この作品は、自然な科学および工学研究審議会のカナダ (レベル) によって賄われていた。ビデオ制作は、教育と学習 (CITL) ニューファウンドランドの記念大学のイノベーション センターと共同で実施されました。

1. 化学薬品および精製された酵素
<ol><li>下記材料を調達: PDHC とブタ心臓起源 (または別の精製されたミトコンドリア フラビン); KGDHCH2O2 (30% 溶液)、ピルビン酸、α-ケトグルタル酸は、+NAD、NADH、CoASH、チアミンピロリン酸 (TPP)、マンニトール、HEPES、ショ糖、グリコールエーテルジアミン四酢酸、3-メチル-2-オキソ吉草酸 (KMV)、スーパーオキシドジスムターゼ (SOD)、西洋ワサビペルオキシダーゼ (HRP)10-アセチル-3, 7-dihydroxyphenoxazine 試薬 (AUR) と消費者物価指数-613。</li>
</ol>2. アッセイ試薬等の計画
<ol><li>96 ウェル プレートに表 1に従って分析を設定します。 注: 各反応の総体積は 200 μ L ストック濃度が調整試薬の 20 μ L を追加できるような各ウェルに。これにより、補因子と基板も分析を開始する前にそれぞれの反応を迅速に追加。</li>
	<li>220 mM マンニトール、70 mM ショ糖、グリコールエーテルジアミン四酢酸、1 mM と 20 mM HEPES (塩酸で pH 7.4) を含む反応バッファーを準備します。 注: これは異なる精製酵素の物性に応じて変更できます。</li>
	<li>Coomassie 染色5または酵素活性アッセイ14,16,17に用いた汚染またはイムノブロットによって浄化された KGDHC または PDHC を確認します。</li>
	<li>すべて試薬作業液濃度が表1 に従って反応バッファーを準備します。</li>
	<li>-20 ° C で 1 mL 因数としてすべての試薬を格納します。ストア ピルビン酸、α-ケトグルタル酸自動酸化と繰り返し凍結融解による自然劣化を防ぐために 100 μ L の因数で-80 ° c。</li>
	<li>AUR 試薬 1 ml の DMSO のマスターの在庫を準備し、反応バッファー 100 μ M に希釈しています。光から保護されたストア。 注: ここでは、100 μ M の作業ソリューションは、すべて 2 〜 3 週間で作った新鮮な光から保護します。</li>
</ol>3. 分析テンプレートを設定します。
<ol><li>デュアル測定機能単色マイクロ プレート リーダーの試金プロシージャを設定します。</li>
	<li>読み取り手順を定義するには、「プロトコル」を選択します。「プロシージャ」をクリックします。</li>
	<li>25 の ° C (図 1 a) に「温度」を設定。</li>
	<li>(図 1 a) 読み取り条件を設定する「運動を開始」をクリックします。時間と読み取り間隔/実験の数を設定します。</li>
	<li>クリックして「読み取り」(図 1 b)。 注: サンプルは、トップの位置からリード 50 のゲインを持つ。時間: 5 分 30 の間隔で読みます。チャネル 1: ら蛍光の励起/蛍光 = 565 nm:600 nm、波長幅 13.5 nm。チャネル 2: NADH 蛍光の励起/蛍光 = 376 nm:450 nm、波長幅 20 nm。</li>
	<li>「読み取り」、[モニター (例えば、A1 A4、B1 B4) に井戸を選択します。</li>
	<li>「キネティック ・ エンド」を選択します。</li>
</ol>4. 標準曲線
<ol><li>試薬を解凍し、必要になるまで氷の上を格納します。</li>
	<li>
NADH 標準曲線 (図 2 a)
	<ol>
<li>NADH の反応バッファーに 0.5 ~ 10 mM の濃度範囲で作業ソリューションを準備します。</li>
		<li>井戸 180 μ L 反応バッファーを含む溶液濃度を作業ごとの NADH から 20 μ 因数を追加します。それぞれ NADH の最終濃度じゃ 0.05 1 mM。</li>
		<li>「読む」をクリックし、、励起/蛍光を設定 = 376 nm:450 nm、波長幅 20 nm。 注: これは読み取り専用エンドポイント - 速度論的パラメーターは必要ではありません。</li>
	</ol>
</li>
	<li>
AUR 標準曲線 (図 2 b)
	<ol>
<li>反応バッファーにおける過酸化水素の株式を働いているを準備します。ストック濃度 200 4,000 nM の範囲です。</li>
		<li>120 μ L の反応液を各ウェルに追加します。</li>
		<li>HRP の 20 μ L を追加 (ストック濃度を作業 = 30 U/mL、井戸に最終濃度 = 3 U/mL) 20、SOD の μ L (ストック濃度を作業 = 250 U/mL、井戸に最終濃度 = 25 U/mL) と AUR の 20 μ L (ストック濃度を作業 = 100 μ M、井戸に最終濃度 = 10 μ M) も含む各反応バッファーにします。</li>
		<li>作業もバッファーとアッセイ試薬を含む各在庫ソリューション各 H2O2の 20 μ L を追加します。最終反応量が 200 μ L と H2O2各ウェル内の最終濃度は 20-400 nM。</li>
		<li>25 ° C でプレート リーダーで 1 分版を孵化させなさい</li>
		<li>「読む」をクリックし、、励起/蛍光を設定 = 565 nm/600 nm、波長幅 13.5 nm。これはエンドポイントは読み取り専用であることに注意してください - 速度論的パラメーターは必要ではありません。</li>
	</ol>
</li>
</ol>5. O2● - /H2 O2リリースと KGDHC と PDHC NADH 生産の測定
<ol><li>それぞれに追加して各作業ソリューションとボリュームの濃度別の試薬の付加の順序表 1を参照してくださいも。</li>
	<li>各ウェルに 40 μ L の反応バッファーを追加します。KMV または CPI 613 を使用して試金、各ウェルにバッファーの 20 μ L を追加します。</li>
	<li>PDHC または KGDHC の 20 μ L を追加 (1 U/mL、ウェルあたり最終濃度の在庫 = 0.1 U/mL) 各ウェルに。</li>
	<li>2 分の 25 ° C で PDHC と KGDHC を孵化させなさい。</li>
	<li>KMV の 20 μ L を追加 (在庫 = 100 mM、最終濃度 = 10 mM) または 20 μ L の消費者物価指数-613 (ストック濃度を作業 = 1.5 mM、最終濃度 150 μ M を =) (表 2)。 注: 試金のための阻害剤が使用されていない場合は、このステップを除外できます。</li>
	<li>25 ° C で 1 分間インキュベートします。 注: 試金のための阻害剤が使用されていない場合は、このステップを除外できます。</li>
	<li>HRP の 20 μ L を追加 (ストック濃度を作業 = 30 台/mL、井戸に最終濃度 = 3 ユニット/mL) と SOD の 20 μ L (ストック濃度を作業 = 250 単位/mL、井戸に最終濃度 = 25 単位/mL) 各ウェルに。</li>
	<li>コエンザイム A の 20 μ L を追加 (ストック濃度を作業 = 最終濃度 1 mM = 0.1 mM)、20 TPP の μ L (ストック濃度を作業 = 3 mM、最終濃度 = 0.3 mM)、および NAD+の 20 μ L (ストック濃度を作業 = 10 mM、最終濃度 = 1 mM) 各 wエル。</li>
	<li>AUR 試薬を保護のアルミ箔カバーから外し、各ウェルに 20 μ L を追加します。</li>
	<li>ピルビン酸の 20 μ L を追加 (ストック濃度 1 〜 100 ミリメートル、試金のための最終的な集中に至る作業 = 0.1 ~ 10 mM) PDHC と α-ケトグルタル酸を含む井戸 (ストック濃度 1 〜 100 ミリメートル、試金のための最終的な集中に至る作業 = 0.1 ~ 10 mM) に井戸は、KGDHC を含みます。</li>
	<li>「読む」運動の測定を開始するをクリックします。</li>
</ol>6. データの分析
<ol><li>キーボードの「CTRL」ボタンを押し、チャンネル 1 (図 3 a) に対応するすべての速度論的測定値を含む 1 つグラフを生成する井戸をクリックします。</li>
	<li>(図 3 b) 異なる時点で測定される相対的な蛍光ユニット (RFU) の生の値の右上隅で表示アイコンに「データ」をクリックします。</li>
	<li>分析 (表 3) の値をエクスポートします。</li>
	<li>2 蛍光チャネルに関連付けられた RFU の値にアクセスする「グラフ」ドロップ ダウン メニューの右上 (図 3 b) をクリックします。</li>
	<li>チャネル 2 の 6.1 に 6.3 の手順を繰り返します。</li>
</ol>

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開示するものがあります。

このプロトコルは有利なので、1) 2 H2O2検出など、抗酸化システム (ROS の他の情報源)、妨げる可能性がありますそうでなければ任意の競合反応を排除) のネイティブ速度の直接評価ROS をフラビン含有ミトコンドリア脱水素酵素、3 によって解放) ネイティブを比較できます ROS リリース 2 つの率またはより精製脱フラビン ベース、4) ROS のリリースと酵素の活性や 5 の率の...

栄養代謝の究極の目標は、アデノシン三リン酸 (ATP) をすることです。哺乳類セルのこれはカーボンで ATP に蓄えられたエネルギーを変換する二重の機能を担った細胞小器官ミトコンドリアで発生します。ATP の生産が始まる炭素がミトコンドリア NADH およびフラビンアデニンジヌクレオチド (FADH2)1の 2 つの電子キャリアを形成によって燃焼させる。NADH および FADH2によって酸化されて複数のサブユニット呼吸鎖複合体 I および II、それぞれ、および解放された電子が複雑な IV1ターミナル電子アクセプター分子酸素 (O2) へフェリーで行きます。熱力学的に有利な「下り坂」電子の移動 O2チェーンの終わりには、intermembrane に陽子の輸出に結合複合体 I、III、および IV スペースします。これは、陽子 (プロトン動機力)、2ATP を作る複雑な V によってタップされてギブス自由エネルギーの一時的な形式の貫通型電気化学的勾配を作成します。ミトコンドリアの電子伝達反応は、ATP の生産に完全には結合されていません。クレブスと呼吸鎖の様々 なポイント、電子途中で O2 ROS3形態にやり取りできます。ミトコンドリアによって生成される最も生物学的に関連する活性酸素は O2● -と H2O2。O2● -ミトコンドリアによって形成された近位の ROS と考えは多くの場合、生産のサイトに対して、自由に関連付けられている O2● -と H2O2の混合物を形成することは明白であります。人工フラビンの根本的な化学グループ4,5です。高いレベルで ROS、危険、酸化遭難6細胞機能に必要な生体成分の損傷です。ただし、低額でミトコンドリア ROS は重要なシグナリングの機能を果たします。例えば、ミトコンドリアから放出される H2O2を制御する T 細胞の活性化、ストレス シグナル (例えば、Nrf2 シグナル伝達経路の誘導)、細胞増殖や分化の誘導に関与しています。インスリン シグナルしリリース、および動作7を供給します。活性酸素のシグナル伝達機能を理解する上でかなりの進歩をしました。ただし、重要な質問はまだ残っているに関してミトコンドリアの酵素として最も重要な源および生産を制御する方法。
いくつかの要因に依存する生産のサイトによって ROS リリース: 1) 濃度の電子の寄付サイト、2) 3) 酸素、および 4) 濃度へのアクセス、電子寄付サイトの酸化還元状態と酸化基板3,の種類8,9. NADH および膜の潜在的な強度の濃度も ROS 生産8,10に影響を与えるようなミトコンドリアの他の要因。たとえば、O2● -/H2O2生産精製 PDHC または KGDHC の率は増加 NADH 可用性5,11に増加します。このシナリオでは、PDHC または KGDHC、O2● -/H2O2生産12サイトの E3サブユニットの流行中心に NADH から電子が逆流です。同様に、NAD+の規定は KGDHC12から ROS リリースを減少、反対の効果を持ちます。したがって、活性酸素産生のサイトからの電子制御の出入りを変更できますどのくらい O2● -/H2O2が形成されます。例えば、PDHC の消費者物価指数-613、アナログ、ほぼ 90% 減少 O2● -/H2O2生産13リポ酸と KGDHC E2サブユニットをブロックします。化学 S glutathionylation 触媒、著者、ジスルフィラム、O2● -/H2O2 PDHC または KGDHC を介して生産の E にグルタチオン抱合をほぼ廃止で同様の結果が得られます2サブユニット14。PDHC の KGDHC E2サブユニットを介して電子の流れをブロックするためのトレードオフ (例えば、複合体 III) の電子輸送鎖によって ROS の生成を減少する NADH の生産の減少であります。これはまた電子輸送鎖によって ATP の出力を減らすことができます。全体的に、活性酸素産生のサイトに電子エントリをブロック O2● -/H2O2生産を制御する非常に効果的な手段をすることができます。
ミトコンドリアは、最大 12 潜在的な O2● -/H2O2ソース6を含めることができます。これらのサイトのほとんどは、フラビン含有 O2● -と H2O2の混合物を生成する酵素。呼吸鎖複合体とミトコンドリアの脱水素酵素機能大容量 O2● -/H2O2のサイトを形成する識別に対して異なる基質と阻害剤の組み合わせの使用を許可しました。異なったティッシュ3。PDHC と KGDHC は、筋肉と肝臓のミトコンドリア13,15発光サイト大容量O2● -/H2O2として示されています。ただし、いくつかの困難のまま O2● -/H2O2異なる基質と阻害剤の組み合わせは、ミトコンドリアと影響の個々 のサイトの潜在的な形成の検討に関して酵素の活動。これは、望ましくない副反応 (例えばO2● -/H2O2興味の酵素以外のサイトでの形成) の存在のために汚染内因性栄養素 (例えば、脂肪酸酸) または細胞内小器官 (例えば、ペルオキシソームO2● -/H2O2を形作る)、選択性を欠いている阻害剤の使用や活性酸素産生を完全に抑制しない化合物の使用。特定の阻害剤ミトコンドリアバイオエナジェティックスと電子流れの方向を変更することも、これは生産の他のサイトから ROS リリースを変更し、結果を混同します。O2●/H2O2リリース ミトコンドリアの個々 のサイトからのための絶対速度 O2● -と H2O2の高濃度のため定量化することは困難です。行列と intermembrane スペースで酵素を除去します。したがって、O2● -/H2O2リリース測定を妨害することができます任意の競合反応の除去することができます大容量 O2● -/H2O2 を識別します。サイトを形成します。
脱水素酵素フラビン依存型精製によって O2● -/H2O2生産と NADH 形成の同時受験可能にする簡単な方法を紹介します。精製された酵素を使用してに、原産 O2● -/H2O2生産率である個々 のフラビン酵素のためのより正確な測定を可能にすると、不要な ROS 形成側の反応と活性酸素分解酵素を除去ことができます。このメソッドを使用して直接比較、O2● -/H2O2 O2● -/H2O の異なる精製脱水素酵素または画面潜在的な部位特異的阻害剤の容量を形成できます。2リリース。最後に、O2● -/H2O2と NADH 生産を同時に測定、酵素と活性酸素放出能の関係のリアルタイム評価できます。

<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

それは、ミトコンドリアが活性酸素種 (ROS) の最大 12 酵素源を含めることができます報告されています。これらのサイトの大半には、フラビン依存型呼吸鎖複合体と脱水素酵素活性酸素 (O2● -) と過酸化水素 (H2O2) の混合物を生成するがあります。ミトコンの個々 のサイトの ROS 産生能の定量の正確さは抗酸化防衛システムとO2● -/H2O2を形成するも副反応の存在のために困難なことができます。ミトコンドリアの生体エネルギーを乱すことができる非特異的阻害剤の使用は、生産の他のサイトから ROS のリリースを変更することにより測定を脅かされる可能性も。精製フラビン結合脱水素酵素によって H2O2リリースとニコチン酸アミドアデニンジヌクレオチド (NADH) 生産の同時測定のための簡単な方法を紹介します。我々 の目的はここで、例として精製されたピルビン酸脱水素酵素複合体 (PDHC) と α-ケトグルタル酸デヒドロゲナーゼ複合体 (KGDHC) のブタ心臓起源を使いました。このメソッドはネイティブによって生産の個々 のサイトの H2O2解放率の正確な測定のため活性酸素と抗酸化システムの他の潜在的な原因を排除することによってできます。さらに、このメソッドは、H2O2リリースと酵素活性と有効性のスクリーニングと活性酸素産生の阻害の選択性の関係の直接比較のためことができます。全体的に、このアプローチを分離ミトコンドリアやグリセリン筋線維でのより高度な実験を行う前に個々 の酵素の ROS リリースのネイティブ速度の詳細な評価を許可できます。

図 3 aは、H2O2と NADH 生産の同時測定の間に精製された KGDHC によって収集された RFU の代表的なトレースを提供します。RFU のデータ各時間間隔は、図 3Bで描かれています。RFU の生データは、分析のため、エクスポートされます。図 2に示した標準曲線から外挿により 5 分間測定間隔ごと...

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

ミトコンドリアは、活性酸素種 (ROS) を生成することができますいくつかのフラビン依存型酵素を含まれています。ROS を監視ミトコンドリアの個々 のサイトからのリリースは不要な副反応のために挑戦です。簡単、安価な精製フラビン酵素とマイクロ プレート蛍光分析を使用して、ROS リリース ネイティブ料金を直接評価法を提案します。

活性酸素・過酸化水素とフラビン含有ミトコンドリア脱水素酵素による NADH 生産の同時測定

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

simultaneous-measurement-superoxidehydrogen-peroxide-nadh-production

Research

JoVE Journal

Biochemistry

10.0K ビュー.  Memorial University of Newfoundland. ミトコンドリアは、活性酸素種 (ROS) を生成することができますいくつかのフラビン依存型酵素を含まれています。ROS を監視ミトコンドリアの個々 のサイトからのリリースは不要な副反応のために挑戦です。簡単、安価な精製フラビン酵素とマイクロ プレート蛍光分析を使用して、ROS リリース ネイティブ料金を直接評価法を提案します。

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

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.

足場リポソームを使用することにより、脂質近位タンパク質 - タンパク質相互作用を再構成します<em&gt;インビトロ</em

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

生化学

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

治療用抗体の実験室規模の産生および精製

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

A Guide to Production, Crystallization, and Structure Determination of Human IKK1/&#945;

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its precursor p100. p52 functions as a homodimer or heterodimer with another NF-&#954;B subunit, RelB. These dimers in turn regulate the expression of hundreds of genes involved in inflammation, cell survival, and cell cycle. IKK1/&#945; primarily remains associated with IKK2/&#946; and NEMO as a ternary complex. However, a small pool of it is also observed as a low molecular weight complex(es). It is unknown if the p100 processing activity is triggered by activation of IKK1/&#945; within the larger or the smaller complex pool. Constitutive activity of IKK1/&#945; has been detected in several cancers and inflammatory diseases. To understand the mechanism of activation of IKK1/&#945;, and enable its use as a drug target, we expressed recombinant IKK1/&#945; in different host systems, such as E. coli, insect, and mammalian cells. We succeeded in expressing soluble IKK1/&#945; in baculovirus infected insect cells, obtaining mg quantities of highly pure protein, crystallizing it in the presence of inhibitors, and determining its X-ray crystal structure. Here, we describe the detailed steps to produce the recombinant protein, its crystallization, and its X-ray crystal structure determination.

I&#954;B Kinase 1/&#945; (IKK1/&#945; CHUK) is a Ser/Thr protein kinase that is involved in a myriad of cellular activities primarily through activation of NF-&#954;B transcription factors. Here, we describe the main steps necessary for the production and crystal structure determination of this protein.

生産、結晶化、および人間 IKK1/α の構造決定へのガイド

A class of extracellular stimuli requires activation of IKK1/&#945; to induce generation of an NF-&#954;B subunit, p52, through processing of its ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

ミトコンドリアの分離と培養細胞におけるミトコンドリアのカルシウム流入の解析

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

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

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

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.

ホルスタイン種乳牛におけるミトコンドリア呼吸を推定する肝ミトコンドリアの酸素消費量とプロトン リーク動態測定

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

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatio-temporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (KD) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

蛍光顕微鏡を使用して競争力のある滴定による転写因子の DNA 結合親和性の高感度測定

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

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

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

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

大腸菌における異種発現による膜トランスポーターの特性評価と膜小胞の産生

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

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

低分子阻害剤スクリーニングのためのNADH結合ATPaseアッセイの半高スループット適応

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 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 IKK&#946;. Upon activation, the IKK complex phosphorylates the I&#954;B molecules leading to NF-&#954;B nuclear translocation and activation of target genes. Inhibition of the NEMO/IKK interaction is an attractive therapeutic paradigm for the modulation of NF-&#954;B pathway activity, making NEMO a target for inhibitors design and discovery. To facilitate the process of discovery and optimization of NEMO inhibitors, we engineered an improved construct of the IKK-binding domain of NEMO that would allow for structure determination of the protein in the apo form and while bound to small molecular weight inhibitors. Here, we present the strategy utilized for the design, expression and structural characterization of the IKK-binding domain of NEMO. The protein is expressed in E. coli cells, solubilized under denaturing conditions and purified through three chromatographic steps. We discuss the protocols for obtaining crystals for structure determination and describe data acquisition and analysis strategies. The protocols will find wide applicability to the structure determination of complexes of NEMO and small molecule inhibitors.

We describe protocols for the structure determination of the IKK-binding domain of NEMO by X-ray crystallography. The methods include protein expression, purification and characterization as well as strategies for successful crystal optimization and structure determination of the protein in its unbound form.