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. Führen Sie die ATP-Hydrolyse-Reaktion mit gereinigtem Protein <ol><li> Bereiten Sie Bestände an alle notwendigen Reagenzien für die Inkubation mit gereinigten Protein. <ol><li> Bereiten 5x HEPES / NaCl / Glycerin (HNG) -Puffer, enthaltend 100 mM HEPES pH 8,5, 65 mM NaCl und 5% Glycerin (oder anderen Assaypuffer wie geeignet). </li><li> Bereiten 100 mM MgCl 2 (oder einem anderen Metall, wenn ATPase Metall abhängig ist) in Wasser. </li><li> Bereiten Sie frische 100 mM ATP in 200 mM Tris Base (nicht einstellen pH-Wert weiter) hoher Reinheit ATP verwendet wird. Aliquotieren und lagern Sie das ATP-Lager bei -20 ° C nicht länger als ein paar Wochen, als ATP im Laufe der Zeit zusammenbrechen wird, und unterlassen das Einfrieren und die ATP-Lager Auftauen. </li><li> Premix MgCl 2 und ATP in einem Verhältnis 1: 1 kurz vor der ATP - Hydrolyse - Reaktion einrichten. </li></ol></li><li> Bereiten Sie und beschriften 1,5-ml-Röhrchen für das Sammeln von Proben in regelmäßigen Abständen während der Reaktion. Bereiten Röhrchen zu sammeln Proben zum Zeitpunkt 0 und zum Zeitpunkt 15, 30,45 und 60 min. HINWEIS: Als Alternative zu sammeln Proben nur zum Zeitpunkt 0 und dem Endpunkt. <ol><li> In 245 ul 1x HNG Puffer in jedes Röhrchen, um Proben von den ATP-Hydrolysereaktionen 01.50 gesammelt zu verdünnen. </li></ol></li><li> Bereiten Sie ein Bad aus Trockeneis und Ethanol schnell Proben Einfrieren der Reaktion zu stoppen. In einem Gummi Eiskübel oder anderen sicheren (nicht Kunststoff) Behälter, fügen Sie mehrere Stücke von Trockeneis und sorgfältig genug 70-100% Ethanol gießen Sie das Trockeneis zu decken. </li><li> Verdünnen gereinigtes Protein in 1x HNG-Puffer, gegebenenfalls (in der Regel 5 bis 10 &amp; mgr; M), und halten Sie auf dem Eis. </li><li> Stellen Sie die ATP-Hydrolyse-Reaktionen auf. <ol><li> In separaten 0,5 - ml - Röhrchen für jede Probe, die folgenden Reagenzien (in dieser Reihenfolge): H 2 O (bis zu einem Gesamtvolumen von 30 &amp; mgr; l), 6 ul 5x HNG oder andere Puffer, 3 ul 100 mM MgCl 2 -ATP Mischung, und 0,25-5 uM Protein. </li><li> Fügen Sie einen Puffer-nur negative Kontrolle Zustand, in dem kein Protein zugesetzt wird.</li></ol></li><li> Entfernen Sie 5 ul aus der Reaktion zum Zeitpunkt 0, verdünnte 01.50 in den vorbereiteten 1,5 ml Röhrchen mit 245 ul HNG-Puffer, und sofort gefrieren die Probe in der Trockeneis / Ethanol-Bad. </li><li> Inkubieren Reaktionen bei 37 ° C ATP Hydrolyse zu ermöglichen, für 1 h auftritt. In jedem Intervall (15, 30, 45 und 60 min), Entfernen 5 ul Aliquots aus dem Reaktions und fügen Röhren markierte Probe enthält HNG-Puffer, wie in Schritt 1.6. </li><li> Am Ende der ATP Hydrolysereaktion bewegen verdünnten Proben auf einen -80 ° C Gefrierschrank für die Lagerung. Um sicherzustellen, dass alle Proben vollständig gefroren sind, warten Sie mindestens 10 Minuten, bevor Sie fortfahren. </li></ol> 2. Inkubieren Proben mit freien Pi mit Nachweisreagenz <ol><li> Auftau verdünnten Proben Aliquots ATP Hydrolysereaktion zu jedem Zeitpunkt enthält, (erhalten aus den Schritten 1.6 und 1.7) bei Raumtemperatur. </li><li> Richten Sie eine 96-Well-Platte mit Proben und Phosphat-Standards. <ol><li> In einer 0,5 ml tusein, verdünnen das Phosphat-Standard (mit Pi Nachweisreagenz versehen) von 800 um bis 40 um 5,5 ul der 800 um Standard zu 104,5 ul HNG Puffer hinzuzufügen. Gut mischen, und fügen Sie 100 ul dieser 40 uM Pi-Standard gut A1 einer 96-Well-Platte. </li><li> In 50 ul HNG-Puffer in Vertiefungen B1-H1 für 1: 1 serielle Verdünnungen des Pi-Standard. </li><li> Entfernen Sie 50 ul 40 uM Pi aus gut A1 und in den 50 &amp; mgr; l Testpuffer in gut B1, mischen, und entfernen Sie 50 ul von gut B1 und in den gut C1, weiterhin Verdünnungen durch gut G1. Entsorgen 50 ul von gut G1 nach dem Mischen jeder Vertiefung, um sicherzustellen, das gleiche Volumen hat. Nun H1 sollte nur Puffer enthalten, um einen Pi-Standard 40-0 uM erstellen. HINWEIS: Wenn ein zusätzlicher Faktor (wie ein Inhibitor) wurde zu den Proben hinzugefügt wurde, erstellen Sie eine Standardkurve, diesen Faktor für Veränderungen in der Phosphatfreisetzung oder Absorption unter diesen Bedingungen zu steuern, enthält. </li><li> In 50 ul jeder sample in zweifacher Ausfertigung an die Platte. In Proben aus dem gleichen Zeitpunkt in Spalten vertikal (Probe 1 Zeit 0 = A2, A3; Probe 2 Mal 0 = B2, B3) und verschiedenen Zeitpunkten in horizontaler Richtung. Auf diese Weise können bis zu 8 Proben und 5 Zeitpunkten pro Platte. </li></ol></li><li> Eine sterile Pipette genug Malachitgrün / Molybdat Pi Nachweisreagenz zu entfernen, um eine Schale zum einfachen Pipettieren mit 100 &amp; mgr; l zu jeder der Vertiefungen, die Proben und Standards (Reagenz benötigt (ml) = 0,1 x Anzahl der Proben und Standards) und fügen mit einer Mehrkanalpipette. gießen Sie das Nachweisreagenz Sie nicht direkt in die Schale, wie Pi Verunreinigung auftreten dürfte. </li><li> durch vorsichtiges Auf- und Abpipettieren eine konsistente Anzahl von Zeiten ohne Einführung von Blasen, vorzugsweise in der Reihenfolge von der letzten Zeitpunkten zu den ersten Zeitpunkten mit einer Mehrkanalpipette, 100 &amp; mgr; l des Pi-Nachweisreagens in jede Vertiefung und mischen. </li><li> Die Platte für 25 Minuten bei Raumtemperatur oder nach der mahersteller der Richtungen. </li></ol> 3. Quantifizieren Ergebnisse eines Mikroplatten-Reader verwenden <ol><li> Die Extinktion der Proben bei 650 nm eine Extinktion Mikrotiterplatten-Lesegerät verwendet wird. </li><li> Machen Sie einen Pi-Standardkurve. Mit einer Grafiksoftware, Graph, der die Absorptionswerte für die Pi-Standardproben gegen die Konzentration, um eine Gleichung zu finden verwendet, um die Menge an Phosphat, die in jeder Probe zu lösen. </li><li> Berechnen Sie die ATPase-Aktivität für jede Probe, indem sie mit dem Phosphat-Standard zu kalibrieren. Phosphat freigesetzt = (OD 650 - Y Intercept) / Steigung. <ol><li> Der Mittelwert der Gesamt Pi aus Duplikate jeder Probe. Subtrahieren Sie die Puffer nur Absorptionswert der Kontrolle von dieser Nummer. Multipliziert mit dem Verdünnungsfaktor (50 in unserem Beispiel). </li><li> Bestimmen Sie die Pi nmol pro umol Protein freigesetzt. Graphische Darstellung dieser Werte für jeden Zeitpunkt in einem kinetischen Test sollte linear Anfällen von mindestens R = 0,99 (Abbildung 1) ergeben; wenn not, kann der Test mit mehr oder weniger Protein oder eine längere oder kürzere Inkubationszeit eingestellt werden. </li></ol></li><li> Stellen die Ergebnisse als nmol Pi / umol Protein / min (2, 3) oder als nmol Pi / &amp; mgr; g Protein / min , falls gewünscht. </li></ol>

<ol>
	<li>Hanson, P. I., Whiteheart, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAA++proteins:+have+engine,+will+work.">AAA+ proteins: have engine, will work.</a> Nat Rev Mol Cell Biol. 6 (7), 519-529 (2005).</li><li>Baker, T. A., Sauer, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClpXP,+an+ATP-powered+unfolding+and+protein-degradation+machine.">ClpXP, an ATP-powered unfolding and protein-degradation machine.</a> Biochimica et Biophysica Acta. 1823 (1), 15-28 (2012).</li><li>Maxson, M. E., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vacuolar-type+H+-ATPase+at+a+glance+-+more+than+a+proton+pump.">The vacuolar-type H+-ATPase at a glance - more than a proton pump.</a> J Cell Sci. 127 (23), 4987-4993 (2014).</li><li>Planet, P. J., Kachlany, S. C., DeSalle, R., Figurski, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+of+genes+for+secretion+NTPases:+Identification+of+the+widespread+tadA+subfamily+and+development+of+a+diagnostic+key+for+gene+classification.">Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification.</a> Proc Natl Acad Sci U S A. 98 (5), 2503-2508 (2001).</li><li>Robien, M. A., Krumm, B. E., Sandkvist, M., Hol, W. G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+of+the+Extracellular+Protein+Secretion+NTPase+EpsE+of+Vibrio+cholerae.">Crystal Structure of the Extracellular Protein Secretion NTPase EpsE of Vibrio cholerae.</a> J Mol Biol. 333 (3), 657-674 (2003).</li><li>Camberg, J. L., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+the+Vibrio+cholerae+type+II+secretion+ATPase+EpsE.">Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE.</a> J Bacteriol. 187 (1), 249-256 (2005).</li><li>Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+II+secretion+and+pathogenesis.">Type II secretion and pathogenesis.</a> Infect Immun. 69 (6), 3523-3535 (2001).</li><li>Brune, M., Hunter, J. L., Corrie, J. E. T., Webb, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct,+Real-time+measurement+of+rapid+inorganic+phosphate+release+using+a+novel+fluorescent+probe+and+its+application+to+actomyosin+subfragment+1+ATPase.">Direct, Real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.</a> Biochemistry. 33 (27), 8262-8271 (1994).</li><li>Carter, S. G., Karl, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inorganic+phosphate+assay+with+malachite+green:+An+improvement+and+evaluation.">Inorganic phosphate assay with malachite green: An improvement and evaluation.</a> J Biochem Biophys Methods. 7 (1), 7-13 (1982).</li><li>Henkel, R. D., VandeBerg, J. L., Walsh, R. 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+microassay+for+ATPase.">A microassay for ATPase.</a> Anal Biochem. 169 (2), 312-318 (1988).</li><li>Harder, K. W., Owen, P., Wong, L. K. H., Aebersold, R., Clark-Lewis, I., Jirik, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+kinetic+analysis+of+the+intracellular+domain+of+human+protein+tyrosine+phosphatase+&#946;+(HPTP+&#946;)+using+synthetic+phosphopeptides.">Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase &#946; (HPTP &#946;) using synthetic phosphopeptides.</a> Biochem J. 298 (2), 395-401 (1994).</li><li>Camberg, J. L., Johnson, T. L., Patrick, M., Abendroth, J., Hol, W. G., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+stimulation+of+EpsE+ATP+hydrolysis+by+EpsL+and+acidic+phospholipids.">Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids.</a> EMBO J. 26 (1), 19-27 (2006).</li><li>McLaughlin, S. H., Smith, H. W., Jackson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+the+weak+ATPase+activity+of+human+Hsp90+by+a+client+protein.">Stimulation of the weak ATPase activity of human Hsp90 by a client protein.</a> J Mol Biol. 315 (4), 787-798 (2002).</li><li>Shiue, S., Kao, K., Leu, W., Chen, L., Chan, N., Hu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XpsE+oligomerization+triggered+by+ATP+binding,+not+hydrolysis,+leads+to+its+association+with+XpsL.">XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL.</a> EMBO J. 25 (7), 1426-1435 (2006).</li><li>Ghosh, A., Hartung, S., van der Does, C., Tainer, J. A., Albers, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaeal+flagellar+ATPase+motor+shows+ATP-dependent+hexameric+assembly+and+activity+stimulation+by+specific+lipid+binding.">Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding.</a> Biochem J. 437 (1), 43-52 (2011).</li><li>Savvides, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VirB11+ATPases+are+dynamic+hexameric+assemblies:+new+insights+into+bacterial+type+IV+secretion.">VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion.</a> EMBO J. 22 (9), 1969-1980 (2003).</li><li>Sanghera, J., Li, R., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+luminescent+ADP-Glo+assay+to+a+standard+radiometric+assay+for+measurement+of+protein+kinase+activity.">Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity.</a> Assay Drug Dev Techn. 7 (6), 615-622 (2009).</li><li>Sherman, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoupling+catalytic+activity+from+biological+function+of+the+ATPase+that+powers+lipopolysaccharide+transport.">Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport.</a> Proc Natl Acad Sci U S A. 111 (13), 4982-4987 (2014).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cofactor+regulation+with+disease-associated+p97/VCP+mutations.">Altered cofactor regulation with disease-associated p97/VCP mutations.</a> Proc Natl Acad Sci U S A. 112 (14), E1705-E1714 (2015).</li><li>Rowlands, M. G., Newbatt, Y. M., Prodromou, C., Pearl, L. H., Workman, P., Aherne, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+assay+for+inhibitors+of+heat-shock+protein+90+ATPase+activity.">High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.</a> Anal Biochem. 327 (2), 176-183 (2004).</li></ol>

The authors have nothing to disclose.

Dies ist ein allgemeines Protokoll für die in vitro ATPase Aktivität von gereinigten Proteinen für die biochemische Charakterisierung zu messen. Dieses Verfahren ist leicht optimiert; beispielsweise die Menge an Protein, Puffer und Salzzusammensetzungen Einstellen der Temperatur und der Variation der Testlänge und Intervalle (einschließlich der Gesamtzahl der Intervalle Erhöhung) können Aktivitäts Quantifizierung verbessern. Im Handel erhältliche Malachitgrün-basierenden Reagenzien sind sehr empfindli...

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

Adenosintriphosphat-hydrolysierende Enzyme oder ATPase, eine entscheidende Rolle in einer Vielfalt von Zellfunktionen spielen. Diese dynamischen Proteine ​​können Energie für die mechanische Arbeit, wie zum Beispiel Proteintransport und Abbau, Transport gelöster Stoffe und zelluläre Bewegungen erzeugen. Das hier beschriebene Protokoll ist ein einfaches Assay zur Messung der in vitro - Aktivität von gereinigtem ATPasen für die funktionelle Charakterisierung. Proteine ​​hydrolysieren ATP in einer Reaktion, die in anorganischen Phosphatfreisetzung resultiert, und die Menge an Phosphat freigesetzt wird dann quantifiziert eines kolorimetrischen Assay. Das hochflexible Protokoll kann eingestellt werden ATPase-Aktivität in kinetische oder Endpunkt-Assays zu messen. Ein repräsentatives Protokoll ist vorgesehen , bezogen auf die Aktivität und Anforderungen EPSE umfasste die AAA + ATPase in Type II Secretion in dem Bakterium Vibrio cholerae. Die Menge an gereinigtem Protein benötigt Aktivität zu messen, die Länge des Tests und der Zeitpunkt und die Anzahl der sampling Intervallen, Puffer und Salzzusammensetzung, der Temperatur, Co-Faktoren, Stimulantien (falls vorhanden), usw. können hier von den beschriebenen abweichen, und somit kann eine gewisse Optimierung erforderlich sein. Dieses Protokoll stellt ein Grundgerüst für die Charakterisierung ATPasen und kann schnell und einfach eingestellt wie erforderlich durchgeführt werden.

Die in vitro Aktivität der ATPase T2S EPSE kann durch gemeinsame Reinigung von EPSE mit der zytoplasmatischen Domäne von EPSL (EPSE-cytoEpsL) und Zugabe der sauren Phospholipid Cardiolipin 12 stimuliert werden. Es ist auch möglich, die Rolle bestimmter EPSE Rückstände in ATP-Hydrolyse zu bestimmen, unter Verwendung dieses Assay Aktivität von Wildtyp (WT) zu varianten Formen des Proteins verglichen. Hier wird die Wirkung der Substitution von zwei Lysinreste in de...

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.

Messung<em&gt; In Vitro</em&gt; ATPase Aktivität für die enzymatische Charakterisierung

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

18.0K Aufrufe.  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.

Serie: 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.

Test der Proteinkinase-Aktivität mit radioaktiv markiertem 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 ...

Forschung

Biochemie

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.

Charakterisierung auf molekularer Ebene mit robusten biochemischen Ansätzen eines neuen Kinase-Proteins

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.

Eine semi-High-Throughput-Anpassung des NADH-gekoppelten ATPase-Assays zum Screening kleiner Molekülinhibitoren

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.

Direkter Nachweis der Acetat-bildenden Aktivität des Enzyms Acetat-Kinase

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

Biologie

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.

Eine Reinigung und In vitro- Aktivität Assay für ein (p) PpGpp Synthestase von Clostridium difficile

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

Immunologie und Infektion

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.

Beurteilung der mitochondrialen Funktionen und Zellviabilität in Renal Überexpression Protein Kinase C-Isoenzyme

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.

Biochemischen Assays zur Analyse von Aktivitäten von ATP-abhängigen Chromatin-Remodeling-Enzyme

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.

In vitro-Test an Phosphatidylethanolamin Methyltransferase-Aktivität zu messen

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.

Dünnschichtchromatographie-basierter Enzymaktivitätsassay: Eine Technik zur Bestimmung der In-vitro-Pyrophosphokinase-Aktivität mittels Dünnschichtchromatographie

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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Dünnschichtchromatographie-basierter Enzymaktivitätsassay: Eine Technik zur Bestimmung der In-vitro-Pyrophosphokinase-Aktivität mittels Dünnschichtchromatographie