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

1. Eseguire ATP idrolisi di reazione con proteina purificata <ol><li> Preparare scorte di tutti i reagenti necessari per l&#39;incubazione con proteina purificata. <ol><li> Preparare tampone 5x HEPES / NaCl / glicerolo (HNG) contenente 100 mM HEPES pH 8,5, 65 mM NaCl, e il 5% glicerolo (o altro tampone a seconda dei casi). </li><li> Preparare 100 mM MgCl 2 (o altro metallo, se ATPasi è in metallo-dipendente) in acqua. </li><li> Preparare fresco 100 mM ATP a 200 mm Tris Base (non regolare il pH ulteriormente) con alta ATP purezza. Aliquota e memorizzare il titolo ATP a -20 ° C per non più di un paio di settimane, come l&#39;ATP si rompe nel corso del tempo, e astenersi dal congelamento e scongelamento del titolo ATP. </li><li> Premix MgCl 2 e ATP in un rapporto 1: 1 appena prima di impostare la reazione di idrolisi dell&#39;ATP. </li></ol></li><li> Preparare ed etichettare provette da 1,5 ml per la raccolta di campioni a intervalli regolari durante la reazione. Preparare le provette per raccogliere campioni al tempo 0 e al tempo 15, 30,45, e 60 min. NOTA: In alternativa, raccogliere campioni solo in fase 0 e il punto finale. <ol><li> Aggiungere 245 microlitri di buffer 1x HNG ad ogni provetta in modo da diluire campioni raccolti dalle reazioni di idrolisi dell&#39;ATP 1:50. </li></ol></li><li> Preparare un bagno di ghiaccio secco ed etanolo per il congelamento rapido campioni per arrestare la reazione. In un secchio di ghiaccio gomma o contenitore altra cassetta di sicurezza (non di plastica), aggiungere alcuni pezzi di ghiaccio secco e versare attentamente abbastanza 70-100% di etanolo per coprire il ghiaccio secco. </li><li> Diluire proteina purificata in un tampone HNG 1x, a seconda dei casi (tipicamente 5-10 micron), e tenere in ghiaccio. </li><li> Impostare le reazioni di idrolisi dell&#39;ATP. <ol><li> In separati 0,5 ml provette per ogni campione, aggiungere le seguenti reagenti (in ordine): H 2 O (fino ad un volume finale di 30 microlitri), 6 microlitri 5x HNG o altro buffer, 3 microlitri 100 mM MgCl 2 -ATP miscela, e 0,25-5 proteine ​​micron. </li><li> Includere un buffer di sola condizione di controllo negativo in cui è stata aggiunta alcuna proteina.</li></ol></li><li> Rimuovere 5 ml dalla reazione al tempo 0, diluire 1:50 nella provetta da 1,5 ml preparato contenente 245 microlitri di buffer HNG, e congelare immediatamente il campione in condizioni di asciutto bagno di ghiaccio / etanolo. </li><li> Incubare le reazioni a 37 ° C per consentire idrolisi dell&#39;ATP a verificarsi per 1 ora. Ad ogni intervallo (15, 30, 45 e 60 min), rimuovere 5 aliquote microlitri dalla reazione e aggiungere al provettoni etichettati contenenti tampone HNG come al punto 1.6. </li><li> Al termine della reazione di idrolisi dell&#39;ATP, spostare campioni diluiti in un congelatore C -80 ° per la conservazione. Per garantire tutti i campioni sono completamente congelati, attendere almeno 10 minuti prima di procedere. </li></ol> 2. I campioni Incubare contenenti libero Pi con Detection Reagent <ol><li> Scongelare i campioni diluiti contenenti aliquote reazione di idrolisi dell&#39;ATP in ogni momento (ottenuti da misure 1.6 e 1.7) a temperatura ambiente. </li><li> Impostare una piastra a 96 pozzetti contenenti i campioni e gli standard di fosfati. <ol><li> In un ml tu 0,5essere, diluire lo standard di fosfato (fornito con il reagente di rilevamento Pi) da 800 micron a 40 micron con l&#39;aggiunta di 5,5 ml di buffer HNG 800 micron standard per 104,5 ml. Mescolare bene e aggiungere 100 ml di questa 40 micron di serie Pi a ben A1 di una piastra a 96 pozzetti. </li><li> Aggiungere tampone HNG 50 microlitri di pozzi B1-H1 per 1: 1 diluizioni seriali dello standard Pi. </li><li> Rimuovere 50 ml di 40 mM Pi dal pozzetto A1 e aggiungere al tampone 50 microlitri nel pozzetto B1, mescolare, e rimuovere 50 ml da ben B1 e aggiungere al ben C1, diluizioni continui attraverso ben G1. Scartare 50 ml da ben G1 dopo la miscelazione per assicurare ogni pozzetto ha lo stesso volume. Ebbene H1 dovrebbe contenere solo buffer per creare uno standard Pi 40 a 0 pM. NOTA: Se un fattore aggiuntivo (come un inibitore) è stato aggiunto ai campioni, creare una curva standard contenente quel fattore di controllo per i cambiamenti nel rilascio fosfato o assorbanza in queste condizioni. </li><li> Aggiungere 50 ml di ogni SAMPLe in duplice copia alla piastra. Aggiungere campioni dallo stesso punto temporale in colonne verticali (campione 1 ora 0 = A2, A3; campione 2 tempo 0 = B2, B3) e differenti tempi orizzontalmente. Questo permette fino a 8 campioni e 5 punti temporali per piastra. </li></ol></li><li> Utilizzare una pipetta sterile per rimuovere abbastanza verde malachite molibdato reagente di rilevamento / Pi aggiungere 100 ml a ciascuno dei pozzetti contenenti campioni e gli standard (reagente necessario (ml) = 0,1 x numero di campioni e gli standard) e aggiungere ad un piatto per una facile pipettaggio usando una pipetta multicanale. Non versare il reagente di rilevamento direttamente nel piatto, come la contaminazione Pi è probabile che si verifichi. </li><li> Usando una pipetta multicanale, aggiungere 100 ml di reagente di rilevamento Pi in ogni pozzetto e mescolare accuratamente pipettando su e giù un consistente numero di volte senza bolle introduzione, preferibilmente nell&#39;ordine da ultimi punti temporali ai primi punti temporali. </li><li> Incubare la piastra per 25 min a temperatura ambiente, o secondo la maindicazioni nufacturer. </li></ol> 3. Risultati quantificare utilizzando un lettore di micropiastre <ol><li> Leggere l&#39;assorbanza dei campioni a 650 nm con un lettore di micropiastre assorbanza. </li><li> Effettuare una curva standard Pi. Utilizzando un software grafici, grafico i valori di assorbanza per i campioni standard Pi funzione della concentrazione al fine di trovare una equazione utilizzata per risolvere per la quantità di fosfato in ciascun campione. </li><li> Calcolare l&#39;ATPasi per ogni campione calibrando con lo standard di fosfato. Fosfato rilasciato = (OD 650 - Y intercetta) / pendio. <ol><li> Nella media del totale Pi da duplicati di ogni campione. Sottrarre lettura di assorbanza del controllo del buffer solo da questo numero. Moltiplicate questo per il fattore di diluizione (50 nel nostro esempio). </li><li> Determinare il Pi nmol rilasciato per proteine ​​micromol. Rappresentazione grafica questi valori per ogni punto temporale in un saggio cinetica dovrebbe produrre attacchi lineari di almeno R = 0,99 (figura 1); Se not, il dosaggio può essere regolato con più o meno proteina o un tempo di incubazione più lungo o più breve. </li></ol></li><li> Rappresentano i risultati come nmol Pi / mol proteina / min (figure 2, 3), o come nmol Pi / mg proteine ​​/ min se desiderato. </li></ol>

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<li>Maxson, M. E., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+vacuolar-type+H%2B-ATPase+at+a+glance+-+more+than+a+proton+pump%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">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/pubmed?term=((Phylogeny+of+genes+for+secretion+NTPases%3A+Identification+of+the+widespread+tadA+subfamily+and+development+of+a+diagnostic+key+for+gene+classification%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Crystal+Structure+of+the+Extracellular+Protein+Secretion+NTPase+EpsE+of+Vibrio+cholerae%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((Molecular+analysis+of+the+Vibrio+cholerae+type+II+secretion+ATPase+EpsE%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">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/pubmed?term=((Type+II+secretion+and+pathogenesis%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">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/pubmed?term=((Direct%2C+Real-time+measurement+of+rapid+inorganic+phosphate+release+using+a+novel+fluorescent+probe+and+its+application+to+actomyosin+subfragment+1+ATPase%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Inorganic+phosphate+assay+with+malachite+green%3A+An+improvement+and+evaluation%5BTitle%5D)+AND+%22J+Biochem+Biophys+Methods%22%5BJournal%5D)">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/pubmed?term=((A+microassay+for+ATPase%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((Characterization+and+kinetic+analysis+of+the+intracellular+domain+of+human+protein+tyrosine+phosphatase+%CE%B2+%28HPTP+%CE%B2%29+using+synthetic+phosphopeptides%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase β (HPTP β) 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/pubmed?term=((Synergistic+stimulation+of+EpsE+ATP+hydrolysis+by+EpsL+and+acidic+phospholipids%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((Stimulation+of+the+weak+ATPase+activity+of+human+Hsp90+by+a+client+protein%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((XpsE+oligomerization+triggered+by+ATP+binding%2C+not+hydrolysis%2C+leads+to+its+association+with+XpsL%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((Archaeal+flagellar+ATPase+motor+shows+ATP-dependent+hexameric+assembly+and+activity+stimulation+by+specific+lipid+binding%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">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/pubmed?term=((VirB11+ATPases+are+dynamic+hexameric+assemblies%3A+new+insights+into+bacterial+type+IV+secretion%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+the+luminescent+ADP-Glo+assay+to+a+standard+radiometric+assay+for+measurement+of+protein+kinase+activity%5BTitle%5D)+AND+%22Assay+Drug+Dev+Techn%22%5BJournal%5D)">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/pubmed?term=((Decoupling+catalytic+activity+from+biological+function+of+the+ATPase+that+powers+lipopolysaccharide+transport%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Altered+cofactor+regulation+with+disease-associated+p97%2FVCP+mutations%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((High-throughput+screening+assay+for+inhibitors+of+heat-shock+protein+90+ATPase+activity%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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.

Questo è un protocollo generale per la misurazione dell&#39;attività di ATPasi vitro di proteine ​​purificate per la caratterizzazione biochimica. Questo metodo può essere facilmente ottimizzato; per esempio, regolando la quantità di proteine, tampone e composizioni di sale, la temperatura, e variando la lunghezza del test e intervalli (compreso l&#39;aumento del numero totale di intervalli) può migliorare l&#39;attività di quantificazione. Disponibili in commercio malachite reagenti verde-based sono molto sensibili, e in grado di rilevare piccole quantità di fosfati (~ 50 pmoli in 100 ml). A causa della sensibilità di questo saggio, è fondamentale utilizzare software usa e getta di plastica, acqua ultrapura, tamponi e reagenti privi di contaminanti fosfati. Dopo proteine ​​purificante, è raccomandato l&#39;esclusione dimensioni o la cromatografia a scambio ionico per migliorare la purezza delle proteine ​​e rimuovere i contaminanti. Per proteine ​​visualizzazione debole ATPasi vitro, stimolanti possono essere aggiunti alla reazione per migliorare el&#39;attività nzymatic. Molti fattori che stimolano l&#39;attività ATPasi sono stati caratterizzati. Ad esempio, cardiolipina e altri lipidi di membrana, proteine ​​clienti di accompagnatori, come Hsp90, e altre proteine ​​coinvolte nel sostegno particolari conformazioni o ambienti in cui ATPases funzione 13-15. Il nostro laboratorio prima caratterizzata Epse purificando monomeri con l&#39;attività ATPasi debole rispetto a ATPases omologhi 6. Abbiamo poi scoperto che quando Epse stata copurified con il dominio citoplasmatico di EPSL, una proteina transmembrana e partner legante di Epse nel sistema T2S, aggiunta di fosfolipidi acidi come cardiolipina alla miscela di reazione aumentata notevolmente l&#39;attività di ATPasi EPSE 12. Questo probabilmente imita le condizioni esperienze Epse a livello della membrana citoplasmatica che possono promuovere oligomerizzazione. Molte tecniche sono state utilizzate per quantificare in vitro ATPasi attività delle proteine ​​purificate. γ- radioattivo <sup&gt; 32 P è stata spesso e con successo realizzato da quantificare idrolisi dell&#39;ATP 14,16, invece, la sicurezza è una preoccupazione e richiede l&#39;approvazione per l&#39;uso in laboratorio di radioattività. Mentre altamente sensibile, la breve emivita di γ- 32 P è anche uno svantaggio. Altri metodi di rilevamento di fosfato commerciali sono disponibili, come quelli che si basano sulla formazione di un prodotto fluorescente. Alcuni di questi metodi sono molto sensibili, ma spesso richiedono l&#39;aggiunta di altri enzimi per la reazione, con conseguente reagenti che sono meno stabili nel tempo. Inoltre, sono disponibili kit che rilevano ADP rilasciato durante idrolisi usando un reagente stabile luminescenza a base, ma questi possono essere meno ideale per ATPasi con bassi livelli di attività 17. Il test qui descritto è costituito da un solo passo di misura rilascio di fosfato, è altamente sensibile, e può tipicamente essere eseguita entro pochi hr. È anche possibile prepare un substrato verde contenente malachite per evitare l&#39;acquisto di un kit da un fornitore commerciale 6,9. Una considerazione prima di intraprendere questo saggio è che il passo tra l&#39;aggiunta del reagente di rilevamento di fosfato e prendendo le letture di assorbanza è relativamente tempo sensibile e deve essere compreso tra 20-30 minuti. Questo protocollo di base può essere utilizzato per determinare il ruolo di stimolanti (come descritto), antagonisti, subunità, domini e residui specifici ATPasi attività 15,16,18,19. Questo test può anche essere esteso per misurare l&#39;attività di fosfatasi o altri enzimi che rilasciano fosfato durante la catalisi. Inoltre, questo metodo può essere applicato a screening ad alto rendimento per gli inibitori ATPasi 20.

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

Adenosina trifosfato enzimi-idrolisi, o ATPasi, svolgono un ruolo critico in una gamma diversificata di funzioni cellulari. Queste proteine ​​dinamici in grado di generare energia per il lavoro meccanico, come il traffico di proteine ​​e il degrado, trasporto dei soluti, e movimenti cellulari. Il protocollo qui descritto è un metodo semplice per misurare l&#39;attività in vitro di ATPasi purificata per la caratterizzazione funzionale. Proteine ​​idrolizzano ATP in una reazione che provoca il rilascio inorganica fosfato e la quantità di fosfato liberato viene poi quantizzati utilizzando un saggio colorimetrico. Questo protocollo altamente adattabile può essere regolata per misurare l&#39;attività ATPasi in saggi cinetici o endpoint. Un protocollo rappresentativo è qui fornito in base all&#39;attività e requisiti di EPSE, AAA + ATPasi coinvolta nella secrezione di tipo II nel batterio Vibrio cholerae. La quantità di proteina purificata necessaria per misurare l&#39;attività, la lunghezza del test e la sincronizzazione e il numero di saintervalli mpling, buffer e la composizione del sale, temperatura, co-fattori, stimolanti (se presente), ecc possono variare da quelli qui descritti, e quindi un po &#39;di ottimizzazione può essere necessario. Questo protocollo fornisce un quadro di base per ATPasi caratterizzano e può essere eseguita rapidamente e facilmente regolato come necessario.

L&#39;attività in vitro del T2S ATPasi Epse può essere stimolato da copurification del Epse con il dominio citoplasmatico di EPSL (Epse-cytoEpsL) e l&#39;aggiunta di acido cardiolipina fosfolipide 12. È anche possibile determinare il ruolo di particolari residui Epse a idrolisi confrontando l&#39;attività di tipo selvatico (WT) per forme varianti della proteina utilizzando questo saggio. Qui, l&#39;effetto di sostituire due residui di lisina nel dominio zinc-binding Epse viene misurata confrontando ATPasi attività di purificato WT EPSE-cytoEpsL alla variante EPSE K417AK419A-cytoEpsL. Nella figura 1, il rilascio di fosfato procede linearmente nel corso di un 1 ora dosaggio cinetica, un requisito per accurata quantificazione dell&#39;attività ATPasi cinetica. Figura 2 mostra i dati dagli stessi campioni di proteine ​​purificate da figura 1 che sono stati analizzati tre volte in duplicato e quantificato in termini diIl tasso medio di rilascio di fosfato. Questi dati dimostrano che K417 e K419 sembrano essere importanti per l&#39;attività Epse ATPasi. Tuttavia, il confronto di non stimolate (non cardiolipina) tassi di attività ATPasi (Figura 3) mostra che K417 e K419 non contribuiscono all&#39;attività basale di Epse ma piuttosto la capacità della proteina di essere stimolato da cardiolipina. Le lisine carica positiva nel dominio zinc-binding Epse possono interagire direttamente con i fosfolipidi a carica negativa, contribuendo così alla stimolazione fosfolipide-mediata di attività EPSE ATPasi. <img alt="Figura 1" src="/files/ftp_upload/54305/54305fig1.jpg" /> Figura 1: fosfato viene rilasciato linearmente in una cinetica ATPasi Assay Un&#39;ora cinetica cardiolipina-stimolata saggio ATPasi confrontando rilascio di fosfato di 0,5 micron WT-Epse cytoEpsL per Epse K417AK419A-cytoEpsL con albumina sierica bovina (BSA) dosati come.un controllo negativo. Dati [quantità di fosfato liberato (asse y) in funzione del tempo (asse x)] sono stati tracciati e sottoposti ad analisi di regressione lineare. La pendenza rappresenta il tasso di idrolisi dell&#39;ATP. Viene visualizzato un grafico rappresentativo con le equazioni di regressione lineare per le tre proteine. <a href="https://www.jove.com/files/ftp_upload/54305/54305fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="figura 2" src="/files/ftp_upload/54305/54305fig2.jpg" /> Figura 2:.. Doppio Lisina mutazioni nel Epse zinco-dominio di legame Ridurre stimolata ATPasi attività Risultati del cinetica saggio ATPasi cardiolipina stimolata 1 ora con le stesse proteine ​​e le condizioni in figura 1 saggi sono stati eseguiti tre volte separate in duplice copia tecnica e la tasso di idrolisi dell&#39;ATP è stato calcolato come fosfato nmol generato al minuto per micron pr<html> otein utilizzando le equazioni di regressione lineare. Vengono visualizzati i risultati medi con errore standard. <a href="https://www.jove.com/files/ftp_upload/54305/54305fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54305/54305fig3.jpg" /> Figura 3: Doppio Lisina mutazioni nel Epse zinco-dominio di legame non interferiscono con stimolate ATPasi attività Overnight (16 ore) endpoint non stimolate (senza cardiolipina) saggio ATPasi a confronto l&#39;attività di 5 micron WT Epse-cytoEpsL per Epse K417AK419A-cytoEpsL con BSA. come controllo negativo. I dosaggi sono stati eseguiti tre volte separate in duplice copia tecnica e risultati medi con errore standard è mostrato. Il livello basale di non stimolato attività Epse ATPasi analizzati nel corso di un periodo di 16 ore è ~ 1.000 volte inferiore a 1 ora cinetica attività cardiolipina-stimolata.e.com/files/ftp_upload/54305/54305fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura.

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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.

misurazione<em&gt; In Vitro</em&gt; Attività ATPasi per enzimatica Caratterizzazione

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

Universidad San Sebastian

Research

JoVE Journal

Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

18.0K Views.  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.

Video: 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.

Analizzare l&#39;attività della proteina con l&#39;ATP radioattivato

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 ...

Ricerca

Biochimica

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.

Caratterizzazione a livello molecolare utilizzando robusti approcci biochimici di una nuova proteina Kinase

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.

Adattamento semi-alto/throughput del saggio ATPase accoppiato NADH per lo screening degli inibitori delle piccole molecole

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.

<meta charset="utf8"></head><body>Saggio delle idrolasi nucleosidiche trifosfato in Toxoplasm gondii e Neospora caninum per lo screening ad alto rendimento utilizzando un braccio robotico

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.

Saggio delle idrolasi nucleosidiche trifosfato in Toxoplasm gondii e Neospora caninum per lo screening ad alto rendimento utilizzando un braccio robotico

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.

Rilevazione diretta della acetato di formazione Attività di acetato enzima chinasi

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

Biologia

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.

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

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

Immunologia e infezioni

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.

La valutazione delle funzioni mitocondriali e la vitalità cellulare in cellule renali proteina chinasi C overexpressing isoenzimi

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.

Saggi biochimici per le attività di analisi ATP-dipendente rimodellamento della cromatina Enzimi

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.

misurazione

Riepilogo

Esplora Altri Video

Analizzare l'attività della proteina con l'ATP radioattivato

Caratterizzazione a livello molecolare utilizzando robusti approcci biochimici di una nuova proteina Kinase

Adattamento semi-alto/throughput del saggio ATPase accoppiato NADH per lo screening degli inibitori delle piccole molecole

Saggio delle idrolasi nucleosidiche trifosfato in Toxoplasm gondii e Neospora caninum per lo screening ad alto rendimento utilizzando un braccio robotico

Saggio delle idrolasi nucleosidiche trifosfato in Toxoplasm gondii e Neospora caninum per lo screening ad alto rendimento utilizzando un braccio robotico

Rilevazione diretta della acetato di formazione Attività di acetato enzima chinasi

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

La valutazione delle funzioni mitocondriali e la vitalità cellulare in cellule renali proteina chinasi C overexpressing isoenzimi

Saggi biochimici per le attività di analisi ATP-dipendente rimodellamento della cromatina Enzimi

In vitro misurare phosphatidylethanolamine metiltransferasi attività