この作品は、KSSのNSF賞＃0920274およびサウスカロライナ州試験場プロジェクト（SC - 1700340）によってサポートされていました。本論文では、クレムソン大学の試験場の技術的貢献番号5929です。

このプロトコルの全体的なスキームを図1に概説されている。 1。標準曲線とアッセイのためのソリューションの準備<ol><li>ヒドロキシルアミン塩酸の2モル/ Lの溶液100 mLを準備します。ヒドロキシルアミン塩酸塩の13.9グラム（MW 69.49グラム/モル）を秤量し、約50 mLの蒸留-脱イオン水（のddH 2 O）に溶解する。水酸化カリウムペレットまたは濃縮液を用いてpHを7.0に...

<ol>
	<li>Bork, P., Sander, C., Valencia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ATPase+domain+common+to+prokaryotic+cell+cycle+proteins,+sugar+kinases,+actin,+and+hsp70+heat+shock+proteins.">An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 7290-7294 (1992).</li><li>Bork, P., Sander, C., Valencia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+evolution+of+similar+enzymatic+function+on+different+protein+folds:+the+hexokinase,+ribokinase,+and+galactokinase+families+of+sugar+kinases.">Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.</a> Protein Sci. 2, 31-40 (1993).</li><li>Buss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urkinase:+structure+of+acetate+kinase,+a+member+of+the+ASKHA+superfamily+of+phosphotransferases.">Urkinase: structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases.</a> J. Bacteriol. 183, 680-686 (2001).</li><li>Hurley, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sugar+kinase/heat+shock+protein+70/actin+superfamily:+implications+of+conserved+structure+for+mechanism.">The sugar kinase/heat shock protein 70/actin superfamily: implications of conserved structure for mechanism.</a> Annu. Rev. Biophys. Biomol. Struct. 25, 137-162 (1996).</li><li>Holmes, K. C., Sander, C., Valencia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+ATP-binding+fold+in+actin,+hexokinase+and+Hsc70.">A new ATP-binding fold in actin, hexokinase and Hsc70.</a> Trends. Cell. Biol. 3, 53-59 (1993).</li><li>Ingram-Smith, C., Martin, S. R., Smith, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetate+kinase:+not+just+a+bacterial+enzyme.">Acetate kinase: not just a bacterial enzyme.</a> Trends. Microbiol. 14, 249-253 (2006).</li><li>Aceti, D. J., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+acetate+kinase+from+acetate-grown+Methanosarcina+thermophila.+Evidence+for+regulation+of+synthesis.">Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis.</a> J. Biol. Chem. 263, 15444-15448 (1988).</li><li>Latimer, M. T., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning,+sequence+analysis,+and+hyperexpression+of+the+genes+encoding+phosphotransacetylase+and+acetate+kinase+from+Methanosarcina+thermophila.">Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.</a> J. Bacteriol. 175, 6822-6829 (1993).</li><li>Ingram-Smith, C., Barber, R. D., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+histidines+in+the+acetate+kinase+from+Methanosarcina+thermophila.">The role of histidines in the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 275, 33765-33770 (2000).</li><li>Miles, R. D., Iyer, P. P., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutational+analysis+of+active+site+residues+in+the+acetate+kinase+from+Methanosarcina+thermophila.">Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 276, 45059-45064 (2001).</li><li>Miles, R. D., Gorrell, A., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+transition+state+analog,+MgADP-aluminum+fluoride-acetate,+in+acetate+kinase+from+Methanosarcina+thermophila.">Evidence for a transition state analog, MgADP-aluminum fluoride-acetate, in acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 277, 22547-22552 (2002).</li><li>Ingram-Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+acetate+binding+pocket+in+the+Methanosarcina+thermophila+acetate+kinase.">Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase.</a> J. Bacteriol. 187, 2386-2394 (2005).</li><li>Gorrell, A., Lawrence, S. H., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+kinetic+analyses+of+arginine+residues+in+the+active+site+of+the+acetate+kinase+from+Methanosarcina+thermophila.">Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 280, 10731-10742 (2005).</li><li>Gorrell, A., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Methanosarcina+thermophila+acetate+kinase+mechanism+by+fluorescence+quenching.">Investigation of the Methanosarcina thermophila acetate kinase mechanism by fluorescence quenching.</a> Biochemistry. 46, 14170-14176 (2007).</li><li>Reeves, R. E., Guthrie, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetate+kinase+(pyrophosphate).+A+fourth+pyrophosphate-dependent+kinase+from+Entamoeba+histolytica.">Acetate kinase (pyrophosphate). A fourth pyrophosphate-dependent kinase from Entamoeba histolytica.</a> Biochem. Biophys. Res. Commun. 66, 1389-1395 (1975).</li><li>Fowler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+and+structural+characterization+of+the+novel+PPi-dependent+acetate+kinase+from+the+parasite+Entamoeba+histolytica.">Kinetic and structural characterization of the novel PPi-dependent acetate kinase from the parasite Entamoeba histolytica.</a> , (2011).</li><li>Lipmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+synthesis+of+acetyl+phosphate.">Enzymatic synthesis of acetyl phosphate.</a> J. Biol. Chem. 155, 55-70 (1944).</li><li>Lipmann, F., Tuttle, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+micromethod+for+determination+of+acyl+phosphates.">A specific micromethod for determination of acyl phosphates.</a> J. Biol. Chem. 159, 21-28 (1945).</li><li>Lipmann, F., Jones, M. E., Black, S., Flynn, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+ATP-CoA-acetate+reaction.">The mechanism of the ATP-CoA-acetate reaction.</a> J. Cell. Physiol. Suppl. 41, 109-112 (1953).</li><li>Rose, I. A., Grunberg-Manago, M., Korey, S. F., Ochoa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+phosphorylation+of+acetate.">Enzymatic phosphorylation of acetate.</a> J. Biol. Chem. 211, 737-756 (1954).</li><li>Allen, S. H., Kellermeyer, R. W., Stjernholm, R. L., Wood, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+enzymes+involved+in+the+proponic+acid+fermentation.">Purification and properties of enzymes involved in the proponic acid fermentation.</a> Journal of Bacteriology. 87, 171-187 (1964).</li><li>Clarke, P. M., Payton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+assay+for+acetate+in+spent+bacterial+culture+supernatants.">An enzymatic assay for acetate in spent bacterial culture supernatants.</a> Anal. Biochem. 130, 402-405 (1983).</li><li>Mukhopadhyay, S., Hasson, M. S., Sanders, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+assay+of+acetate+kinase+activity:+measurement+of+inorganic+phosphate+release+generated+by+hydroxylaminolysis+of+acetyl+phosphate.">A continuous assay of acetate kinase activity: measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate.</a> Bioorg. Chem. 36, 65-69 (2008).</li><li>Nakajima, H., Suzuki, K., Imahori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+acetate+kinase+from+Bacillus+stearothermophilus.">Purification and properties of acetate kinase from Bacillus stearothermophilus.</a> J. Biochem. 84, 193-203 (1978).</li></ol>

このアッセイにおけるアセチルリン酸の検出は、ヒドロキシルアミン塩酸塩と塩化第二鉄溶液の濃度と酸性度の十分な濃度に依存している。アッセイ体積の変化は、これらのコンポーネントの両方の見直しが必要になります。酵素反応は、ここで説明し、37℃60℃実行ヒドロキシル終端を持つC℃で5分間で行った。これより高い温度は、アセチルヒドロキに残っているアセチルリン酸塩の迅速?...

<table border="1"><tr><td width="160"> 試薬の名前 </td><td width="108"> 会社 </td><td width="134"> カタログ番号 </td><td width="103"> コメント </td></tr><tr><td>アセチルリン酸</td><td>シグマアルドリッチ</td><td> 01409 </td><td>リチウム塩（97％） </td></tr><tr><td>ナトリウム一塩基性リン酸（脱水） </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td>ナトリウム二塩基性リン酸（無水） </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td>塩化マグネシウム（六水和物） </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td>トリスベース</td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td>塩化第二鉄（六水和物） </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td>トリクロロ酢酸</td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td>ヒドロキシルアミン塩酸塩</td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td> 5&#39; -二リン酸アデノシンナトリウム塩</td><td>シグマアルドリッチ</td><td> A2754 </td><td></td></tr><tr><td> Biomate III分光光度計</td><td> ThermoFisher </td><td> 142982082 </td><td>標準的なUV /可視分光光度計</td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

酢酸キナーゼ、酢酸、砂糖キナーゼ- Hsp70の-アクチン（ASKHA）酵素のスーパーファミリー1-5のメンバーは、基質としてATPを利用したアセチルリン酸への酢酸の可逆的リン酸化を担当しています。酢酸キナーゼは、 古細菌の一属で見つかった、 細菌のユビキタスであり、またEukarya 6の微生物中に存在する。最もよく特徴付け酢酸キナーゼは、メタンガスを作り出す細菌Methanosarcinaサーモ 7月14日からということです。唯一のリン酸塩形成アセチル方向にATPない私を PPに利用できますが、酢酸キナーゼは、 赤痢アメーバ 、アメーバ赤痢の病原体から分離されており、これまでこの属15,16でのみ発見されました。 アセチルリン酸形成の方向では、酢酸キナーゼ活性は、通常、最初リップマンによって記述された、ヒドロキアッセイを用いて測定され17-20、ADPへのATPの変換が酵素のピルビン酸キナーゼ、乳酸脱水素酵素21,22、またはアセチルリン酸塩の製品との反応後の無機リン酸の放出を測定するアッセイによりNAD +にNADHの酸化に結合される結合アッセイヒドロキシルアミン23と。反対の活動は、酢酸を形成する方向は、ADPからNADPの還元+酵素のヘキソキナーゼとグルコース-6 -リン酸脱水素酵素24によりNADPHにするためにATPの形成を結合することによって測定されます。 ここでは、カップリング酵素を必要としない酢酸の形成の方向に酢酸キナーゼ活性を検出するための方法を説明しますが、代わりにアセチルリン酸の消費量の直接測定に基づいています。酵素反応の後、残りのアセチルリン酸塩は、ヒドロキアッセイ用として、分光光度法で測定することができる第二鉄ヒドロキ複合体に変換されます。このように、STとは異なり、ADPからATPの生産に依存しているこの方向のandard結合アッセイは、この直接的なアッセイは、ATPまたはPP iを生成する酢酸キナーゼのために使用することができます。

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

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

酢酸キナーゼ活性の測定方法について説明する。このアッセイは異なるホスホリルアクセプターで、酢酸を形成する方向に酢酸キナーゼの酵素活性と反応速度を決定するための直接的な反応を利用している。さらに、この方法は、酵素を利用する他のアセチルリン酸塩またはアセチル- CoAを検定するために利用することができます。

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 ...

Research

JoVE Journal

Biology

Detection of Protein Ubiquitination

タンパク質のユビキチン化の検出

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

プロテアーゼ活性を測定するためにプロテアーゼ蛍光検出キットの使用

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

ELIME（免疫磁気電気化学的にリンクされた酵素）マイコトキシンの検出方法

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

トランスジェニックの生産アフリカツメガエル制限酵素を介した統合と核移植によって

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

一酸化窒素とスピントラップを使用して、細胞からの電子常磁性共鳴分光法によるスーパーオキシドアニオンラジカルの検出

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

FRETはレシオメトリックで細胞周期のキナーゼおよびホスファターゼの活動を監視

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Assaying the Kinase Activity of LRRK2 in vitro

Assaying the Kinase Activity of LRRK2 in vitro

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 ...

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

土壌中の微生物の残留決定のためのAldononitrile酢酸誘導体化グルコサミンとムラミン酸のGCベースの検出

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Measurement of Larval Activity in the Drosophila Activity Monitor

幼虫活性の測定<em&gt;ショウジョウバエ</em&gt;アクティビティモニタ

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Studying the Activity of Neuropeptides and Other Regulators of the Excretory System in the Adult Mosquito

成虫の蚊における排泄系の神経ペプチドおよび他の調節因子の活性の研究

Studies of insect physiology, particularly in those species that are vectors of pathogens causing disease in humans and other vertebrates, provide the ...