måle<em> In Vitro</em> ATPase aktivitet for 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
Adenosin trifosfat-hydrolyserende enzymer, eller ATPaser, spille en avgjørende rolle i et mangfoldig utvalg av cellulære funksjoner. Disse dynamiske proteiner kan generere energi til mekanisk arbeid, slik som protein menneskehandel og degradering, oppløst stoff transport, og cellulære bevegelser. Protokollen er beskrevet her er en grunnleggende undersøkelse for å måle in vitro-aktiviteten av rensede ATPaser for funksjonell karakterisering. Proteiner hydrolyserer ATP i en reaksjon som fører til uorganisk fosfat frigjøring, og mengden av fosfat frigjort blir deretter kvantifisert ved hjelp av en kolorimetrisk analyse. Denne svært tilpasningsdyktig protokollen kan tilpasses for å måle ATPase-aktivitet i kinetiske eller endepunkt analyser. En representant protokollen er gitt her er basert på aktiviteten og krav EPSE, AAA + ATPase involvert i Type II Utskillelse i bakterien Vibrio cholerae. Mengden av renset protein som er nødvendig for å måle aktivitet, lengden av analysen og timingen og antallet sampling intervaller, buffer og salt sammensetning, temperatur, co-faktorer, sentralstimulerende midler (hvis noen), etc. kan variere fra det som er beskrevet her, og dermed noen optimalisering kan være nødvendig. Denne protokollen gir en grunnleggende rammeverk for å karakterisere ATPaser og kan utføres raskt og enkelt kan justeres etter 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
Dette er en generell protokoll for å måle in vitro-ATPase-aktiviteten av rensede proteiner for biokjemisk karakterisering. Denne metoden er lett optimaliseres; for eksempel kan justere mengden av protein, en buffer og saltsammensetninger, temperatur og å variere analysen lengde og intervaller (inkludert øke det totale antall intervaller) bedre aktivitet kvantifisering. Kommersielt tilgjengelige malakittgrønt-baserte reagenser er meget følsomme, og kan detektere små mengder av fritt fosfat (~ 50 pmol i 10…
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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