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 whic...

<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. R., Hernandez, V. J., Vinella, D., Curtiss, R., Neidhardt, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Escherichia coli and Salmonella : cellular and molecular biology. , 1458-1496 (1996).</li><li>Magnusson, L. U., Farewell, A., Nystrom, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp:+a+global+regulator+in+Escherichia+coli.">ppGpp: a global regulator in Escherichia coli.</a> Trends in Microbiology. 13, 236-242 (2005).</li><li>Phan, A. P., Ngo, T. T., Lenhoff, 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=Spectrophotometric+assay+for+lysine+decarboxylase.">Spectrophotometric assay for lysine decarboxylase.</a> Anal Biochem. 120, 193-197 (1982).</li><li>Park, Y. 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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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.

Watch this Scientific Journal Video about An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase at JoVE.com

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 ...

an-assay-for-measuring-activity-escherichia-coli-inducible-lysine

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14.1K visualizações.  University of Toronto. A atividade da descarboxilase induzível lisina é monitorado pela reação do substrato L-lisina e cadaverina o produto com 2,4,6 trinitrobenzensulfonic-ácido para formar adutos que têm solubilidade diferencial em tolueno.