Name: an assay for measuring the activity of escherichia coli inducible lysine decarboxyase
Uploaded: 2010-12-19T17:00:00
Description: Watch this Scientific Journal Video about An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase at JoVE.com

Wij danken dr. Michael Cashel (National Institutes of Health, Bethesda, MA, USA) voor het sturen van bacteriestammen, plasmiden, en de nodige protocollen. Wij danken dr. John Glover (Vakgroep Biochemie, Universiteit van Toronto) voor het gebruik van de SpecraMax plaat lezer. Groot-Brittannië is de ontvanger van een National Sciences and Engineering Research Council of Canada (NSERC) Postdoctorale Scholarship, een Canadese Institutes of Health Research Strategic Training Program in de Structurele Biologie van membraaneiwitten Gekoppeld aan de ziekte, en een Universiteit van Toronto Open Fellowship. Dit werk werd ondersteund door een subsidie ​​van de Canadian Institutes of Health Research (MOP-67210) aan WAH.

1) Reagentia en apparatuur <ol><li> De eerste, de voorbereiding van de volgende drie oplossingen: 1 ml oplossing A die bestaat uit 8 mM L-lysine, 100 mM natrium-2 - (N-morfolino) ethanesulphonic zuur (MES) pH 6,5, 0,2 mM nucleotide, waarbij de nucleotide is ofwel: guanosine difosfaat (BBP), guanosine trifosfaat (GTP), guanosine 5&#39;-difosfaat, 3&#39;-difosfaat (ppGpp), of guanosine 5&#39;-trifosfaat, 3&#39;-difosfaat (pppGpp), 0,1 mM pyridoxal-5&#39;-fosfaat (PLP), en 1 mM β-mercapto-ethanol / (β-ME). 1 ml oplossi...

<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. A., A, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+between+the+Bacterial+Acid+Stress+and+Stringent+Responses+Revealed+by+the+Structure+of+the+Inducible+Lysine+Decarboxylase.">Linkage between the Bacterial Acid Stress and Stringent Responses Revealed by the Structure of the Inducible Lysine Decarboxylase.</a> EMBO Journal. 30, 931-944 (2011).</li></ol>

In de lysine decarboxylase-test, is TNBS reageerde met de primaire amines van L-lysine en cadaverine naar TNP-lysine en TNP-cadaverine adducten (figuur 1) te vormen. Door de aanwezigheid van het carbonzuur groep op TNP-lysine, dit adduct blijft oplosbaar in water, terwijl de TNP-cadaverine, ontbreekt het carbonzuur-groep, in staat is partitioneren in tolueen 11. Dit type test kan breder worden ingezet op andere soorten aminozuren waar het verlies van een carbonzuur groep optreedt tijdens de reactie. Dit gebeu...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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an assay for measuring the activity of escherichia coli inducible lysine decarboxyase

Escherichia coli is een enterische bacterie die in staat is te groeien over een breed bereik van pH waarden (pH 5 - 9) 1 en, ongelooflijk, is in staat om extreem zuur spanningen onder passage door de maag zoogdieren, waar de pH-waarde kan vallen overleven tot een zo laag als pH 1 tot 2 februari. In staat te stellen een breed assortiment van zure pH te overleven, E. coli beschikt over vier verschillende induceerbare aminozuur decarboxylasen dat decarboxyleren hun substraat aminozuren in een proton-afhankelijke manier dus ook de interne pH verhogen. De decarboxylasen zijn de glutaminezuur decarboxylasen Gada en GadB 3, de arginine decarboxylase Adia 4, van de lysine decarboxylase LdcI 5, 6 en de ornithinedecarboxylase SPEF 7. Al deze enzymen maken gebruik van pyridoxal-5&#39;-fosfaat als een co-factor 8 en werken samen met binnen-membraan substraat-product antiporters dat decarboxylering producten te verwijderen om de externe medium in ruil voor verse ondergrond 2. In het geval van LdcI, is de lysine-cadaverine antiporter Cadb genoemd. Onlangs hebben we de X-ray kristalstructuur van LdcI tot 2,0 Å, en ontdekten we een nieuwe klein-molecule gebonden aan de strenge LdcI reactie regulator guanosine 5&#39;-difosfaat, 3&#39;-difosfaat (ppGpp) 14. De strenge reactie treedt op wanneer exponentieel groeiende cellen ervaring voedingsstof deprivatie of een van een aantal andere belastingen 9. Als gevolg hiervan, cellen produceren ppGpp die leidt tot een signaalcascade culminerend in de verschuiving van exponentiële groei naar stationaire fase groei 10. We hebben aangetoond dat ppGpp is een specifieke remmer van LdcI 14. Hier beschrijven we de lysine decarboxylase assay, gewijzigd ten opzichte van de test is ontwikkeld door Phan et al.. 11, die we hebben gebruikt om de activiteit van LdcI en het effect van pppGpp / ppGpp op die activiteit te bepalen. De LdcI decarboxyleringsreactie verwijdert de α-carboxy groep van L-lysine en produceert kooldioxide en de polyamine cadaverine (1,5-diaminopentane) 5. L-lysine en cadaverine kan worden gereageerd met 2,4,6-trinitrobenzensulfonic zuur (TNBS) bij hoge pH-waarde N, N&#39;-bistrinitrophenylcadaverine (TNP-cadaverine) en N, N&#39;-bistrinitrophenyllysine (TNP-lysine) te genereren, respectievelijk 11. Het TNP-cadaverine kan worden gescheiden van de TNP-lysine als de eerste is oplosbaar in organische oplosmiddelen, zoals tolueen, terwijl de laatste is niet (zie figuur 1). Het lineaire bereik van de test werd bepaald met behulp van empirisch gezuiverd 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

De activiteit van de induceerbare lysine decarboxylase wordt gecontroleerd door de reactie van het substraat L-lysine en het product cadaverine met 2,4,6-trinitrobenzensulfonic zuur te adducten dat differentiële oplosbaarheid hebben in tolueen vorm.

Een test voor het meten van de activiteit van de Escherichia coli Induceerbare 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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An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

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