Meten<em> In Vitro</em> ATPase activiteit voor enzymatische Karakterisatie
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
Adenosine-trifosfaat hydrolyserende enzymen, of ATPasen, spelen een cruciale rol in een breed scala van cellulaire functies. Deze dynamische eiwitten kunnen energie op te wekken voor het mechanisch werk, zoals eiwitten mensenhandel en degradatie, transport van opgeloste stoffen, en cellulaire bewegingen. De hier beschreven protocol is een eenvoudige test voor het meten van de in vitro activiteit van gezuiverde ATPasen voor functionele karakterisatie. Eiwitten hydrolyseren ATP in een reactie die resulteert in anorganisch fosfaat afgifte en de hoeveelheid fosfaat vrijgemaakt wordt vervolgens gekwantificeerd onder toepassing van een colorimetrische assay. Deze zeer flexibele protocol kan worden aangepast om ATPase-activiteit te meten in kinetische of eindpunt assays. Een representatief protocol is hier voorzien gebaseerd op de activiteit en vereisten van Epse, de AAA + ATPase betrokken type II Uitscheiding in de bacterie Vibrio cholerae. De hoeveelheid gezuiverde eiwit nodig te meten, lengte van de test en het tijdstip en het aantal sampling intervallen, buffer en zoutsamenstelling, temperatuur, co-factoren, stimulerende middelen (indien aanwezig), etc. kunnen afwijken van de hier beschreven en aldus sommige optimalisering nodig zijn. Dit protocol voorziet in een basiskader voor het karakteriseren ATPasen en kan snel worden uitgevoerd en gemakkelijk als nodig aangepast.
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
Dit is een algemeen protocol voor het meten van ATPase-activiteit in vitro van gezuiverde eiwitten voor biochemische karakterisatie. Deze werkwijze is eenvoudig geoptimaliseerd; bijvoorbeeld het aanpassen van de hoeveelheid proteïne, buffer en zout samenstellingen, temperatuur, en het variëren van de lengte en assay intervallen (inclusief de bevordering totaal aantal intervallen) kan activiteit kwantificering verbeteren. In de handel verkrijgbare malachietgroen-gebaseerde reagentia zijn zeer gevoelig en kunne…
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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