Name: measuring in vitro atpase activity for enzymatic characterization
Uploaded: 2016-08-23T14:00:00.000+00:00
Duration: 7 min 38 s
Description: Watch this Scientific Journal Video about Measuring In Vitro ATPase Activity for Enzymatic Characterization at JoVE.com

The authors would like to acknowledge funding from a National Institutes of Health grant RO1AI049294 (to M. S.).

1.精製タンパク質とATP加水分解反応を行います<ol><li>精製タンパク質とのインキュベーションのために必要なすべての試薬の株式を準備します。 <ol><li> 100mMのHEPES pHは8.5、65 mMのNaClおよび5％グリセロール（またはその他の適切なアッセイバッファー）を含む5倍HEPES /食塩/グリセロール（HNG）バッファを準備します。 </li><li> （ATPアーゼは、金属依存している場合、または他の金属）を水に100のMgCl 2を準備します。 </li><li>高純度のATPを用いて、200 mMトリス塩基（さらにpHを調整しない）で新鮮な100mMのATPを準備します。 ATPは時間をかけて分解し、そしてATPストックを凍結し、解凍を控えるよう、もはや数週間以上のために-20℃でATPストックをアリコートし、保管しません。 </li><li>ちょうどATP加水分解反応をセットアップする前に1：1の比率でプレミックスのMgCl 2およびATP。 </li></ol></li><li>反応の間、一定の間隔でサンプルを収集するために1.5ミリリットルのチューブを準備し、ラベルを付けます。時間0で、かつ時間15、30でサンプルを収集するためのチューブを準備し、45、および60分。 注：別の方法として、唯一の時間0およびエンドポイントでサンプルを収集します。 <ol><li> ATP加水分解反応1時50分から採取された試料を希釈するために、各チューブに245μlの1×HNGバッファを追加します。 </li></ol></li><li>すぐに反応を停止したサンプルを凍結するためにドライアイスとエタノールのバスを準備します。ゴムアイスバケットまたは他の安全な（非プラスチック）容器の中で、ドライアイスのいくつかの部分を追加し、慎重にドライアイスをカバーするのに十分な70から100パーセントのエタノールを注ぎます。 </li><li> （通常5〜10μM）必要に応じて、1×HNG緩衝液中で精製したタンパク質を希釈し、そして氷上に保ちます。 </li><li> ATP加水分解反応を設定します。 <ol><li>各サンプルの別々の0.5ミリリットルのチューブでは、以下の試薬 ​​を順に追加：H（30μlの最終容量まで）2 O、6μlの5倍HNGまたは他のバッファー、3μlの100のMgCl 2 -ATP混合物を、および0.25から5μMタンパク質。 </li><li>何のタンパク質を添加していないされているバッファのみの負の制御条件が含まれます。</li></ol></li><li> 、時間0での反応から5μLを削除する245μlのHNG緩衝液を含む準備された1.5mlチューブに1:50に希釈し、直ちにドライアイス/エタノール浴中でサンプルを凍結します。 </li><li> ATP加水分解は、1時間に発生できるようにするために、37℃で反応をインキュベートします。各区間（15、30、45、および60分）で、反応から5μlのアリコートを削除し、ステップ1.6のようにHNG緩衝液を含む標識試料管に追加します。 </li><li> ATPの加水分解反応の終了時に、貯蔵のために-80℃の冷凍庫に希釈したサンプルを移動させます。すべての試料が完全に凍結されていることを確認するには、続行する前に少なくとも10分を待ちます。 </li></ol>検出試薬で無料のPiを含む2.インキュベートサンプル<ol><li>各時点でのATP加水分解反応のアリコートを含む融解希釈した試料は、室温で（ステップ1.6および1.7から得られました）。 </li><li>サンプルおよびリン酸塩標準を含む96ウェルプレートをセットアップ。 <ol><li> 0.5ミリリットルのTUで104.5μlのHNGバッファに標準800μMの5.5μLを添加することにより、40μMに800μMから（Piの検出試薬に付属）、リン酸標準を希釈し、こと。よく混合し、96ウェルプレートのウェルA1に、この40μMPiを標準の100μlを添加します。 </li><li>パイ規格の1の連続希釈：1のためのウェルB1-H1に50μlのHNGのバッファを追加します。 </li><li>よくA1から40μMPiを50μlのを削除し、よくB1に50μlのアッセイバッファーに追加し、混ぜ、よくB1から50μLを削除してもG1を介して、よくC1に継続的な希釈液を追加します。各ウェルが同じボリュームを持って確保するために混合した後も、G1から50μLを捨てます。まあH1は40から0μMにPiの標準を作成するための唯一のバッファが含まれている必要があります。 注：追加の要因は、（このような阻害剤など）をサンプルに添加されている場合は、それらの条件の下でリン酸放出または吸光度の変化を制御するために、その要因を含む標準曲線を作成します。 </li><li>各SAMPの50μLを追加プレートに二重にル。水平方向と異なる時間点、垂直列に同じ時点からのサンプル（サンプル2時間0 = B2、B3サンプル1時間0 = A2、A3）を追加します。これは、プレートあたり最大8個のサンプルと5つの時点を可能にします。 </li></ol></li><li>サンプルおよび標準を含むウェル（試薬必要（ミリリットル）=サンプルおよび標準の0.1 x個）のそれぞれに100μlを添加するのに十分なマラカイトグリーン/モリブデン酸Piの検出試薬を除去するために、滅菌ピペットを使用して、簡単にピペット操作のための皿に追加マルチチャンネルピペットを使用して。 Piの汚染が発生する可能性があるとして、皿に直接検出試薬をかけないでください。 </li><li>マルチチャンネルピペットを用いて、好ましくは、第1の時点への最後の時点から順に、慎重に気泡を導入することなく、ピペッティングと時間の一貫した数ダウンすることにより、各ウェルにPiの検出試薬の100μlを添加し、混合します。 </li><li>室温で25分間プレートをインキュベートし、またはミリアンペアに従ってnufacturerの指示。 </li></ol>マイクロプレートリーダーを使用した3定量結果<ol><li>吸光マイクロプレートリーダーを用いて650 nmでサンプルの吸光度を読みます。 </li><li> Piの標準曲線を作成します。グラフ作成ソフトウェアを使用して、各試料中のリン酸量を解くために使用される式を見つけるために、濃度に対するPiの標準サンプルについて吸光度値をグラフ。 </li><li>リン酸標準で校正することにより、各サンプルのATPase活性を計算します。リン酸解放=（OD 650 - Y切片）/傾き。 <ol><li>各サンプルの重複からの総パイを平均します。この番号からバッファのみの対照の吸光度の読み取りを引きます。希釈係数（この例では50）で、これを乗算します。 </li><li>マイクロモルのタンパク質当たりに放出されるnmolのPiを決定します。動態アッセイにおいて、各時点のためにこれらの値をグラフ化することは、少なくともR = 150（ 図1）の線形フィットをもたらすべきです。 n個の場合OT、アッセイは、多かれ少なかれタンパク質又はより長いまたはより短いインキュベーション時間で調整することができます。 </li></ol></li><li>所望であれば、ナノモルのPi /モルタンパク質/分（ 図2、図 3）、またはナノモルPI /μgタンパク質/分として結果を表します。 </li></ol>

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The authors have nothing to disclose.

これは、生化学的な特徴付けのために精製されたタンパク質のin vitroでのATPase活性に測定するための一般的なプロトコルです。この方法は、容易に最適化されています。例えば、蛋白質、緩衝液および塩組成物の量、温度を調整し、アッセイ長さ（間隔の合計数を増やすなど）の間隔を変化させると、活性定量を改善することができます。市販のマラカイトグリーンベースの試?...

ATPases are integral enzymes in many processes across all kingdoms of life. ATPases act as molecular motors that use the energy of ATP hydrolysis to power such diverse reactions as protein trafficking, unfolding, and assembly; replication and transcription; cellular metabolism; muscle movement; cell motility; and ion pumping1-3. Some ATPases are transmembrane proteins involved in transporting solutes across membranes, others are cytoplasmic and may be associated with a biological membrane such as the plasma membrane or those of organelles.
AAA+ ATPases (ATPases associated with various cellular activities) make up a large group of ATPases that share some sequence and structural conservation. These proteins contain conserved nucleotide binding motifs such as Walker-A and -B boxes and form oligomers (generally hexamers) in their active state1. Large conformational changes in these proteins upon nucleotide binding have been characterized among diverse members of the AAA+ family. EpsE is a AAA+ ATPase and member of the bacterial Type II/IV secretion subfamily of NTPases4-6. EpsE powers Type II Secretion (T2S) in Vibrio cholerae, the causative agent of cholera. The T2S system is responsible for the secretion of a wide variety of proteins, such as the virulence factor cholera toxin that causes profuse watery diarrhea when V. cholerae colonizes the human small intestine7. 
Techniques for quantitating in vitro ATPase activity are varied, but commonly measure phosphate release using colorimetric, fluorescent, or radioactive substrates8-11. We describe a basic method for determining in vitro ATPase activity of purified proteins using a colorimetric assay based on a commercially available malachite green-containing substrate that measures liberated inorganic phosphate (Pi). At low pH, malachite green molybdate forms a complex with Pi and the level of complex formation can be measured at 650 nm. This simple and sensitive assay may be used to functionally characterize new ATPases and to evaluate the roles of potential activators or inhibitors, to determine the importance of domains and/or specific residues, or to assess the effect of particular treatments on enzymatic activity.

<table><tbody><tr><td>HEPES buffer</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>BP358-212</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td></td></tr><tr><td>Adenosine triphosphate (ATP)</td><td>Fisher</td><td>BP41325</td><td></td></tr><tr><td>96-well plates (clear, flat-bottom)</td><td>VWR</td><td>82050-760</td><td></td></tr><tr><td>BIOMOL Green</td><td>Enzo Life Sciences</td><td>BML-AK111</td><td>Preferred phosphate detection reagent. Caution: irritant.</td></tr><tr><td>Microplate reader</td><td></td><td></td><td>BioTek Synergy or comparable</td></tr><tr><td>Prism 5</td><td>GraphPad Software</td><td></td><td></td></tr></tbody></table>

measuring in vitro atpase activity for enzymatic characterization

アデノシン三リン酸加水分解酵素、またはATPアーゼは、細胞機能の多様な配列に重要な役割を果たしています。これらの動的なタンパク質は、タンパク質輸送および分解、溶質輸送、および細胞の動きとして、機械的な作業のためのエネルギーを生成することができます。ここで説明するプロトコルは、機能的な特徴付けのために精製されたATPアーゼのインビトロ活性を測定するための基本的なアッセイです。タンパク質は、無機リン酸の放出をもたらす反応でATPを加水分解し、遊離したリン酸塩の量を、比色アッセイを用いて定量されま​​す。この高度に適応プロトコルは、運動またはエンドポイントアッセイにおいてATPアーゼ活性を測定するために調整することができます。代表的なプロトコルは活動とEPSEの要件に基づいて、ここで提供され、AAA + ATPアーゼは、細菌コレラ菌にタイプIIの分泌に関与します。活性を測定するために必要な精製されたタンパク質の量は、アッセイの長さおよびSAのタイミングおよび数mpling間隔は、緩衝液および塩組成、温度、補因子、刺激剤（もしあれば）、 等は、ここで説明したものと異なる場合があり、したがって、いくつかの最適化が必要であってもよいです。このプロトコルは、特徴付けるATPアーゼのための基本的な枠組みを提供し、迅速に行われ、容易に、必要に応じて調整することができます。

T2S ATPアーゼEPSEのin vitro活性は、細胞質EPSLのドメイン（EPSE-cytoEpsL）と酸性リン脂質カルジオリピン12を加えてEPSEの同時精製によって刺激することができます。このアッセイを用いてタンパク質の変異形態を、野生型（WT）の活性を比較することにより、ATP加水分解の特定EPSE残基の役割を決定することも可能です。ここで、EPSE亜鉛結合ドメイン内の2つ?...

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Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

計測<em&gt;インビトロ</em酵素特性評価のための&gt; ATPase活性

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

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Measuring In Vitro ATPase Activity for Enzymatic Characterization

18.0K ビュー.  University of Michigan. We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

ビデオ: Measuring In Vitro ATPase Activity for Enzymatic Characterization

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

放射性標識ATPによるタンパク質キナーゼ活性のアッセイ

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

生化学

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

新しいキナーゼタンパク質の堅牢な生化学的アプローチを用いて分子レベルでの特性解析

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

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 ...

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

酵素酢酸キナーゼのアセテート形成活性の直接検出

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

生物学

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

浄化、体外(p) ppGppクロストリジウム ・ ディフィシルから合成酵素の活性

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

免疫・感染学

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

腎細胞内のミトコンドリアの機能と細胞生存率の評価を過剰発現するプロテインキナーゼCアイソザイム

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

ATP依存性クロマチンリモデリング酵素の活性を分析するための生化学的アッセイ

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

インビトロアッセイではホスファチジルメチルトランスフェラーゼ活性を測定します

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

Source: Pokhrel, A., et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018).
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.

薄層クロマトグラフィーに基づく酵素活性アッセイ:薄層クロマトグラフィーを使用してin vitroピロホスホキナーゼ活性を決定する技術

Source: Pokhrel, A., et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018).
This ...

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放射性標識ATPによるタンパク質キナーゼ活性のアッセイ

新しいキナーゼタンパク質の堅牢な生化学的アプローチを用いて分子レベルでの特性解析

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

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

酵素酢酸キナーゼのアセテート形成活性の直接検出

浄化、体外(p) ppGppクロストリジウム・ディフィシルから合成酵素の活性

腎細胞内のミトコンドリアの機能と細胞生存率の評価を過剰発現するプロテインキナーゼCアイソザイム

ATP依存性クロマチンリモデリング酵素の活性を分析するための生化学的アッセイ

インビトロアッセイではホスファチジルメチルトランスフェラーゼ活性を測定します

薄層クロマトグラフィーに基づく酵素活性アッセイ:薄層クロマトグラフィーを使用してin vitroピロホスホキナーゼ活性を決定する技術

計測

要約

さらに動画を探す

この動画の章

放射性標識ATPによるタンパク質キナーゼ活性のアッセイ

新しいキナーゼタンパク質の堅牢な生化学的アプローチを用いて分子レベルでの特性解析

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

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

酵素酢酸キナーゼのアセテート形成活性の直接検出

浄化、体外(p) ppGppクロストリジウム ・ ディフィシルから合成酵素の活性

腎細胞内のミトコンドリアの機能と細胞生存率の評価を過剰発現するプロテインキナーゼCアイソザイム

ATP依存性クロマチンリモデリング酵素の活性を分析するための生化学的アッセイ

インビトロアッセイではホスファチジルメチルトランスフェラーゼ活性を測定します

薄層クロマトグラフィーに基づく酵素活性アッセイ:薄層クロマトグラフィーを使用してin vitroピロホスホキナーゼ活性を決定する技術

浄化、体外(p) ppGppクロストリジウム・ディフィシルから合成酵素の活性