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 empfindlich und können geringe Mengen an freiem Phosphat (~ 50 pmol in 100 ul) zu erkennen. Wegen dieser Empfindlichkeit des Assays ist es entscheidend, Einweg-Kunststoff-Geschirr, Reinstwasser, Puffer und Reagenzien zu verwenden, ohne Phosphat zu kontaminieren. Nach der Reinigung von Proteinen, Grßenausschluß oder Ionenaustauschchromatographie wird empfohlen Proteinreinheit zu verbessern und Verunreinigungen zu entfernen. Für Proteine ​​schwach in vitro ATPase - Aktivität anzeigt, Stimulantien können der Reaktion zugesetzt werden , e zu verbessernnzymatic Aktivität. Viele Faktoren, die ATPase-Aktivität stimulieren, charakterisiert. Zum Beispiel, Cardiolipin und andere Membranlipide, Client - Proteine ​​von Chaperonen wie Hsp90 und anderen beteiligten Proteine ​​bei der Unterstützung von bestimmten Konformationen oder Umgebungen , in denen ATPasen funktionieren 13-15. Unser Labor charakterisiert erste Epse durch Monomere mit schwachen ATPase - Aktivität im Vergleich zu homologe ATPase 6 zu reinigen. Wir entdeckten , daß , wenn später EPSE mit der zytoplasmatischen Domäne von EPSL, einem Transmembranprotein und der Bindungspartner EPSE im T2S System Zugabe von sauren Phospholipide wie Cardiolipin zu der Reaktionsmischung stark die ATPase - Aktivität von EPSE um 12 gemeinsam gereinigten wurde. Diese wahrscheinlich ahmt die Bedingungen Epse Erfahrungen bei der Cytoplasmamembran, die Oligomerisierung fördern kann. Viele Techniken wurden verwendet , um in vitro ATPase Aktivität der gereinigten Proteine ​​zu quantifizieren. Radioaktive γ- <sup&gt; 32 P wurde häufig und erfolgreich umgesetzt ATP - Hydrolyse 14,16 jedoch zu quantifizieren, ist Sicherheit ein Anliegen und bedarf der Genehmigung für den Einsatz im Labor von Radioaktivität. Während hochempfindlich, ist die kurze Halbwertszeit von γ- 32 P auch ein Nachteil. Andere kommerzielle Phosphatnachweisverfahren zur Verfügung, wie jene, die auf der Bildung eines fluoreszierenden Produkts verlassen. Einige dieser Methoden sind auch sehr empfindlich, aber erfordern häufig die Zugabe anderer Enzyme zur Reaktions, wodurch Reagenzien, die weniger stabil sind, im Laufe der Zeit. Darüber hinaus Kits zur Verfügung, die während ADP ATP - Hydrolyse unter Verwendung eines stabilen Lumineszenz-Reagens freigesetzt erfassen, aber diese weniger ideal sein kann für ATPasen mit geringer Aktivität 17. Der Test hier beschriebene besteht aus nur einem Phosphatfreisetzung Meßschritt, ist sehr empfindlich und kann in der Regel innerhalb von wenigen Stunden durchgeführt werden. Es ist auch möglich, prepare ein Malachitgrün haltiges Substrat zu vermeiden , ein Kit von einem kommerziellen Anbieter 6,9 Kauf. Eine Überlegung, bevor Sie diesen Test Bezeichnung ist, dass der Schritt zwischen dem Hinzufügen der Phosphat-Nachweisreagenz und unter Extinktionsablesungen ist relativ zeitempfindlich und müssen zwischen 20-30 min. Dieses Basisprotokoll kann verwendet werden , um die Rolle von Stimulanzien zu bestimmen (wie beschrieben), Antagonisten, Untereinheiten, Domänen und spezifische Reste in ATPase Aktivität 15,16,18,19. Dieser Test kann auch die Aktivität von Phosphatasen und andere Enzyme zur Messung erweitert werden, die während der Katalyse Phosphat freizusetzen. Zusätzlich kann dieses Verfahren zum Hochdurchsatz - Screening für ATPase - Inhibitoren 20 angewendet werden.

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

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 der EPSE Zink-Bindungsdomäne wird durch einen Vergleich ATPase-Aktivität von gereinigtem WT EPSE-cytoEpsL zum EPSE K417AK419A-cytoEpsL Variante gemessen. In Figur 1 verläuft Phosphatfreisetzung linear über den Verlauf einer 1 Std kinetischer Assay eine Anforderung für eine genaue Quantifizierung von kinetischer ATPase Aktivität. 2 zeigt Daten aus der gleichen gereinigten Proteinproben wie in Abbildung 1, die dreimal in zweifacher Ausfertigung getestet wurden und quantifiziert bezüglichdie mittlere Geschwindigkeit der Phosphatfreisetzung. Diese Daten zeigen, dass die K417 und K419 erscheinen für Epse ATPase-Aktivität wichtig. Der Vergleich von unstimulierten (keine Cardiolipin) ATPase Aktivitätsraten (Figur 3) zeigt , dass K417 und K419 nicht EPSE die basale Aktivität beitragen , sondern auf die Fähigkeit des Proteins , das durch Cardiolipin stimuliert werden. Die positiv geladenen Lysine in der EPSE Zink-Bindungsdomäne kann mit den negativ geladenen Phospholipiden direkt interagieren, was zu dem Phospholipid-vermittelte Stimulation der EPSE ATPase-Aktivität beiträgt. <img alt="Abbildung 1" src="/files/ftp_upload/54305/54305fig1.jpg" /> Abbildung 1: Phosphat wird freigegeben linear in einer Kinetic ATPase Assay Eine Stunde kinetische Cardiolipin-stimulierte ATPase - Assay verglichen Phosphatfreisetzung von 0,5 uM WT Epse-cytoEpsL zu Epse K417AK419A-cytoEpsL mit Rinderserumalbumin (BSA) getestet , wie.eine negative Kontrolle. Daten [Menge an freigesetztem Phosphat (y-Achse) gegen die Zeit (x-Achse)] wurden aufgetragen, und auf der linearen Regressionsanalyse unterzogen. Die Steigung stellt die Rate der ATP-Hydrolyse. Eine repräsentative Graph mit linearen Regressionsgleichungen für die drei Proteine ​​gezeigt. <a href="https://www.jove.com/files/ftp_upload/54305/54305fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www.jove.com/files/ftp_upload/54305/54305fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/54305/54305fig2.jpg" /> Fig . 2:. Doppel Lysine Mutations in EPSE Zink-bindende Domäne stimulierte ATPase - Aktivität reduzieren Ergebnisse der 1 hr kinetische Cardiolipin-stimulierte ATPase - Assay mit denselben Proteinen und Bedingungen wie in Abbildung 1 Die Tests wurden drei getrennte Male in technischen zweifach durchgeführt , und die Rate der ATP-Hydrolyse wurde als nmol Phosphat pro Minute erzeugt, berechnet pro uM pr<html> otein lineare Regressionsgleichungen. Die mittleren Ergebnisse mit Standardfehler angezeigt werden. <a href="https://www.jove.com/files/ftp_upload/54305/54305fig2large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://www.jove.com/files/ftp_upload/54305/54305fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/54305/54305fig3.jpg" /> Abbildung 3: Doppel Lysine Mutationen in Epse Zink-Bindungsdomäne nicht stören Nichtstimulierte ATPase Aktivität über Nacht (16 h) Endpunkt nicht stimulierte (kein Cardiolipin) ATPase Assay , um die Aktivität von 5 &amp; mgr; M WT Epse-cytoEpsL zu Epse K417AK419A-cytoEpsL mit BSA zu vergleichen. als negative Kontrolle. Die Tests wurden durchgeführt, drei getrennte Male in technischen Duplikat und mittleren Ergebnisse mit Standardfehler dargestellt. Das Grundniveau der unstimulierten Epse ATPase-Aktivität über einen Zeitraum 16 Stunden getestet ist ~ 1000-fach niedriger als 1 Stunde kinetische Cardiolipin-stimulierte Aktivität.e.com/files/ftp_upload/54305/54305fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte hier klicken, um eine größere Version dieser Figur zu sehen.

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Measuring In Vitro ATPase Activity for Enzymatic Characterization

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

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

Nanyang Technological University

Research

JoVE Journal

Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

17.9K 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

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.

Ein Assay zur Messung der Aktivität von Escherichia coli Inducible Lysin 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 ...

Forschung

Biologie

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.

Untersuchen der Kinase-Aktivität von LRRK2 In-vitro-</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.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

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.

Mess-Cation Transport von Na, K-und H, K-ATPase in<em&gt; Xenopus</em&gt; Oozyten mittels Atomabsorptionsspektrometrie: Eine Alternative zu Radioisotopen Assays

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.

Mess Glutathion-induzierte Fütterung Antwort in Hydra

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.

Funktionelle Rekonstitution und Kanalaktivitätsmessungen von gereinigtem Wildtyp und mutierten CFTR-Protein

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.

Verfahren zur Messung der Aktivität von Deubiquitinierungsenzyme in Zelllinien und Gewebeproben

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.

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 ...

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.

Messung der Laktase enzymatische Aktivität in das Lehrlabor

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.

In-vitro- Sumoylierung Assay SUMO E3 Ligase Tätigkeit zu studieren

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