Name: measuring in vitro atpase activity for enzymatic characterization
Uploaded: 2016-08-23T14:00:00
Description: Watch this Scientific Journal Video about Measuring In Vitro ATPase Activity for Enzymatic Characterization at JoVE.com

The authors would like to acknowledge funding from a National Institutes of Health grant RO1AI049294 (to M. S.).

1. Perform ATP Hydrolysis Reaction with Purified Protein
<ol>
	<li>Prepare Stocks of All the Necessary Reagents for Incubation with Purified Protein.
	<ol>
		<li>Prepare 5x HEPES/NaCl/glycerol (HNG) buffer containing 100 mM HEPES pH 8.5, 65 mM NaCl, and 5% glycerol (or other assay buffer as appropriate).</li>
		<li>Prepare 100 mM MgCl2 (or other metal, if ATPase is metal-dependent) in water.</li>
		<li>Prepare fresh 100 mM ATP in 200 mM Tris Base (do not adjust pH further) using high purity ATP. Aliquot and ...

<ol>
	<li>Hanson, P. I., Whiteheart, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAA++proteins:+have+engine,+will+work.">AAA+ proteins: have engine, will work.</a> Nat Rev Mol Cell Biol. 6 (7), 519-529 (2005).</li><li>Baker, T. A., Sauer, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClpXP,+an+ATP-powered+unfolding+and+protein-degradation+machine.">ClpXP, an ATP-powered unfolding and protein-degradation machine.</a> Biochimica et Biophysica Acta. 1823 (1), 15-28 (2012).</li><li>Maxson, M. E., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vacuolar-type+H+-ATPase+at+a+glance+-+more+than+a+proton+pump.">The vacuolar-type H+-ATPase at a glance - more than a proton pump.</a> J Cell Sci. 127 (23), 4987-4993 (2014).</li><li>Planet, P. J., Kachlany, S. C., DeSalle, R., Figurski, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+of+genes+for+secretion+NTPases:+Identification+of+the+widespread+tadA+subfamily+and+development+of+a+diagnostic+key+for+gene+classification.">Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification.</a> Proc Natl Acad Sci U S A. 98 (5), 2503-2508 (2001).</li><li>Robien, M. A., Krumm, B. E., Sandkvist, M., Hol, W. G. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microassay+for+ATPase.">A microassay for ATPase.</a> Anal Biochem. 169 (2), 312-318 (1988).</li><li>Harder, K. W., Owen, P., Wong, L. K. H., Aebersold, R., Clark-Lewis, I., Jirik, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+kinetic+analysis+of+the+intracellular+domain+of+human+protein+tyrosine+phosphatase+&#946;+(HPTP+&#946;)+using+synthetic+phosphopeptides.">Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase &#946; (HPTP &#946;) using synthetic phosphopeptides.</a> Biochem J. 298 (2), 395-401 (1994).</li><li>Camberg, J. L., Johnson, T. L., Patrick, M., Abendroth, J., Hol, W. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+the+weak+ATPase+activity+of+human+Hsp90+by+a+client+protein.">Stimulation of the weak ATPase activity of human Hsp90 by a client protein.</a> J Mol Biol. 315 (4), 787-798 (2002).</li><li>Shiue, S., Kao, K., Leu, W., Chen, L., Chan, N., Hu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XpsE+oligomerization+triggered+by+ATP+binding,+not+hydrolysis,+leads+to+its+association+with+XpsL.">XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL.</a> EMBO J. 25 (7), 1426-1435 (2006).</li><li>Ghosh, A., Hartung, S., van der Does, C., Tainer, J. A., Albers, S. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VirB11+ATPases+are+dynamic+hexameric+assemblies:+new+insights+into+bacterial+type+IV+secretion.">VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion.</a> EMBO J. 22 (9), 1969-1980 (2003).</li><li>Sanghera, J., Li, R., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+luminescent+ADP-Glo+assay+to+a+standard+radiometric+assay+for+measurement+of+protein+kinase+activity.">Comparison of the luminescent ADP-Glo assay to a standard radiometric assay for measurement of protein kinase activity.</a> Assay Drug Dev Techn. 7 (6), 615-622 (2009).</li><li>Sherman, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoupling+catalytic+activity+from+biological+function+of+the+ATPase+that+powers+lipopolysaccharide+transport.">Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport.</a> Proc Natl Acad Sci U S A. 111 (13), 4982-4987 (2014).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cofactor+regulation+with+disease-associated+p97/VCP+mutations.">Altered cofactor regulation with disease-associated p97/VCP mutations.</a> Proc Natl Acad Sci U S A. 112 (14), E1705-E1714 (2015).</li><li>Rowlands, M. G., Newbatt, Y. M., Prodromou, C., Pearl, L. 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This is a general protocol for measuring in vitro ATPase activity of purified proteins for biochemical characterization. This method is easily optimized; for example, adjusting the amount of protein, buffer and salt compositions, temperature, and varying the assay length and intervals (including increasing the total number of intervals) can improve activity quantitation. Commercially available malachite green-based reagents are highly sensitive, and can detect small amounts of free phosphate (~50 pmol in 100 &#1...

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.

<table border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">HEPES buffer</td>
			<td style="width:128px;">Fisher</td>
			<td style="width:108px;">BP310-500</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine triphosphate (ATP)</td>
			<td>Fisher</td>
			<td>BP41325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well plates (clear, flat-bottom)</td>
			<td>VWR</td>
			<td>82050-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOMOL Green</td>
			<td>Enzo Life Sciences</td>
			<td>BML-AK111</td>
			<td>Preferred phosphate detection reagent. Caution: irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td></td>
			<td></td>
			<td>BioTek Synergy or comparable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism 5</td>
			<td colspan="2">GraphPad Software</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring in vitro atpase activity for enzymatic characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

The in vitro activity of the T2S ATPase EpsE can be stimulated by copurification of EpsE with the cytoplasmic domain of EpsL (EpsE-cytoEpsL) and addition of the acidic phospholipid cardiolipin12. It is also possible to determine the role of particular EpsE residues in ATP hydrolysis by comparing activity of wild type (WT) to variant forms of the protein using this assay. Here, the effect of substituting two lysine residues in the EpsE zinc-binding domain is measured by...

Watch this Scientific Journal Video about Measuring In Vitro ATPase Activity for Enzymatic Characterization at JoVE.com

Measuring In Vitro ATPase Activity for Enzymatic Characterization

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.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

The overall goal of this procedure is to quantitate the in vitro ATPase activity of purified proteins for functional characterization. This method can be used to characterize new putative ATPases, evaluate the effective potential activators or inhibitors and to determine the contribution of particular domains or residues to ATPase activity. The main advantage of this technique is that it is a simple, sensitive method for measuring in vitro ATPase activity and can be easily optimized.Prepare stocks of all the necessary reagents for incubation with purified protein as instructed in the text protocol. Premix 100 millimolar magnesium chloride and 100 millimolar ATP at a one to one ratio just before setting up the ATP hydrolysis reaction. Prepare and label 1.5 milliliter tubes for collecting samples at regular intervals throughout the reaction.Prepare tubes to collect samples at times zero as well as at times of 15, 30, 45 and 60 minutes. Add 245 microliters of 1x HNG buffer to each tube to dilute the samples collected from the ATP hydrolysis reactions at a dilution of one to 50. Next, prepare a bath of dry ice and ethanol for quickly freezing samples to stop the reaction.In a rubber ice bucket or other safe container, add several piece of dry ice and carefully pour enough 70%to 100%ethanol to cover the dry ice. Dilute the purified protein in 1X HNG buffer and keep it on ice. Set up the ATP hydrolysis reactions in the prepared 0.5 milliliter tubes by adding a pre-calculated volume of water to reach a final reaction volume of 30 microliters.Followed by six microliters of 5X HNG or other buffer, three microliters of 100 millimolar magnesium chloride ATP mixture and 0.25 to five micromolar protein. Incubate a buffer only negative control condition in which no protein is added. Next, remove five microliters from the reaction at times zero.Then dilute the aliquot one to 50 in the prepared 1.5 milliliter tube containing 245 microliters of HNG buffer. Immediately freeze the sample in the dry ice ethanol bath. Incubate reactions at 37 degrees Celsius to allow ATP hydrolysis to occur for one hour.At each interval remove five microliter aliquots from the reaction and add to labeled sample tubes containing HNG buffer as before. At the end of the ATP hydrolysis reaction, move diluted samples to a 80 degree Celsius freezer for storage. To ensure all samples are completely frozen, wait at least 10 minutes before proceeding.Thaw the diluted samples containing ATP hydrolysis reaction aliquots from each time point at room temperature. Next, prepare a 96 well plate containing samples and phosphate standards. In a 0.5 milliliter tube dilute the phosphate standard from 800 micromolar to 40 micromolar by adding 5.5 microliters of the standard to 104.5 microliters of HNG buffer.Mix well and add 100 microliters of this 40 micromolar phosphate standard to well A1 of the 96 well plate. Add 50 microliters of HNG buffer to wells B1 through H1 for one to one serial dilutions of the phosphate standard. Remove 50 microliters of 40 micromolar phosphate from well A1, add it to the 50 microliters of assay buffer in well B1 and mix.Then remove 50 microliters from well B1 and add it to well C1, continuing dilutions through well G1.Discard 50 microliters from well G1 after mixing to ensure each well has the same volume. Well H1 should contain only buffer to create a phosphate standard from 40 to zero micromolar. Next, add 50 microliters of each sample in duplicate to the plate.Add samples from the same time point in columns vertically and from different time points horizontally. This allows for up to eight samples and five time points per plate. Use a sterile pipette to remove enough malachite green meliptate phosphate detection reagent to add 100 microliters to each of the wells containing samples and standards.Add the detection reagent to a dish for easy pipetting using a multi-channel pipette. Do not pour the detection reagent directly into the dish as phosphate contamination is likely to occur. Using a multi-channel pipette, add 100 microliters of the phosphate detection reagent to each well and mix carefully by pipetting up and down a consistent number of times without introducing bubbles, working in order from the last time point to the first time point.Leave the plate at room temperature for 25 minutes or according to the manufacturer's directions. To quantitate the results, read the absorbance of the samples at 650 nanometers using an absorbance micro plate reader. Representative results of kinetic and endpoint ATPases are shown, in which the activity of wild type and mutant forms of the type two secretion ATPase/EPSE is determined in the presence and absence of a stimulant cardiolipin.Shown here are the results of a kinetic cardiolipin-stimulated ATPase, demonstrating the linear phosphate release over time for wild type EPSE and a double lysine mutant, with bovine serum albumin serving as a negative control. These data can be represented as the amount of phosphate released per minute divided by the protein concentration in order to quantitate and compare the ATPase activity of each protein. The data indicate that two lysine residues, K417 and K419, contribute to the cardiolipin-stimulated ATPase activity of EPSE.Of comparison of the ATPase activity of the same proteins without cardiolipin-stimulation shows that these two lysine residues do not contribute to the low basal activity of EPSE, but rather to the ability of the protein to be stimulated by cardiolipin. After watching this video you should have a good understanding of how to quickly and accurately quantitate the ATPase activity of purified proteins. While attempting this procedure it is important to consider the sensitivity of the phosphate detection reagent.To avoid phosphate contamination, we recommend using disposable plastic-ware and ultra-pure water and performing size exclusion or ion exchange chromatography following protein purification.

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