misurazione<em> In Vitro</em> Attività ATPasi per enzimatica Caratterizzazione
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
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'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'attività ATPasi in saggi cinetici o endpoint. Un protocollo rappresentativo è qui fornito in base all'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'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 '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.
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
Questo è un protocollo generale per la misurazione dell'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'aumento del numero totale di intervalli) può migliorare l'attività di quantificazione. Disponibili in commercio malachite reagenti verde-ba…
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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