Name: quantification of coenzyme a in cells and tissues
Uploaded: 2019-09-27T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Coenzyme A in Cells and Tissues at JoVE.com

著者らは、BridgeBio LLCの子会社であるCoAセラピューティクス社、国立衛生研究所補助金GM34496、およびアメリカのレバノン・シリア関連慈善団体が提供する研究に対する資金提供を認めている。

このプロトコルで言及された動物の手順は、プロトコル323および556に従って行われ、特にセントジュード小児研究病院機関動物ケアおよび使用委員会によって承認されました。
1. ソリューションの準備
注:すべての溶液に対して、および手順に記載されている場合は、超純水を使用してください。
<ol><li>水中に水酸化カリウム(KOH)1mを調調します。</...

<ol>
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M., Leonardi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Nudt7+decreases+bile+acid+levels+and+peroxisomal+fatty+acid+oxidation+in+the+liver.">Overexpression of Nudt7 decreases bile acid levels and peroxisomal fatty acid oxidation in the liver.</a> Journal of Lipid Research. 60, 1005-1019 (2019).</li><li>Shumar, S. 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=Induction+of+neuron-specific+degradation+of+Coenzyme+A+models+pantothenate+kinase-associated+neurodegeneration+by+reducing+motor+coordination+in+mice.">Induction of neuron-specific degradation of Coenzyme A models pantothenate kinase-associated neurodegeneration by reducing motor coordination in mice.</a> PLoS ONE. 10, 0130013 (2015).</li><li>Zano, S. P., Pate, C., Frank, M., Rock, C. O., Jackowski, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+a+genetic+deficiency+in+pantothenate+kinase+1+using+phosphopantothenate+replacement+therapy.">Correction of a genetic deficiency in pantothenate kinase 1 using phosphopantothenate replacement therapy.</a> Molecular Genetics and Metabolism. 16, 281-288 (2015).</li><li>Sharma, L. K., 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=A+therapeutic+approach+to+pantothenate+kinase+associated+neurodegeneration.">A therapeutic approach to pantothenate kinase associated neurodegeneration.</a> Nature Communications. 9, 4399 (2018).</li><li>Alvarez-Cordoba, M., 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=Pantothenate+Rescues+Iron+Accumulation+in+Pantothenate+Kinase-Associated+Neurodegeneration+Depending+on+the+Type+of+Mutation.">Pantothenate Rescues Iron Accumulation in Pantothenate Kinase-Associated Neurodegeneration Depending on the Type of Mutation.</a> Molecular Neurobiology. 56, 3638-3656 (2019).</li><li>Arber, C., 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=iPSC-derived+neuronal+models+of+PANK2-associated+neurodegeneration+reveal+mitochondrial+dysfunction+contributing+to+early+disease.">iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease.</a> PLoS One. 12, 0184104 (2017).</li><li>Di Meo, I., 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=Acetyl-4'-phosphopantetheine+is+stable+in+serum+and+prevents+phenotypes+induced+by+pantothenate+kinase+deficiency.">Acetyl-4'-phosphopantetheine is stable in serum and prevents phenotypes induced by pantothenate kinase deficiency.</a> Scientific Reports. 7, 11260 (2017).</li><li>Nishikawa, T., Edelstein, D., Brownlee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+missing+link:+a+single+unifying+mechanism+for+diabetic+complications.">The missing link: a single unifying mechanism for diabetic complications.</a> Kidney International Supplements. 77, 26-30 (2000).</li><li>Tsuchiya, Y., Pham, U., Gout, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+measuring+CoA+and+CoA+derivatives+in+biological+samples.">Methods for measuring CoA and CoA derivatives in biological samples.</a> Biochemical Society Transactions. 42, 1107-1111 (2014).</li><li>Demoz, A., Netteland, B., Svardal, A., Mansoor, M. A., Berge, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+Detection+of+Tissue+Coash+and+Long-Chain+Acyl-Coa+by+Reversed-Phase+High-Performance+Liquid-Chromatography+after+Precolumn+Derivatization+with+Monobromobimane.">Separation and Detection of Tissue Coash and Long-Chain Acyl-Coa by Reversed-Phase High-Performance Liquid-Chromatography after Precolumn Derivatization with Monobromobimane.</a> Journal of Chromatography. 635, 251-256 (1993).</li><li>Shimada, K., Mitamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+Thiol-Containing+Compounds.">Derivatization of Thiol-Containing Compounds.</a> Journal of Chromatography B. 659, 227-241 (1994).</li><li>Minkler, P. E., Kerner, J., Ingalls, S. T., Hoppel, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+isolation+procedure+for+short-,+medium-,+and+long-chain+acyl-coenzyme+A+esters+from+tissue.">Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue.</a> Analytical Biochemistry. 376, 275-276 (2008).</li><li>Newton, G. L., Fahey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+biothiols+by+bromobimane+labeling+and+high-performance+liquid+chromatography.">Determination of biothiols by bromobimane labeling and high-performance liquid chromatography.</a> Methods in Enzymology. 251, 148-166 (1995).</li><li>Radkowsky, A. E., Kosower, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimanes+.17.+(Haloalkyl)-1,5-Diazabicyclo[3.3.0]Octadienediones+(Halo-9,10-Dioxabimanes)+-+Reactivity+toward+the+Tripeptide+Thiol,+Glutathione.">Bimanes .17. (Haloalkyl)-1,5-Diazabicyclo[3.3.0]Octadienediones (Halo-9,10-Dioxabimanes) - Reactivity toward the Tripeptide Thiol, Glutathione.</a> Journal of the American Chemical Society. 108, 4527-4531 (1986).</li><li>Zhang, Y. M., 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=Chemical+knockout+of+pantothenate+kinase+reveals+the+metabolic+and+genetic+program+responsible+for+hepatic+coenzyme+A+homeostasis.">Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis.</a> Chemistry &amp; Biology. 14, 291-302 (2007).</li><li>Tokutake, Y., Onizawa, N., Katoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toyoda+A,Chohnan+S.+Coenzyme+A+and+its+thioester+pools+in+fasted+and+fed+rat+tissues.">Toyoda A,Chohnan S. Coenzyme A and its thioester pools in fasted and fed rat tissues.</a> Biochemical and Biophysical Research Communications. 402, 158-162 (2010).</li><li>Shibata, K., Nakai, T., Fukuwatari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+high-performance+liquid+chromatography+determination+of+coenzyme+A,+dephospho-coenzyme+A,+and+acetyl-coenzyme+A+in+normal+and+pantothenic+acid-deficient+rats.">Simultaneous high-performance liquid chromatography determination of coenzyme A, dephospho-coenzyme A, and acetyl-coenzyme A in normal and pantothenic acid-deficient rats.</a> Analytical Biochemistry. 430, 151-155 (2012).</li><li>Chohnan, S., Takamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+micromethod+for+measurement+of+CoASH+and+its+use+in+measuring+the+intracellular+levles+of+CoASH+and+short+chain+acyl-CoAs+in+Escherichia+coli+K12+cells.">A simple micromethod for measurement of CoASH and its use in measuring the intracellular levles of CoASH and short chain acyl-CoAs in Escherichia coli K12 cells.</a> Agricultural and Biological Chemistry. 55, 87-94 (1991).</li></ol>

ここでは、吸光度または蛍光出力検出器を備えたHPLCを持つ多くのラボでアクセス可能な幅広い線形検出を持つ細胞および動物組織の全CoAを定量するための信頼性の高いステップバイステップの手順を示します。あるいは、質量分析はCoAおよびCoAチオエステルを評価するための一般的な技術であるが、計測器のコストとデータの方法論および解釈の開発に必要な専門知識のために広く利用でき...

コエンザイムA(CoA)は、すべての生物に不可欠な共因子であり、パントテン酸(パントテン酸の塩)またはビタミンB5とも呼ばれるパントテン酸から合成されます。CoAは、酢酸やコハク酸などの短鎖酸を含む有機酸の主要な細胞内担体であり、プロピオン酸やメチルマロン酸などの分岐鎖酸、パルミテートやオレエートなどの長鎖脂肪酸、非常に長鎖脂肪酸等多価不飽和脂肪酸、及びバルプロ酸などの異生物製剤。有機酸は、中間代謝1における100以上の反応における基質としての使用を可能にするために、CoAと酵素的にチオスターリンケージを形成する。CoAチオエステルはまた、アロステリックレギュレータおよび転写活性化剤である。細胞全体のCoA供給が3、4を規制されていることは今では2を評価されています。したがって、CoAの利用可能性は制限され、CoAの欠乏は、CoA生合成5に影響を与える遺伝的障害によって例示されるように、壊滅的なことができます。パントテナテキナーゼは、CoA生合成(図1)およびパントテナテキナーゼ関連神経変性、PKANと呼ばれる、PANK2遺伝子6の突然変異によって引き起こされる第一歩を触媒する。COASYN遺伝子によってコードされるCoA合成酵素は、CoA生合成(図1)およびCOASYタンパク質関連神経変性(CoPAN)の最後の2つのステップを触媒し、COASYN遺伝子7の突然変異によって引き起こされる。PKANとCoPANの両方は、脳内の鉄蓄積に関連する神経変性疾患とCoA欠乏症の下で疾患病理の下に継承されています。
全CoAの細胞レベルは組織8によって異なり、全CoAは様々な生理学的、病理学的および薬理学的状態の下で増加または減少することができる。肝臓CoAは、給餌状態から断食状態9への燃料切り替え中に増加し、レプチン欠損肥満マウス10では肝臓CoAレベルが異常に高い。肝臓CoAは、慢性エタノール摂取に応答して減少する 11.Pank2ノックアウトマウスモデルにおける脳CoAレベルは周産期中にうつ病化されるが、成人期の後の脳CoA含有量は野生型レベルと同等であり、開発12の間に適応的CoA応答を示す。トランスジェネシスまたは遺伝子送達法による組織CoA含有量の操作は、代謝および神経機能に影響を与える13,14,15.PKANまたはCoPANのための潜在的な治療法の前臨床開発は、有効性の指標として細胞または組織CoA測定を含む16,17,18,19,20.これらすべての条件とその代謝または機能的結果の評価には、CoA全体の測定のための定量的方法が必要です。
生物学的サンプル中のCoAを測定するための正確で信頼性の高いアッセイは、多くのラボで技術的な課題です。残念ながら、無傷の細胞におけるCoAまたはCoAチオエステルを評価または定量化するためのプローブはないが、天然CoAチオエステルの類似体は、酵素21を利用したCoAエステルの研究において機械的プローブとして広く用いられている。CoAの変換は、遊離スルフヒドリル(-SH)部分を用いて、CoAチオエステル(またはその逆)に対して、異なる環境への転移中および細胞リシス中に細胞または動物組織において急速である。多数のアシルCoA合成酵素およびアシル-CoAチオエステラーゼは、CoAプール内の相互変換を仲介し、CoAチオエステルを基質として利用する追加の酵素は、化学的または物理的に消毒されるまで生物学的サンプルにおいて活性を維持する。手段。アシルトランスフェラーゼによるCoAからカルニチンへのアシル基のオフロードは、CoA/CoAチオエステル分布を変化させることができる反応のネットワーク内の一例です。放射性トレーサーは、細胞内のCoA合成率を測定するために使用することができます。生体試料中のCoAおよびCoA誘導体を測定するための現在の方法は22を検討され、結合酵素分光光アッセイ、高圧液体クロマトグラフィーおよび質量分析ベースの手順が含まれる。しかし、これらの方法は、多くの場合、特定のCoA分子種に焦点を当てており、全CoAプールの変動に盲目である。結合された酵素アッセイは、一般に、検出感度が低いため、より大量の入力材料を必要とし、線形性の範囲が限られています。
当研究室では、培養細胞および動物組織におけるCoA全体の定量化に関する信頼性の高い手順を開発しました。この戦略には、CoA種のスペクトル全体を維持および分析する努力をするのではなく、サンプル調製中に無料のCoAのみを得るために、すべてのCoAチオエステルの加水分解が含まれます。この手順は、高圧液体クロマトグラフィー(HPLC)に続くサンプル調製、CoA誘導体化、精製および同定、および吸収性による導出CoAの定量化のための個々の公開方法をまとめたものである。蛍光検出23,24,25.この手順を用いて得られたCoAの決定は、CoA規則の理解とCoA欠陥の治療のための治療アプローチの開発を可能にしました。

<table>
	<tbody>
		<tr>
			<td>2-(2-pyridyl)-ethyl silica gel SPE column</td>
			<td>Millipore-Sigma</td>
			<td>54127-U</td>
			<td></td>
		</tr>
		<tr>
			<td>coenzyme A</td>
			<td>Avanti Polar Lipids</td>
			<td>870700</td>
			<td></td>
		</tr>
		<tr>
			<td>Gemini C18 3 &#956;m 100 &#197; HPLC column</td>
			<td>Phenomenex</td>
			<td>00F-4439-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>monobromobimane</td>
			<td>ThermoFisher Scientific</td>
			<td>M-1378</td>
			<td></td>
		</tr>
		<tr>
			<td>Omni-Tip probe tissue disrupter</td>
			<td>Omni International</td>
			<td>32750H</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher</td>
			<td>S37440</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerGen 125 motorized rotor stator homogenizer</td>
			<td>ThermoFisher Scientific</td>
			<td>NC0530997</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin-X centrifuge tube filter</td>
			<td>CoStar</td>
			<td>8161</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma-HCl</td>
			<td>Fisher</td>
			<td>T395-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters 2475 fluorescence detector</td>
			<td>Waters</td>
			<td>2475</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters 2489 UV-Vis detector</td>
			<td>Waters</td>
			<td>2489</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters e2695 separations module</td>
			<td>Waters</td>
			<td>e2695</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of coenzyme a in cells and tissues

新しい研究は、細胞補酵素A(CoA)供給が成長、代謝および生存に有害な影響で制限されることができることを明らかにしました。細胞CoAの測定は、その比較的低い存在量と、順番に、多数の代謝反応に関与するCoAチオエステルへの自由CoAの動的変換による課題です。サンプル調製中に潜在的な落とし穴をナビゲートし、多くの生物医学研究室での使用に適した幅広い線形検出でアッセイを得る方法が記載されています。

培養細胞および組織における全CoAの検出のための比較的高速かつ信頼性の高い方法は、mBBrを用いてCoAのチオールを蛍光剤に誘導し、その後、逆相HPLCを用いて誘導体化CoA-バイマンを精製することによって開発された。標準曲線が最初に生成され、CoA-bimane標準の既知および増加量が個別に注入され、CoA-bimaneクロマトグラムのピーク下の領域が入力CoA-bimaneの関数としてプロットされます(<strong c...

Watch this Scientific Journal Video about Quantification of Coenzyme A in Cells and Tissues at JoVE.com

Quantification of Coenzyme A in Cells and Tissues

この方法は、培養細胞および動物組織からのサンプル調製、試料中のコエンザイムAの抽出および誘導体化、続いて誘導体化されたコエンザイムAの精製および定量のための高圧液体クロマトグラフィーを記載する。吸光度または蛍光検出。

細胞および組織におけるコエンザイムAの定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

コエンザイムAは、略してCoAと呼ばれ、細胞代謝の重要な補因子です。定量的測定は、動物モデルの栄養状態とホルモン状態で起こる変化を明らかにします。この方法は、CoA誘導体およびフリーCoAを含むセルラーCoAの総量を、簡単で信頼性の高い方法で定量化します。脳鉄蓄積を伴う神経変性のいくつかのタイプは、CoA生合成経路の遺伝子の突然変異に関連している。すべての生物は生存、成長、および機能のためにCoAを必要とするので、この方法は、任意の生物学的システムに適用することができます。初めての研究者は、サンプルを絶対に冷たく保ち、すぐに水酸化カリウムに移す必要があるサンプル調製の最初のステップに苦労するかもしれません。水酸化カリウムに移す前にバッチ内のサンプル数を制限し、実験サンプルを操作する前に転送を練習します。多くの研究者は、後の生化学的分析のための生物学的サンプルの調製において固相抽出に精通していない。まず、培養皿を並行してインキュベートし、生化学的分析用に2つ、生存可能な細胞数を決定するための1つをインキュベートする。培養が後期サブコンフルエント密度に近づくと、例えば、ヒトHepG2/C3A細胞は6〜8倍の密度に成長し、6個またはHEK 293T細胞が100ミリメートル皿当たり約1.3倍の密度に成長し、培養中の接着細胞を収穫する。その後、同じ培養皿に氷冷水を1ミリリットル加えます。皿の中の冷たい水に細胞を削り、400マイクロリットルの0.25モル水酸化カリウムと1.5ミリリットルの水を含むガラス試験管に細胞懸濁液を移し、pHを12以上に持ち込みます。サスペンションを高く10秒間ボルテックスして激しく混ぜます。その後、パラフィンフィルムでしっかりと覆い、水浴で振ることなく1時間摂氏55度でインキュベートします。次に、1モルトリズマ塩酸塩溶液160マイクロリットルと100ミリモルmBBrの10マイクロリットルを加えます。高い渦を10秒間混ぜます。これにより、pHは約8にして、フリーCoAでmBBr反応をサポートします。チューブをパラフィンフィルムで覆い、MBBrがCoAのチオールと反応するように暗闇の中で室温で2時間インキュベートします。2時間後、100マイクロリットルの酢酸を加え、10秒間ボルテックスで混ぜて反応を止めます。沈殿はチューブに形成されます。次に、2,000回gで遠心分離機を15分間用いた。沈殿した細胞の破片を除去するには、固体相抽出カラムのクリーンアップのためにガラス試験管に上清を移して保存する。分析を開始する前に、ローター・ステータ・ホモジナイザーによるプローブの挿入と組織破壊用に設計されたガラス試験管に、水酸化1ミリモルの2ミリリットルを加えます。チューブは使用するまで氷の上に置いてください。凍結した組織片を素早く秤量し、サンプルごとに湿った重量を記録します。ガラス管に組織を移し、30秒間組織を均質化する。組織の完全な解凍を避けてください。0.25モルの水酸化カリウムを500マイクロリットル加えます。10秒間高い渦を、氷の上に保ちます。これにより、12以上のサンプルのpHを加水分解してCoAチオエステルを加水分解し、合計CoAを生成します。パラフィンフィルムでチューブを覆います。培養細胞の調製および組織の調製は、この手順の中で最も重要なステップである。CoAの分解を防ぐために、高水酸化カリウムを迅速に添加することが重要です。加水分解を支持するために振ることなく、水浴で2時間摂氏55度でサンプルをインキュベートします。次に、1モルのトリズマ塩酸塩150マイクロリットルと100ミリモルmBBrの10マイクロリットルを加え、無料CoAでmBBr反応を支持するためにpHを約8に持ち込む。10秒間高い渦。チューブをパラフィンフィルムで覆い、暗い状態で室温で2時間インキュベートし、すべてのCoAがmBBrで誘導体化されるようにします。その後、100マイクロリットルの酢酸を加え、渦を10秒間高く加えて反応を止めます。遠心分離機を15分間15分間gの2,000回でペレットにし、上澄み物をガラス試験管に移して固相抽出カラムをクリーンアップする。室温では、1ミリリットルの使い捨て2-2-ピリジルエチルシリカゲルカラムを1ミリリットルの洗浄バッファーで平衡化し、ピリジル官能基がプロトン化され、アニオン交換体として機能することを確認します。次いで、試料上清をカラムにピペットし、溶出物を回収する。カラムを1ミリリットルの洗浄バッファーで2回、1ミリリットルの水で1回洗浄して、未留保種を除去します。次に、別のガラス試験管に、CoA-bimaneを回収するために1ミリリットルの溶出バッファーでカラムを2回洗浄する。窒素ガスの下でチューブ内のCoA-ビマネサンプルを乾燥させます。シールは、完全にチューブをカバーし、マイナス20度で保存します。HPLC分析の準備ができたら、300マイクロリットルの水でサンプルを再懸濁し、高い渦を10秒間ボルテックスして激しく混ぜます。再懸濁したサンプルを遠心分離管フィルターに移し、遠心分離機を5,000回gで10分間移動して沈殿物を除去します。次いで、ろ過したサンプルを、HPLC注入に適したガラスバイアルに移す。本研究では、代表的なHPLCプロファイルは、C18 HPLCカラム上のCoA-バイマネ標準の保持時間を示す。CoA-ビマネ標準の増加量を個別に注入し、CoA-ビマネクロマトグラムのピーク下の領域を入力CoA-bimaneの関数としてプロットした。標準曲線は、393ナノメートルの波長でCoA-ビマネの吸光度の大きさを反射した。CoA-bimaneの蛍光は、393ナノメートルの励起波長と470ナノメートルの発光波長でも反射した。マウス肝臓またはヒト培養C3A細胞の後のHPLC分離および典型的な検出プロファイルは、ここでは、溶出プログラムを使用して11〜12分間の保持時間を有する赤色のピークによって示される。mBBrとの反応が検出可能な結果を得られない場合、Trizma-HClの量はpHを約8に下げるために調整する必要がある。それは、バイマネ誘導体としてスルヒドリルまたはチオエステル部分を含む追加の細胞分子を検出することが可能です。例えば、グルタチオンビマネは、HPLC法で検出することができる。この技術は、他の研究者が2型糖尿病の動物モデルなどの代謝不均衡に関連するCoA機能不全の潜在的な役割を調査することを可能にする。研究者は、固相抽出に使用される有機溶媒を扱う際に、個人用保護具を使用する必要があります。

quantification-of-coenzyme-a-in-cells-and-tissues

Research

JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

RNAの測定方法<em&gt; N</em<sup&gt; 6</sup細胞や組織における&gt; -methyladenosine変更

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

生化学

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

ニトロセルロース結合アッセイを用いて細菌ヒスチジンキナーゼ自己リン酸化の定量化

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

豊かさと充電転送エシェリヒア属大腸菌の Rna のレベルの定量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

培養色素脱失活動の定量化

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

多重共免疫沈殿を用いたタンパク質相互作用ネットワークダイナミクスの定量化

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

細胞および組織におけるグルコース取り込みの間接的尺度としての細胞外グルコース枯渇 Ex vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

mtDNAの合成と分布のハイスループット定量

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.

著者スポットライト:ミトコンドリアDNA合成と分布のハイスループット画像ベースの定量化  

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.

ミトコンドリアDNA合成と分布のハイスループット画像ベースの定量

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