We thank Dr. Dr. Michael Cashel (National Institutes of Health, Bethesda, MA, USA) for sending us bacterial strains, plasmids, and necessary protocols. We thank Dr. John Glover (Department of Biochemistry, University of Toronto) for use of the SpecraMax plate reader. UK is the recipient of a National Sciences and Engineering Research Council of Canada (NSERC) Postgraduate Scholarship, a Canadian Institutes of Health Research Strategic Training Program in the Structural Biology of Membrane Proteins Linked to Disease, and a University of Toronto Open Fellowship. This work was supported by a grant from the Canadian Institutes of Health Research (MOP-67210) to WAH.

1) Reagents and Equipment
<ol>
 <li>First, prepare the following three solutions: 1 mL of Solution A which is made up of 8 mM L-lysine, 100 mM sodium 2-(N-Morpholino)ethanesulphonic acid (MES) pH 6.5, 0.2 mM nucleotide, where the nucleotide is either: guanosine diphosphate (GDP), guanosine triphosphate (GTP), guanosine 5'-diphosphate,3'-diphosphate (ppGpp), or guanosine 5'-triphosphate,3'-diphosphate (pppGpp), 0.1 mM pyridoxal 5'-phosphate (PLP), and 1 mM &beta;-mercaptoethanol/ (&beta;-ME). 1 mL of Solution B which consists of 100 mM sodium MES pH 6.5, 0.1 mM PLP, 1 mM &beta;-ME, and 50 nM LdcI. LdcI was purified as described in Snider et al.6 and Kanjee et al. 14. 
 1 mL of Solution C which is identical to Solution B but does not contain LdcI.</li>
 <li>Prepare 100 mL of the stop solution consisting of 1 M sodium carbonate (10.6 g/100 mL) and aliquot 50 &mu;L into each well of a 96-well polystyrene plate using a multi-channel pipette. Add 30 &mu;L of water to the plate and then cover. Note that the sum of the volume of water added and the volume of sample extracted during the enzyme reaction must equal 50 &mu;L. In this case, 20 &mu;L of enzyme reaction sample will be removed.</li>
 <li>Prepare 5 mL of TNBS solution at 10 mM by diluting 294 &mu;L of 5% (w/v) TNBS stock with 4706 &mu;L water, wrap in aluminium foil and keep on ice.</li>
 <li>Equilibrate the Eppendorf ThermoStat Plus at 37&deg;C. The ThermoStat Plus has 24 wells in 6 columns. See Figure 2 for schematic. Label 1.5 mL Eppendorf tubes indicating the identity of the nucleotide that will be tested (one per column): GTP (column A), GDP (column B), ppGpp (column C), pppGpp (column D), no nucleotide (column E), and protein samples (column F).</li>
 <li>Rack several boxes of 200 &mu;L tips such that each alternate row is omitted giving a total of four rows of tips. This is necessary for the use of the multi-channel pipette with the ThermoStat Plus heatblock during the enzyme assay.</li>
 <li>Equilibrate Digital Heatblock (VWR) at 42&deg;C.</li>
 <li>Place a 96-well 2.0 mL polypropylene plate on ice to cool.</li>
</ol>
2) LdcI Assay
<ol>
 <li>Aliquot 50 &mu;L of Solution A with the appropriate nucleotide to 1.5 mL Eppendorf tubes in each of the five columns of the Eppendorf ThermoStat.</li>
 <li>Add 330 &mu;L of Solution B to three tubes in column F and add 330 &mu;L of Solution C (the no-protein control) to the final tube in column F.</li>
 <li>Equilibrate the solutions at 37&deg;C for five minutes. Tip: after 5 minutes, cut off the caps of the tubes in the center of the heating-block to prevent them from interfering with the multichannel pipette to be used next.</li>
 <li>Using a 5-50 &mu;L VWR multi-channel pipette, transfer 50 &mu;L from the tubes in column F into each of the tubes in columns A-E. Start the timer as soon as the first tube is mixed.</li>
 <li>At 2, 4, and 6 minutes remove 20 &mu;L of sample using the multi-channel pipette and add to the stop solution.</li>
 <li>Note: samples in stop solution can be covered in plastic wrap and frozen at -20&deg;C for subsequent processing. The plates should be thawed at room temperature before reaction with TNBS.</li>
</ol>
3) TNBS Reaction and Color Development
<ol>
 <li>Add 50 &mu;L of 10 mM TNBS solution to the stop solution using the multi-channel pipette and then incubate at 42&deg;C for 6 minutes. The solution will turn a dark yellow/orange color as the TNBS reacts with the lysine and the cadaverine.</li>
 <li>After 6 minutes, cool the plate on ice to slow down the reaction.</li>
 <li>Remove 100 &mu;L of sample using a VWR 20-200 &mu;L multi-channel pipette and place in the 2 mL deep well plate that was cooled on ice. Transfer ice-bucket to the fumehood.</li>
 <li>Add 500 &mu;L toluene to each well using a HandyStep (Brand) repeat pipettor and 12.5 mL pipette tip (Plastibrand).</li>
 <li>Wipe away any excess toluene using a kimwipe and cover the plate with strips of packaging tape. Make sure to press down firmly to obtain a good seal. Cover the 96-well plate with a flat lid and then shake vigorously for 1 minute and 30 seconds.</li>
 <li>Leave the solutions to settle for 5 minutes. The TNP-cadaverine is now in the upper toluene phase, while the TNP-lysine remains water soluble (see Figure 1).</li>
 <li>Remove 200 &mu;L of toluene using the 20-200 &mu;L multi-channel pipette into a 96-well plate for reading. Note: make sure to check that each sample being removed is clear and does not have any of the bottom aqueous phase. Note: use barrier tips to prevent damage to multi-channel pipette by the toluene.</li>
 <li>Read absorbance at 340 nm in a SpectraMax 340 plate reader.</li>
 <li>To clean the quartz plate, rinse out the toluene with water and place the quartz plate in a large glass tray in the fume hood. Pour over 100 mL of a 7:3 mixture of 70% (v/v) nitric acid:95% (v/v) ethanol and cover the quartz plate with a second glass tray. Warning: the reaction is highly exothermic and sometimes explosive wear appropriate protection and ensure that the fume hood sash is set to the lower 6" level. After cooling, wash out nitric acid with water and then 95% ethanol.</li>
</ol>
4) Representative Results
1)Cadaverine Standard Curve (Figure 3) 
 The assay was performed as described but without any L-lysine or LdcI and instead various concentrations of cadaverine were used to empirically determine the linear range of the assay. The assay was linear to an OD340 of 0.25, corresponding to ~ 22 nmoles of cadaverine (Figure 3).
2)Activity of LdcI (Figure 4) 
 The activity of LdcI alone was determined to be 153.5 (&plusmn; 18.1) nmoles cadaverine min-1 &mu;g LdcI-1 at pH 6.5. The activity of LdcI is unaffected in the presence of 100 &mu;M GTP or GDP but is strongly inhibited (>10-fold) in the presence of pppGpp and ppGpp (Figure 4).
<img src="/files/ftp_upload/2094/2094fig1.jpg" alt="Figure 1" /> 
 Figure 1. Schematic diagram of the LdcI reaction. The decarboxylation reaction of L-lysine to generate CO2 and cadaverine is shown as well as the subsequent reaction with TNBS at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N'-bistrinitrophenyllysine (TNP-lysine). Based on Phan et al.11
<img src="/files/ftp_upload/2094/2094fig2.jpg" alt="Figure 2" /> 
 Figure 2. Setup of Eppendorf Thermostat. The layout of the samples in the 24-well Eppendorf ThermoStat is shown along with description of the steps of the assay (indicated in bold). Arrows indicate the transfer of reaction solutions according to the protocol. Removal of the reaction solution to the stop solution in the 96-well plate is also indicated.
<img src="/files/ftp_upload/2094/2094fig3.jpg" alt="Figure 3" /> 
 Figure 3. Cadaverine Standard Curve. A plot of absorbance at 340 nm versus nmoles of cadaverine is shown. Error bars represent the standard deviation of at least three independent measurements. The line of best fit is also shown and has an R2 of 0.996.
<img src="/files/ftp_upload/2094/2094fig4.jpg" alt="Figure 4" /> 
 Figure 4. LdcI assay results. A plot of the rate of LdcI activity (in nmoles cadaverine produced min-1 &mu;g-1 LdcI) is shown in the presence of 100 &mu;M GTP, GDP, pppGpp, ppGpp and in the absence of nucleotide. The stringent response nucleotides (p)ppGpp are capable of significantly inhibiting LdcI activity. The error bars represent the standard deviation of at least six independent measurements.

<ol>
	<li>Gale, E. F., Epps, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+pH+of+the+medium+during+growth+on+the+enzymic+activities+of+bacteria+(Escherichia+coli+and+Micrococcus+lysodeikticus)+and+the+biological+significance+of+the+changes+produced.">The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced.</a> Biochem J. 36, 600-618 (1942).</li><li>Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+acid+resistance:+tales+of+an+amateur+acidophile.">Escherichia coli acid resistance: tales of an amateur acidophile.</a> Nat Rev Microbiol. 2, 898-907 (2004).</li><li>Castanie-Cornet, M. P., Penfound, T. A., Smith, D., Elliott, J. F., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+acid+resistance+in+Escherichia+coli.">Control of acid resistance in Escherichia coli.</a> J Bacteriol. 181, 3525-3535 (1999).</li><li>Iyer, R., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine-agmatine+antiporter+in+extreme+acid+resistance+in+Escherichia+coli.">Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli.</a> J Bacteriol. 185, 6556-6561 (2003).</li><li>Sabo, D. L., Boeker, E. A., Byers, B., Waron, H., Fischer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+physical+properties+of+inducible+Escherichia+coli+lysine+decarboxylase.">Purification and physical properties of inducible Escherichia coli lysine decarboxylase.</a> Biochemistry. 13, 662-670 (1974).</li><li>Snider, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+a+distinctive+complex+between+the+inducible+bacterial+lysine+decarboxylase+and+a+novel+AAA++ATPase.">Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase.</a> J Biol Chem. 281, 1532-1546 (2006).</li><li>Kashiwagi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+the+genes+for+putrescine+transport+protein+and+ornithine+decarboxylase+at+16+min+on+Escherichia+coli+chromosome.">Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome.</a> J Biol Chem. 266, 20922-20927 (1991).</li><li>Schneider, G., Kack, H., Lindqvist, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+manifold+of+vitamin+B6+dependent+enzymes.">The manifold of vitamin B6 dependent enzymes.</a> Structure. 8, 1-6 (2000).</li><li>Cashel, M., Gentry, D. 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K., Bearson, B., Bang, S. H., Bang, I. S., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+pH+crisis,+lysine+decarboxylase+and+the+acid+tolerance+response+of+Salmonella+typhimurium.">Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium.</a> Mol Microbiol. 20, 605-611 (1996).</li><li>Merrell, D. S., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cadA+gene+of+Vibrio+cholerae+is+induced+during+infection+and+plays+a+role+in+acid+tolerance.">The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance.</a> Mol Microbiol. 34, 836-849 (1999).</li><li>Kanjee, U., Gutsche, I., Alexopoulos, E., Zhao, B., Bakkouri, M. E. l., Thibault, G., Liu, K., Ramachandran, S., Snider, J., Pai, E. F., Houry, W. 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No conflicts of interest declared.

In the lysine decarboxylase assay, TNBS is reacted with the primary amines of L-lysine and cadaverine to form TNP-lysine and TNP-cadaverine adducts (Figure 1). Due to the presence of the carboxylic acid group on TNP-lysine, this adduct remains soluble in water while the TNP-cadaverine, lacking the carboxylic acid group, is capable of partitioning into toluene11. This type of assay can be utilized more broadly on other types of amino acids where the loss of a carboxylic acid group occurs during the reaction. Th...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2,4,6-trinitrobenzensulfonic acid </td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2297</td>
<td>&nbsp;</td>
<tr>
<td>0.1 mM pyridoxal 5'-phosphate (PLP)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P9255</td>
<td>&nbsp;</td>
<tr>
<td>Cadaverine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>D22606</td>
<td>&nbsp;</td>
<tr>
<td>96 well polystyrene plates</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>96-well quartz plate</td>
<td>&nbsp;</td>
<td>Hellma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>VWR Digital Heatblock</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ThermoStat Plus</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2.0 mL 96-well polypropylene plates</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>P-DW-20-C</td>
<td>&nbsp;</td>
<tr>
<td>Handy Step Repeat Pipettor</td>
<td>&nbsp;</td>
<td>Brand</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>12.5 mL Repeat Pipettor Tips</td>
<td>&nbsp;</td>
<td>Brand (Plastibrand)</td>
<td>702378</td>
<td>&nbsp;</td>
<tr>
<td>SpectraMax 340PC Plate Reader</td>
<td>&nbsp;</td>
<td>SpectraMax</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an assay for measuring the activity of escherichia coli inducible lysine decarboxyase

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.

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An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

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.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

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 ...

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14.1K Views.  University of Toronto. 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.