Måling<em> In vitro</em> ATPase-aktivitet til 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
Adenosintriphosphat-hydrolyserende enzymer eller ATPaser, spiller en kritisk rolle i en bred vifte af cellulære funktioner. Disse dynamiske proteiner kan generere energi til mekanisk arbejde, såsom protein handel og nedbrydning, stoftransport og cellulære bevægelser. Protokollen beskrevet her er en grundlæggende assay til måling af in vitro-aktiviteten af oprensede ATPaser til funktionel karakterisering. Proteiner hydrolyserer ATP i en reaktion, der resulterer i uorganisk phosphat frigivelse, og mængden af phosphat frigivet derefter kvantificeret under anvendelse af et kolorimetrisk assay. Denne meget fleksibel protokol kan justeres til at måle ATPase-aktivitet i kinetiske eller endpoint assays. En repræsentativ protokol gives her baseret på aktiviteten og krav i Epse, AAA + ATPase involveret i type II Sekretion i bakterien Vibrio cholerae. Mængden af oprenset protein er nødvendig for at måle aktivitet, længde af assayet samt tidspunktet for og antallet af sampling intervaller, puffer og salt sammensætning, temperatur, cofaktorer, stimulanser (hvis nogen), etc. kan variere fra de her beskrevne, og således nogle optimering kan være nødvendig. Denne protokol giver en grundlæggende ramme for karakteriserer ATPaser og kan udføres hurtigt og nemt justeres efter 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 generel protokol til måling in vitro ATPase aktivitet af oprensede proteiner til biokemisk karakterisering. Denne metode er let optimeres; for eksempel kan justere mængden af protein, puffer og salt sammensætninger, temperatur og varierende assayet længde og intervaller (herunder forøge det samlede antal intervaller) at forbedre aktivitet kvantificering. Kommercielt tilgængelige malakitgrønt-baserede reagenser er meget følsomme, og kan detektere små mængder af frit phosphat (~ 50 pmo…
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 | ||
References
- Hanson, P. I., Whiteheart, S. W. AAA+ proteins: have engine, will work. Nat Rev Mol Cell Biol. 6 (7), 519-529 (2005).
- Baker, T. A., Sauer, R. T. ClpXP, an ATP-powered unfolding and protein-degradation machine. Biochimica et Biophysica Acta. 1823 (1), 15-28 (2012).
- Maxson, M. E., Grinstein, S. The vacuolar-type H+-ATPase at a glance – more than a proton pump. J Cell Sci. 127 (23), 4987-4993 (2014).
- Planet, P. J., Kachlany, S. C., DeSalle, R., Figurski, D. H. Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc Natl Acad Sci U S A. 98 (5), 2503-2508 (2001).
- Robien, M. A., Krumm, B. E., Sandkvist, M., Hol, W. G. J. Crystal Structure of the Extracellular Protein Secretion NTPase EpsE of Vibrio cholerae. J Mol Biol. 333 (3), 657-674 (2003).
- Camberg, J. L., Sandkvist, M. Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE. J Bacteriol. 187 (1), 249-256 (2005).
- Sandkvist, M. Type II secretion and pathogenesis. Infect Immun. 69 (6), 3523-3535 (2001).
- Brune, M., Hunter, J. L., Corrie, J. E. T., Webb, M. R. Direct, Real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry. 33 (27), 8262-8271 (1994).
- Carter, S. G., Karl, D. W. Inorganic phosphate assay with malachite green: An improvement and evaluation. J Biochem Biophys Methods. 7 (1), 7-13 (1982).
- Henkel, R. D., VandeBerg, J. L., Walsh, R. A. A microassay for ATPase. Anal Biochem. 169 (2), 312-318 (1988).
- Harder, K. W., Owen, P., Wong, L. K. H., Aebersold, R., Clark-Lewis, I., Jirik, F. R. Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase β (HPTP β) using synthetic phosphopeptides. Biochem J. 298 (2), 395-401 (1994).
- Camberg, J. L., Johnson, T. L., Patrick, M., Abendroth, J., Hol, W. G., Sandkvist, M. Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids. EMBO J. 26 (1), 19-27 (2006).
- McLaughlin, S. H., Smith, H. W., Jackson, S. E. Stimulation of the weak ATPase activity of human Hsp90 by a client protein. J Mol Biol. 315 (4), 787-798 (2002).
- Shiue, S., Kao, K., Leu, W., Chen, L., Chan, N., Hu, N. XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. EMBO J. 25 (7), 1426-1435 (2006).
- Ghosh, A., Hartung, S., van der Does, C., Tainer, J. A., Albers, S. V. Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding. Biochem J. 437 (1), 43-52 (2011).
- Savvides, S. N., et al. VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion. EMBO J. 22 (9), 1969-1980 (2003).
- Sanghera, J., Li, R., Yan, J. Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity. Assay Drug Dev Techn. 7 (6), 615-622 (2009).
- Sherman, D. J., et al. Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport. Proc Natl Acad Sci U S A. 111 (13), 4982-4987 (2014).
- Zhang, X., et al. Altered cofactor regulation with disease-associated p97/VCP mutations. Proc Natl Acad Sci U S A. 112 (14), E1705-E1714 (2015).
- Rowlands, M. G., Newbatt, Y. M., Prodromou, C., Pearl, L. H., Workman, P., Aherne, W. High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity. Anal Biochem. 327 (2), 176-183 (2004).
