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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+Structure+of+the+Extracellular+Protein+Secretion+NTPase+EpsE+of+Vibrio+cholerae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+the+Vibrio+cholerae+type+II+secretion+ATPase+EpsE.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+II+secretion+and+pathogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct,+Real-time+measurement+of+rapid+inorganic+phosphate+release+using+a+novel+fluorescent+probe+and+its+application+to+actomyosin+subfragment+1+ATPase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inorganic+phosphate+assay+with+malachite+green:+An+improvement+and+evaluation.">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/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. G., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+stimulation+of+EpsE+ATP+hydrolysis+by+EpsL+and+acidic+phospholipids.">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/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. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaeal+flagellar+ATPase+motor+shows+ATP-dependent+hexameric+assembly+and+activity+stimulation+by+specific+lipid+binding.">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/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. H., Workman, P., Aherne, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+assay+for+inhibitors+of+heat-shock+protein+90+ATPase+activity.">High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.</a> Anal Biochem. 327 (2), 176-183 (2004).</li></ol>

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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17.8K ビュー.  University of Michigan. 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.Pr...

ビデオ: 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...

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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 &#181;L per well. The platform should be applicable to virtually any ADP producing...

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This video demonstrates an in vitro assay to quantify neutrophil serine protease activity in sputum samples. The sample containing secreted protease is incubated with a fluorescent reporter bearing a recognition motif. Cleavage of the motif by the protease enables individual fluorescence emission by the donor and acceptor fluorophore of the reporter, indicating protease activity in the...

An In Vitro Assay for Measuring Neutrophil Serine Protease Activity Using a Fluorescent Reporter

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Measuring Enzymatic Stability by Isothermal Titration Calorimetry

The thermal stability of enzyme activity is readily measured by isothermal titration calorimetry (ITC). Most protein stability assays currently used measure protein unfolding, but do not provide information about enzymatic activity. ITC enables direct determination of the effect of enzyme modifications on the stability of enzyme...

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We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic...

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Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction...

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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...

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isolation of f1-atpase from the parasitic protist trypanosoma brucei

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic...

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

Next, resuspend heat-killed bacterial cells at a concentration of 0.5 grams wet weight in 30 milliliters of RBB solution. For purified peptidoglycan, resuspend the peptidoglycan at a concentration of 0.3 grams wet weight and 30 milliliters of RBB...

measuring calpain activity in fixed and living cells by flow cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow...

caffeine extraction, enzymatic activity and gene expression of caffeine synthase from plant cell suspensions

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this...

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

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.

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...