞中的琥珀酸盐氧化和富马酸盐还原（SDH逆转）。数据代表平均± SEM;N = 3 每个条件。使用 GraphPad Prism 中的未配对 t 检验表示 p < 0.05。缩写：SDH = 琥珀酸氧化;SDH 逆转 = 富马酸盐减少。 <a href="https://www.jove.com/files/ftp_upload/65184/65184fig07large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65184/65184fig08v2.jpg"> 图 8：基于 LCMS 的 MitoSox 检测超氧化物。 在tBuOOH±NAC存在下，从用MitoSox处理的Caki1细胞中分离的2-OH-Mito E2+ 的提取离子色谱图30分钟。缩写：LCMS =液相色谱-质谱;NAC = N-乙酰半胱氨酸。 <a href="https://www.jove.com/files/ftp_upload/65184/65184fig08large.jpg" target="_blank">请点击此处查看此图的大图。</a>
表1：本协议中使用的试剂、缓冲液和培养基的组成。<a href="https://www.jove.com/files/ftp_upload/65184/65184_Table%201%20Reaction%20Compositions_Revision%20(2).xlsx" target="_blank">请按此下载此表格。</a>

Watch this Scientific Journal Video about Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals at JoVE.com

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

在这里，我们提出了一个测定汇编，以直接测量哺乳动物细胞中的线粒体功能，而与它们消耗分子氧的能力无关。

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

我们的研究重点是线粒体如何感知和适应代谢压力源。一个关键的代谢应激源是缺氧，当细胞缺氧时就会发生缺氧。我们正在开发更好的检测方法来研究低氧环境中的线粒体功能。采用更高分辨率的质谱法研究细胞和组织代谢物水平。我们使用稳定同位素示踪研究来追踪特定代谢物的命运，并发现与健康和患病哺乳动物生理学相关的新代谢途径。研究小组发现，哺乳动物线粒体可以在低氧环境中维持关键功能。在机械上，他们使用富马酸盐作为电子传输链的替代电子权杖。该协议汇编了几种分析方法，研究人员可以使用这些测定来更好地评估哺乳动物细胞中的线粒体功能。重要的是，这些方案可用于研究缺氧时的线粒体功能，因为经典的呼吸测量实验是不够的。

oxygen-independent-assays-to-measure-mitochondrial-function-in-mammals

Research

JoVE Journal

Biochemistry

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

2.2K 次观看.  University of Massachusetts Chan Medical School. 我们的研究重点是线粒体如何感知和适应代谢压力源。一个关键的代谢应激源是缺氧，当细胞缺氧时就会发生缺氧。我们正在开发更好的检测方法来研究低氧环境中的线粒体功能。采用更高分辨率的质谱法研究细胞和组织代谢物水平。我们使用稳定同位素示踪研究来追踪特定代谢物的命运，并发现与健康和患病哺乳动物生理学相关的新代谢途径。研究小组发现，哺乳动?...

视频: 作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 &#176;C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

蛋白质tRNA琼脂糖凝胶延迟测定分析<em&gt;ñ</em<sup&gt; 6</sup&gt; - 磺酰氨基甲酰腺苷TcdA功能

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

用天然电泳，凝胶电泳和电解法分析线粒体电子传递链的超复合物

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

荷尔斯坦奶牛肝线粒体耗氧量和质子泄漏动力学评价线粒体呼吸

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

通过高分辨率呼吸测量法评估坐骨神经的线粒体功能

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

mtDNA 合成和分布的高通量定量

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

作者聚焦:基于图像的高通量线粒体 DNA 合成和分布定量  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

分析果蝇的线粒体生理学和生物能量学

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

作者聚焦：使用黑腹果蝇探索线粒体功能和化学毒性 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...