Name: Measuring In Vitro ATPase Activity for Enzymatic Characterization (Video) | JoVE Journal
Uploaded: 2016-08-23T14:00:00.000+00:00
Duration: 7 min 38 s
Description: Learn to quantitate in vitro ATPase activity of purified proteins using a simple, sensitive biochemical assay. This method enables functional characterization of ATPases and evaluation of potential activators or inhibitors.

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5 minutes

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 store the ATP stock at -20 &#176;C for no longer than a few weeks, as ATP will break down over time, and refrain from freezing and thawing the ATP stock.</li>
		<li>Premix MgCl2 and ATP at a 1:1 ratio just before setting up the ATP hydrolysis reaction.</li>
	</ol>
	</li>
	<li>Prepare and label 1.5 ml tubes for collecting samples at regular intervals throughout the reaction. Prepare tubes to collect samples at time 0 and at time 15, 30, 45, and 60 min. 
	NOTE: As an alternative, collect samples only at time 0 and the endpoint.
	<ol>
		<li>Add 245 &#181;l 1x HNG buffer to each tube in order to dilute samples collected from the ATP hydrolysis reactions 1:50.</li>
	</ol>
	</li>
	<li>Prepare a bath of dry ice and ethanol for quickly freezing samples to stop the reaction. In a rubber ice bucket or other safe (non-plastic) container, add several pieces of dry ice and carefully pour enough 70-100% ethanol to cover the dry ice.</li>
	<li>Dilute purified protein in 1x HNG buffer, as appropriate (typically 5-10 &#181;M), and keep on ice.</li>
	<li>Set up the ATP hydrolysis reactions.
	<ol>
		<li>In separate 0.5 ml tubes for each sample, add the following reagents (in order): H2O (up to a final volume of 30 &#181;l), 6 &#181;l 5x HNG or other buffer, 3 &#181;l 100 mM MgCl2-ATP mixture, and 0.25-5 &#181;M protein.</li>
		<li>Include a buffer-only negative control condition in which no protein is added.</li>
	</ol>
	</li>
	<li>Remove 5 &#181;l from the reaction at time 0, dilute 1:50 in the prepared 1.5 ml tube containing 245 &#181;l HNG buffer, and immediately freeze the sample in the dry ice/ethanol bath.</li>
	<li>Incubate reactions at 37 &#176;C to allow ATP hydrolysis to occur for 1 hr. At each interval (15, 30, 45, and 60 min), remove 5 &#181;l aliquots from the reaction and add to labeled sample tubes containing HNG buffer as in step 1.6.</li>
	<li>At the end of the ATP hydrolysis reaction, move diluted samples to a -80 &#176;C freezer for storage. To ensure all samples are completely frozen, wait at least 10 min before proceeding.</li>
</ol>
2. Incubate Samples Containing Free Pi with Detection Reagent
<ol>
	<li>Thaw diluted samples containing ATP hydrolysis reaction aliquots at each time point (obtained from steps 1.6 and 1.7) at room temperature.</li>
	<li>Set up a 96-well plate containing samples and phosphate standards.
	<ol>
		<li>In a 0.5 ml tube, dilute the phosphate standard (provided with Pi detection reagent) from 800 &#181;M to 40 &#181;M by adding 5.5 &#181;l of the 800 &#181;M standard to 104.5 &#181;l HNG buffer. Mix well, and add 100 &#181;l of this 40 &#181;M Pi standard to well A1 of a 96-well plate.</li>
		<li>Add 50 &#181;l HNG buffer to wells B1-H1 for 1:1 serial dilutions of the Pi standard.</li>
		<li>Remove 50 &#181;l of 40 &#181;M Pi from well A1 and add to 50 &#181;l assay buffer in well B1, mix, and remove 50 &#181;l from well B1 and add to well C1, continuing dilutions through well G1. Discard 50 &#181;l from well G1 after mixing to ensure each well has the same volume. Well H1 should contain only buffer to create a Pi standard from 40 to 0 &#181;M. 
		NOTE: If an additional factor (such as an inhibitor) has been added to the samples, create a standard curve containing that factor to control for changes in phosphate release or absorbance under those conditions.</li>
		<li>Add 50 &#181;l of each sample in duplicate to the plate. Add samples from the same time point in columns vertically (sample 1 time 0 = A2, A3; sample 2 time 0 = B2, B3) and different time points horizontally. This allows for up to 8 samples and 5 time points per plate.</li>
	</ol>
	</li>
	<li>Use a sterile pipet to remove enough malachite green/molybdate Pi detection reagent to add 100 &#181;l to each of the wells containing samples and standards (reagent needed (ml) = 0.1 x number of samples and standards) and add to a dish for easy pipetting using a multichannel pipet. Do not pour the detection reagent directly into the dish, as Pi contamination is likely to occur.</li>
	<li>Using a multichannel pipet, add 100 &#181;l of the Pi detection reagent to each well and mix by carefully pipetting up and down a consistent number of times without introducing bubbles, preferably in order from the last time points to the first time points.</li>
	<li>Incubate the plate for 25 min at room temperature, or according to the manufacturer&#39;s directions.</li>
</ol>
3. Quantitate Results Using a Microplate Reader
<ol>
	<li>Read the absorbance of the samples at 650 nm using an absorbance microplate reader.</li>
	<li>Make a Pi standard curve. Using a graphing software, graph the absorbance values for the Pi standard samples versus concentration in order to find an equation used to solve for the amount of phosphate in each sample.</li>
	<li>Calculate the ATPase activity for each sample by calibrating with the phosphate standard. Phosphate released = (OD650 - Y intercept)/slope.
	<ol>
		<li>Average the total Pi from duplicates of each sample. Subtract the buffer-only control&#39;s absorbance reading from this number. Multiply this by the dilution factor (50 in our example).</li>
		<li>Determine the nmol Pi released per &#181;mol protein. Graphing these values for each time point in a kinetic assay should yield linear fits of at least R = 0.99 (Figure 1); If not, the assay may be adjusted with more or less protein or a longer or shorter incubation time.</li>
	</ol>
	</li>
	<li>Represent the results as nmol Pi/&#181;mol protein/min (Figures 2, 3), or as nmol Pi/&#181;g protein/min if desired.</li>
</ol>

<ol><li>Hanson, P. I., Whiteheart, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAA%2B+proteins%3A+have+engine%2C+will+work%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((ClpXP%2C+an+ATP-powered+unfolding+and+protein-degradation+machine%5BTitle%5D)+AND+%22Biochimica+et+Biophysica+Acta%22%5BJournal%5D)">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/pubmed?term=((The+vacuolar-type+H%2B-ATPase+at+a+glance+-+more+than+a+proton+pump%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">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/pubmed?term=((Phylogeny+of+genes+for+secretion+NTPases%3A+Identification+of+the+widespread+tadA+subfamily+and+development+of+a+diagnostic+key+for+gene+classification%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal+Structure+of+the+Extracellular+Protein+Secretion+NTPase+EpsE+of+Vibrio+cholerae%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Crystal Structure of the Extracellular Protein Secretion NTPase EpsE of Vibrio cholerae.</a> J Mol Biol. 333 (3), 657-674 (2003).</li>
<li>Camberg, J. L., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+analysis+of+the+Vibrio+cholerae+type+II+secretion+ATPase+EpsE%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE.</a> J Bacteriol. 187 (1), 249-256 (2005).</li>
<li>Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+II+secretion+and+pathogenesis%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Type II secretion and pathogenesis.</a> Infect Immun. 69 (6), 3523-3535 (2001).</li>
<li>Brune, M., Hunter, J. L., Corrie, J. E. T., Webb, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct%2C+Real-time+measurement+of+rapid+inorganic+phosphate+release+using+a+novel+fluorescent+probe+and+its+application+to+actomyosin+subfragment+1+ATPase%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Direct, Real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.</a> Biochemistry. 33 (27), 8262-8271 (1994).</li>
<li>Carter, S. G., Karl, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inorganic+phosphate+assay+with+malachite+green%3A+An+improvement+and+evaluation%5BTitle%5D)+AND+%22J+Biochem+Biophys+Methods%22%5BJournal%5D)">Inorganic phosphate assay with malachite green: An improvement and evaluation.</a> J Biochem Biophys Methods. 7 (1), 7-13 (1982).</li>
<li>Henkel, R. D., VandeBerg, J. L., Walsh, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microassay+for+ATPase%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">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/pubmed?term=((Characterization+and+kinetic+analysis+of+the+intracellular+domain+of+human+protein+tyrosine+phosphatase+%CE%B2+%28HPTP+%CE%B2%29+using+synthetic+phosphopeptides%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase β (HPTP β) 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. G., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synergistic+stimulation+of+EpsE+ATP+hydrolysis+by+EpsL+and+acidic+phospholipids%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids.</a> EMBO J. 26 (1), 19-27 (2006).</li>
<li>McLaughlin, S. H., Smith, H. W., Jackson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulation+of+the+weak+ATPase+activity+of+human+Hsp90+by+a+client+protein%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">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/pubmed?term=((XpsE+oligomerization+triggered+by+ATP+binding%2C+not+hydrolysis%2C+leads+to+its+association+with+XpsL%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Archaeal+flagellar+ATPase+motor+shows+ATP-dependent+hexameric+assembly+and+activity+stimulation+by+specific+lipid+binding%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Archaeal flagellar ATPase motor shows ATP-dependent hexameric assembly and activity stimulation by specific lipid binding.</a> Biochem J. 437 (1), 43-52 (2011).</li>
<li>Savvides, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VirB11+ATPases+are+dynamic+hexameric+assemblies%3A+new+insights+into+bacterial+type+IV+secretion%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+the+luminescent+ADP-Glo+assay+to+a+standard+radiometric+assay+for+measurement+of+protein+kinase+activity%5BTitle%5D)+AND+%22Assay+Drug+Dev+Techn%22%5BJournal%5D)">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/pubmed?term=((Decoupling+catalytic+activity+from+biological+function+of+the+ATPase+that+powers+lipopolysaccharide+transport%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Altered+cofactor+regulation+with+disease-associated+p97%2FVCP+mutations%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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. H., Workman, P., Aherne, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+screening+assay+for+inhibitors+of+heat-shock+protein+90+ATPase+activity%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.</a> Anal Biochem. 327 (2), 176-183 (2004).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>HEPES buffer</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>BP358-212</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td></td></tr><tr><td>Adenosine triphosphate (ATP)</td><td>Fisher</td><td>BP41325</td><td></td></tr><tr><td>96-well plates (clear, flat-bottom)</td><td>VWR</td><td>82050-760</td><td></td></tr><tr><td>BIOMOL Green</td><td>Enzo Life Sciences</td><td>BML-AK111</td><td>Preferred phosphate detection reagent. Caution: irritant.</td></tr><tr><td>Microplate reader</td><td></td><td></td><td>BioTek Synergy or comparable</td></tr><tr><td>Prism 5</td><td>GraphPad Software</td><td></td><td></td></tr></tbody></table>

measuring in vitro atpase activity for enzymatic characterization

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...

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.

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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Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([γ-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Assaying Protein Kinase Activity with Radiolabeled ATP

There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.

In Vitro Enzyme Measurement to Test Pharmacological Chaperone Responsiveness in Fabry and Pompe Disease

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 µL per well. The platform should be applicable to virtually any ADP producing enzyme.

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

Learn to quantitate in vitro ATPase activity of purified proteins using a simple, sensitive biochemical assay. This method enables functional characterization of ATPases and evaluation of potential activators or inhibitors.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

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