Mesure<em> In vitro</em> Activité ATPase pour Enzymatic Caractérisation
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
enzymes triphosphate-hydrolyser adénosine, ou ATPases, jouent un rôle essentiel dans un large éventail de fonctions cellulaires. Ces protéines dynamiques peuvent générer de l'énergie pour le travail mécanique, comme le trafic de protéines et la dégradation, le transport de soluté, et les mouvements cellulaires. Le protocole décrit ici est un dosage de base pour mesurer l'activité in vitro des ATPases purifiés pour la caractérisation fonctionnelle. Les protéines hydrolysent l'ATP dans une réaction qui entraîne la libération de phosphate inorganique, et la quantité de phosphate libéré est ensuite quantifié en utilisant un dosage colorimétrique. Ce protocole hautement adaptable peut être ajustée pour mesurer l'activité d'ATPase dans les essais cinétiques ou le terminal. Un protocole représentatif est fourni ici sur la base de l'activité et les exigences de Epse, l'AAA + ATPase impliqué dans Type II Sécrétion dans la bactérie Vibrio cholerae. La quantité de protéine purifiée nécessaire pour mesurer l'activité, la durée de l'essai et le moment et le nombre de saintervalles mpling, tampon et de la composition de sel, température, co-facteurs, les stimulants ( le cas échéant), etc. peuvent varier de ceux décrits ici, et donc une certaine optimisation peuvent être nécessaires. Ce protocole fournit un cadre de base pour ATPases caractérisant et peut être effectuée rapidement et facilement ajustée si nécessaire.
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
Ceci est un protocole général pour la mesure de l'activité ATPase in vitro de protéines purifiées pour la caractérisation biochimique. Cette méthode est facilement optimisée; par exemple, en ajustant la quantité de protéines, un tampon et des compositions de sel, la température, et à faire varier la durée de l'essai et les intervalles (y compris l'augmentation du nombre total d'intervalles) peut améliorer l'activité de quantification. réactifs verts à base de malach…
Disclosures
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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