Name: simultaneous measurement of superoxide/hydrogen peroxide and nadh production by flavin-containing mitochondrial dehydrogenases
Uploaded: 2018-02-24T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases at JoVE.com

这项工作由加拿大自然科学和工程研究委员会 (NSERC) 资助。视频制作是与纽芬兰纪念大学教与学创新中心 (CITL) 合作进行的。

1. 化学物质和纯化酶
<ol><li>采购以下材料: 猪心源的 PDHC 和 KGDHC (或另一种纯化的线粒体 flavoenzyme);H2O2 (30% 解决方案), 丙酮酸、αα-、NAD+、NADH、CoASH、磷酸硫胺 (TPP)、甘露醇、HEPES、蔗糖、EGTA、3-甲基2羰基戊酸 (KMV)、超氧化物歧化酶 (SOD)、辣根过氧化物酶 (HRP)、10-乙酰基 37-dihydroxyphenoxazine 试剂 (AUR) 和 CPI-613。</li>
</ol>2. 计划化验及试剂制备
<ol><li>根据96...

<ol>
	<li>Mailloux, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teaching+the+fundamentals+of+electron+transfer+reactions+in+mitochondria+and+the+production+and+detection+of+reactive+oxygen+species.">Teaching the fundamentals of electron transfer reactions in mitochondria and the production and detection of reactive oxygen species.</a> Redox Biol. 4, 381-398 (2015).</li><li>Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forty+years+of+Mitchell's+proton+circuit:+From+little+grey+books+to+little+grey+cells.">Forty years of Mitchell's proton circuit: From little grey books to little grey cells.</a> Biochim Biophys Acta. 1777 (7-8), 550-556 (2008).</li><li>Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+generation+of+superoxide+and+hydrogen+peroxide+as+the+source+of+mitochondrial+redox+signaling.">Mitochondrial generation of superoxide and hydrogen peroxide as the source of mitochondrial redox signaling.</a> Free Radic Biol Med. 100, 14-31 (2016).</li><li>Massey, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+molecular+oxygen+by+flavins+and+flavoproteins.">Activation of molecular oxygen by flavins and flavoproteins.</a> J Biol Chem. 269 (36), 22459-22462 (1994).</li><li>Mailloux, R. J., Gardiner, D., O'Brien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Oxoglutarate+dehydrogenase+is+a+more+significant+source+of+O2(.-)/H2O2+than+pyruvate+dehydrogenase+in+cardiac+and+liver+tissue.">2-Oxoglutarate dehydrogenase is a more significant source of O2(.-)/H2O2 than pyruvate dehydrogenase in cardiac and liver tissue.</a> Free Radic Biol Med. 97, 501-512 (2016).</li><li>Sies, H., Berndt, C., Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+Stress.">Oxidative Stress.</a> Annu Rev Biochem. 86, 715-748 (2017).</li><li>Kuksal, N., Chalker, J., Mailloux, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review.+Progress+in+understanding+the+molecular+oxygen+paradox+-+function+of+mitochondrial+reactive+oxygen+species+in+cell+signaling.">Review. Progress in understanding the molecular oxygen paradox - function of mitochondrial reactive oxygen species in cell signaling.</a> Biol Chem. , (2017).</li><li>Murphy, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+mitochondria+produce+reactive+oxygen+species.">How mitochondria produce reactive oxygen species.</a> Biochem J. 417 (1), 1-13 (2009).</li><li>Wong, H. S., Dighe, P. A., Mezera, V., Monternier, P. A., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+superoxide+and+hydrogen+peroxide+from+specific+mitochondrial+sites+under+different+bioenergetic+conditions.">Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions.</a> J Biol Chem. , (2017).</li><li>Harper, M. E., Green, K., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+cellular+energy+transduction+and+its+implications+for+obesity.">The efficiency of cellular energy transduction and its implications for obesity.</a> Annu Rev Nutr. 28, 13-33 (2008).</li><li>Ambrus, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+reactive+oxygen+species+by+human+and+bacterial+pyruvate+and+2-oxoglutarate+dehydrogenase+multienzyme+complexes+reconstituted+from+recombinant+components.">Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components.</a> Free Radic Biol Med. 89, 642-650 (2015).</li><li>Tretter, L., Adam-Vizi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+reactive+oxygen+species+in+the+reaction+catalyzed+by+alpha-ketoglutarate+dehydrogenase.">Generation of reactive oxygen species in the reaction catalyzed by alpha-ketoglutarate dehydrogenase.</a> J Neurosci. 24 (36), 7771-7778 (2004).</li><li>Slade, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+the+superoxide/hydrogen+peroxide+forming+and+quenching+potential+of+mouse+liver+mitochondria.">Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria.</a> Biochim Biophys Acta. 1861 (8), 1960-1969 (2017).</li><li>O'Brien, M., Chalker, J., Slade, L., Gardiner, D., Mailloux, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+S-glutathionylation+alters+superoxide/hydrogen+peroxide+emission+from+pyruvate+dehydrogenase+complex.">Protein S-glutathionylation alters superoxide/hydrogen peroxide emission from pyruvate dehydrogenase complex.</a> Free Radic Biol Med. 106, 302-314 (2017).</li><li>Quinlan, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+2-oxoacid+dehydrogenase+complexes+in+mitochondria+can+produce+superoxide/hydrogen+peroxide+at+much+higher+rates+than+complex+I.">The 2-oxoacid dehydrogenase complexes in mitochondria can produce superoxide/hydrogen peroxide at much higher rates than complex I.</a> J Biol Chem. 289 (12), 8312-8325 (2014).</li><li>Mailloux, R. J., Craig Ayre, D., Christian, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mitochondrial+reactive+oxygen+species+production+by+GSH+mediated+S-glutathionylation+of+2-oxoglutarate+dehydrogenase.">Induction of mitochondrial reactive oxygen species production by GSH mediated S-glutathionylation of 2-oxoglutarate dehydrogenase.</a> Redox Biol. 8, 285-297 (2016).</li><li>Mailloux, R. J., Gardiner, D., O'Brien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Oxoglutarate+dehydrogenase+is+a+more+significant+source+of+O2(.-)/H2O2+than+pyruvate+dehydrogenase+in+cardiac+and+liver+tissue.">2-Oxoglutarate dehydrogenase is a more significant source of O2(.-)/H2O2 than pyruvate dehydrogenase in cardiac and liver tissue.</a> Free Radic Biol Med. 97, 501-512 (2016).</li><li>Ambrus, A., Adam-Vizi, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dihydrolipoamide+dehydrogenase+(E3)+deficiency:+Novel+insights+into+the+structural+basis+and+molecular+pathomechanism.">Human dihydrolipoamide dehydrogenase (E3) deficiency: Novel insights into the structural basis and molecular pathomechanism.</a> Neurochem Int. , (2017).</li><li>Young, A., Gardiner, D., Brosnan, M. E., Brosnan, J. T., Mailloux, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+levels+of+formate+activate+mitochondrial+superoxide/hydrogen+peroxide+release+from+mouse+liver+mitochondria.">Physiological levels of formate activate mitochondrial superoxide/hydrogen peroxide release from mouse liver mitochondria.</a> FEBS Lett. 591 (16), 2426-2438 (2017).</li><li>Fisher-Wellman, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+glutathione+depletion+reveals+a+novel+role+for+the+pyruvate+dehydrogenase+complex+as+a+key+H2O2-emitting+source+under+conditions+of+nutrient+overload.">Mitochondrial glutathione depletion reveals a novel role for the pyruvate dehydrogenase complex as a key H2O2-emitting source under conditions of nutrient overload.</a> Free Radic Biol Med. 65, 1201-1208 (2013).</li><li>Kussmaul, L., Hirst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+superoxide+production+by+NADH:ubiquinone+oxidoreductase+(complex+I)+from+bovine+heart+mitochondria.">The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria.</a> Proc Natl Acad Sci U S A. 103 (20), 7607-7612 (2006).</li><li>Huang, L. S., Borders, T. M., Shen, J. T., Wang, C. J., Berry, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+mitochondrial+respiratory+complex+II+from+chicken+heart:+a+membrane-protein+complex+diffracting+to+2.0.">Crystallization of mitochondrial respiratory complex II from chicken heart: a membrane-protein complex diffracting to 2.0.</a> A. Acta Crystallogr D Biol Crystallogr. 61 (Pt 4), 380-387 (2005).</li><li>Yeh, J. I., Chinte, U., Du, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+glycerol-3-phosphate+dehydrogenase,+an+essential+monotopic+membrane+enzyme+involved+in+respiration+and+metabolism.">Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism.</a> Proc Natl Acad Sci U S A. 105 (9), 3280-3285 (2008).</li></ol>

该协议是有利的, 因为, 1) 它消除了任何可能干扰 H2O2检测 (如, 抗氧化剂系统或其他 ROS 来源), 2) 的相互竞争的反应, 提供直接评估的本地率ros 释放的黄素线粒体脱氢酶, 3) 允许比较的本地 ros 释放率的两个或更多的纯化黄素脱氢酶, 4) 可以允许直接比较 ros 释放率和酶活性, 和 5)允许筛选活性氧释放的选择性竞争抑制剂。我们的小组和其他人在以前的出版物中使用这种方法来?...

营养代谢的最终目的是使三磷酸腺苷 (ATP)。在哺乳动物细胞中, 这发生在线粒体, 双 membraned 细胞器, 将储存在碳中的能量转化为 ATP。ATP 的产生是在碳被形成两个电子载体、NADH 和黄素腺嘌呤二核苷酸 (FADH2)1的线粒体燃烧时开始的。NADH 和 FADH2分别由多亚基呼吸复合体 I 和 II 氧化, 并且被解放的电子被运到终端电子受体分子氧 (O2) 在复杂 IV1。热力学上有利的 "下坡" 转移电子到O 2 在链子的末端被结合对质子的出口入膜间隙空间由复合体 I, III 和 iv。这创造了质子的跨膜电化学梯度 (质子动力), 一种临时形式的吉布斯自由能, 被复杂的 V 挖掘, 使 ATP2。线粒体中的电子转移反应与 ATP 生产不完全耦合。在克雷布斯周期和呼吸链的不同点, 电子可以过早地与 O2相互作用, 形成 ROS3。线粒体产生的最生物相关的 ROS 为 O2和 H 2O2。尽管 O2通常被认为是由线粒体形成的近端 ROS, 但现在明显的是, 生产地点形成了 O2和 H 2O2的混合物, 这与自由黄素假肢组的激进化学4,5。在高水平, ROS 可能是危险的, 破坏细胞功能所需的生物成分, 导致氧化窘迫6。然而, 在低数量, 线粒体 ROS 实现重要的信号功能。例如, 从线粒体释放的 H2O2被牵连到控制 T 细胞活化、应力信号 (例如、诱导 Nrf2 信号通路)、诱导细胞增殖和分化,胰岛素信号和释放, 和喂养行为7。在了解 ROS 信号功能方面取得了相当大的进展。然而, 对于线粒体中的酶作为最重要的来源和如何控制生产, 仍然存在重要的问题。
ROS 的释放由一个地点生产取决于几个因素: 1) 电子捐赠站点的集中, 2) 电子捐赠站点的氧化还原状态, 3) 获得氧气, 和 4) 氧化基底的浓度和类型3,8,9. 在线粒体中, 其他因素, 如 NADH 的浓度和膜电位强度也影响 ROS 生产8,10。例如, O2/H2O2生产的速率由纯化的 PDHC 或 KGDHC 增加, NADH 可用性5,11。在这种情况下, 电子从 NADH 向后流向 PDHC 或 KGDHC E3亚基中的时尚中心, O2/H2O2生产12的站点。同样, 提供的 NAD+具有相反的效果, 减少了 KGDHC12中的 ROS 释放。因此, 控制来自 ROS 生产场所的电子进入或退出可以改变多少 O2/H2O2的形成。例如, 用 CPI-613 (硫辛酸模拟) 阻止 PDHC 或 KGDHC 的 E2亚基, 在 O2/H2O2生产13中, 结果几乎减少90%。类似的结果可以获得与化学 s-glutathionylation 催化剂, 胍和双硫醒, 几乎废除O2/H2O 2 生产由 PDHC 或 KGDHC 通过共轭谷胱甘肽到 E2亚基14。通过 PDHC 和 KGDHC E2亚基阻断电子流的权衡是 NADH 产量的减少, 这降低了电子传输链 (例如, 复杂 III) 的 ROS 形成。这也可以减少电子传输链的 ATP 输出。总的来说, 阻止电子进入 ROS 生产场所可以是控制 O2/H2o2生产的高效方法。
线粒体最多可包含 12个潜在 O2/H2O2源6。这些站点中的大多数都是黄素的酶, 它们生成了 O2和 H 2O2的混合物。使用不同的基质和抑制剂组合, 可以确定哪些呼吸复合体和线粒体脱氢酶作为高容量 O 2/H 2 O 2 形成站点在不同的组织3。PDHC 和 KGDHC 已被证明为高容量 O2/H2O2在肌肉和肝脏线粒体13,15发射站点。但是, 在检查 O2/H2O2形成线粒体中单个站点的潜力以及不同基质和抑制剂组合对其产生的影响方面仍存在一些困难酶的活性。这是由于存在不需要的副作用 (例如, 形成 O2/H2O2由非兴趣酵素以外的站点), 污染内源营养素 (例如,脂肪酸) 或细胞器(例如,, 过氧化物酶体也形成O2-/H2O2), 使用缺乏选择性的抑制剂和/或使用不完全抑制 ROS 生产的化合物。某些抑制剂还能改变线粒体的生物能学和电子流的方向, 这就改变了 ROS 从其他生产场所释放出来的活性和混淆结果。o2/H2O2 从单个站点释放的绝对速率也难以量化由于 O2-和 H2o2消除基质中的酶和膜间隙空间。因此, 消除可能干扰 o2/H2O2发布测量的任何相互竞争的反应, 在识别高容量 O2/2o2 时, 都很有用。形成站点。
在这里, 我们提出一个简单的方法, 允许同时检查 O2/H2O2生产和 NADH 形成由纯化的黄素依赖脱氢酶。通过使用纯化的酶, 不需要的 ros 形成的副作用和 ros 降解酶可以消除, 允许更准确地测量本地 O2/H2o2生产率为个别 flavoenzymes。此方法可用于直接比较 o2/H2O2的不同纯化脱氢酶的形成能力, 或为 o 2-/h 2 o 筛选潜在的站点特定抑制剂。2版本。最后, 测量 O2/H2O2和 NADH 生产同时可以对酶活性与 ROS 释放能力之间的关系进行实时评估。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pyruvate dehydrogenase complex</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P7032-10UN</td>
			<td style="width:167px;">purified flavoenzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">alpha-ketoglutarate dehydrogenase complex</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">K1502-20UN</td>
			<td>purified flavoenzyme</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30% hydrogen peroxide solution</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">HX0640-5</td>
			<td>reagent, standard curves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NAD+</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">N0632-1G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NADH</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">N4505-100MG</td>
			<td>reagent, standard curves</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pyruvate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P2256-5G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">alpha-ketoglutarate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">75892-25G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CoASH</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">C3019-25MG</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">thiamine pyrophosphate</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">C8754-1G</td>
			<td>reagent, activity/ROS release assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mannitol</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">M4125-100G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">H3375-25G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sucrose</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">S7903-250G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGTA</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">E3889-10G</td>
			<td>buffer component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KMV</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">198978-5G</td>
			<td>reagent, ROS release inhibitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CPI-613</td>
			<td style="width:71px;">Santa Cruz</td>
			<td style="width:119px;">sc-482709</td>
			<td>reagent, ROS release inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SOD</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">S9697-15KU</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">horseradish peroxidase</td>
			<td style="width:71px;">SIGMA</td>
			<td style="width:119px;">P8375-1KU</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amplex Ultra Red</td>
			<td style="width:71px;">Thermofisher</td>
			<td style="width:119px;">A36006</td>
			<td>reagent, ROS release detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Biotech Synergy 2 microplate reader</td>
			<td style="width:71px;">BioTek Instruments</td>
			<td style="width:119px;"></td>
			<td>microplate reader for assays</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Gen5 software</td>
			<td style="width:71px;">BioTek Instruments</td>
			<td style="width:119px;"></td>
			<td>software, used for collection of raw RFU</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Graphpad Prism</td>
			<td style="width:71px;">Graphpad software</td>
			<td style="width:119px;"></td>
			<td>software, data analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microsoft EXCEL</td>
			<td style="width:71px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>software, data analysis</td>
		</tr>
	</tbody>
</table>

simultaneous measurement of superoxide/hydrogen peroxide and nadh production by flavin-containing mitochondrial dehydrogenases

据报道, 线粒体可以包含多达12种酶源的活性氧 (ROS)。大多数这些网站包括黄素依赖的呼吸复合体和脱氢酶, 产生超氧化物的混合物 (O 2) 和过氧化氢 (H 2 O 2).由于抗氧化剂防御系统的存在和侧面反应, 也形成 O2/H2O2, 因此, 对孤立线粒体中单个站点的 ROS 产生潜能的准确量化可能会有挑战性。使用可破坏线粒体生物能学的非特异性抑制剂也可以通过改变其他生产场所的 ROS 释放来降低测量结果。在这里, 我们提出了一个简单的方法, 同时测量 H2O2释放和烟酰胺腺嘌呤二核苷酸 (NADH) 生产的纯化黄素链脱氢酶。为了我们的目的, 我们用纯化的丙酮酸脱氢酶复合物 (PDHC) 和αα-脱氢酶复合物 (KGDHC) 的猪心源为例。此方法允许通过消除其他潜在的 ROS 和抗氧化剂系统的来源, 精确测量本机 H2O2的发布速率。此外, 该方法还可以直接比较 H2O2释放与酶活性之间的关系, 以及筛选活性抑制剂对 ROS 生产的有效性和选择性。总的来说, 这种方法可以在进行更复杂的实验时, 对单独的线粒体或透肌纤维进行更精密的试验, 从而对个体酶的原生氧释放率进行深入评估。

图 3A为在同时测量 H2O2和 NADH 生产过程中收集的 RFU 提供了一个具有代表性的跟踪。图 3B中描述了每个时间间隔的原始 RFU 数据。然后导出原始 RFU 数据以进行分析。通过从图 2中显示的标准曲线推断, 可以确定在 5 min 期间的每个测量间隔内形成的 NADH 和 H2O2的绝对量。在<stro...

Watch this Scientific Journal Video about Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases at JoVE.com

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

线粒体含有几种黄素依赖的酶, 可以产生活性氧 (ROS)。由于不需要的副作用, 监测线粒体中单个部位的 ROS 释放具有挑战性。我们提出了一种简单, 廉价的方法直接评估的本地率的 ROS 释放使用纯化 flavoenzymes 和微板块荧光。

含黄素线粒体脱氢酶同时测定超氧化物/过氧化氢和 NADH 生产

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Research

JoVE Journal

Biochemistry

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

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

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

Laboratory Scale Production and Purification of a Therapeutic Antibody

实验室规模的生产和治疗性抗体的纯化

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

线粒体钙在分离线粒体和培养细胞中的流入分析

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

用最小组织量测定果蝇透纤维中线粒体耗氧量

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

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

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 ...

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

荧光显微镜竞争滴定法测定转录机-dna 结合感的高灵敏度

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Production, Crystallization, and Structure Determination of the IKK-binding Domain of NEMO

NEMO IKK绑定域的生产、结晶和结构确定

NEMO is a scaffolding protein which plays an essential role in the NF-&#954;B pathway by assembling the IKK-complex with the kinases IKK&#945; and ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

通过TIRF显微镜 在体外 同时可视化交联和单微管的动力学

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...