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 border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">HEPES buffer</td>
			<td style="width:128px;">Fisher</td>
			<td style="width:108px;">BP310-500</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine triphosphate (ATP)</td>
			<td>Fisher</td>
			<td>BP41325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well plates (clear, flat-bottom)</td>
			<td>VWR</td>
			<td>82050-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOMOL Green</td>
			<td>Enzo Life Sciences</td>
			<td>BML-AK111</td>
			<td>Preferred phosphate detection reagent. Caution: irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td></td>
			<td></td>
			<td>BioTek Synergy or comparable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism 5</td>
			<td colspan="2">GraphPad Software</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?...

Watch this Scientific Journal Video about Measuring In Vitro ATPase Activity for Enzymatic Characterization at JoVE.com

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 ...

measuring-in-vitro-atpase-activity-for-enzymatic-characterization

Research

JoVE Journal

Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

17.9K 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

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

の活性を測定するアッセイ大腸菌誘導性リジンDecarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

生物学

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2のキナーゼ活性を測定する in vitroで</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

のNa、KとH、K-ATPaseのインによって陽イオン輸送を測定する<em&gt;アフリカツメガエル</em原子吸光法による&gt;卵母：アイソトープアッセイの代替

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

ハイドラ内グルタチオンによって誘発される摂食応答を測定する

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

細胞株および組織試料中の脱ユビキチン化酵素の活性を測定する方法

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

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 ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

教育実習でラクターゼ酵素活性の測定

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

体外相撲 E3 リガーゼ活性を研究する sumo 化アッセイ

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...