Mätning<em> In Vitro</em> ATPas aktivitet för enzymatisk karakterisering
Summary
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.
Abstract
Adenosintrifosfat-hydrolyserande enzymer, eller ATPaser, spelar en avgörande roll i en mångfald av cellulära funktioner. Dessa dynamiska proteiner kan generera energi för mekaniskt arbete, såsom protein trafficking och nedbrytning, transport av lösta ämnen, och cellulära rörelser. Protokollet som beskrivs här är en grundläggande analys för mätning av aktiviteten av renade ATPaser in vitro för funktionell karakterisering. Proteiner hydrolyserar ATP i en reaktion som resulterar i oorganiskt fosfat release, och mängden fosfat som frigöres därefter kvantifierades med användning av en kolorimetrisk analys. Denna mycket anpassningsprotokoll kan justeras för att mäta ATPas-aktivitet i kinetiska eller slutpunktsanalyser. Ett representativt protokoll tillhandahålls här baserat på aktiviteten och krav Epse, AAA + ATPas inblandade i typ II Sekre i bakterien Vibrio cholerae. Mängden renat protein som behövs för att mäta aktivitet, längd analysen och tidpunkten och antalet sampling intervaller, buffert och saltkomposition, temperatur, co-faktorer, stimulantia (om någon), etc. kan variera från de som beskrivs här, och därmed viss optimering kan vara nödvändig. Detta protokoll ger en grundläggande ram för kännetecknande ATPaser och kan utföras snabbt och enkelt justeras vid behov.
Introduction
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.
Protocol
Representative Results
Discussion
Detta är ett allmänt protokoll för att mäta in vitro ATPas-aktiviteten hos renade proteiner för biokemisk karakterisering. Denna metod kan enkelt optimeras; till exempel, kan justera mängden protein, buffert och saltkompositioner, temperatur och varierande analys längd och intervall (däribland ökade det totala antalet intervall) förbättra aktivitet kvantifiering. Kommersiellt tillgängliga malakitgrönt baserade reagens är mycket känsliga och kan detektera små mängder av fri fosfat (~ 50 pmol i 1…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge funding from a National Institutes of Health grant RO1AI049294 (to M. S.).
Materials
| HEPES buffer | Fisher | BP310-500 | |
| Sodium chloride | Fisher | BP358-212 | |
| Magnesium chloride | Fisher | BP214-500 | |
| Adenosine triphosphate (ATP) | Fisher | BP41325 | |
| 96-well plates (clear, flat-bottom) | VWR | 82050-760 | |
| BIOMOL Green | Enzo Life Sciences | BML-AK111 | Preferred phosphate detection reagent. Caution: irritant. |
| Microplate reader | BioTek Synergy or comparable | ||
| Prism 5 | GraphPad Software | ||
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