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

我们感谢博士博士迈克尔卡舍尔（，贝塞斯达，MA，美国国家卫生研究院）向我们发送菌株，质粒和必要的协议。我们感谢SpecraMax酶标仪约翰格洛弗（博士，加拿大多伦多大学生物化学系）。英国是一个国家科学与工程研究理事会，加拿大（NSERC）的研究生奖学金，加拿大在与疾病相关的膜蛋白的结构生物学研究所的卫生研究战略培训计划，及多伦多大学打开奖学金获得者。这项工作是支持由授予的加拿大卫生研究院（澳门币67210）往华。

1）试剂和仪器<ol><li>首先，准备以下三种解决方案：1 mL的溶液A，它是由L -赖氨酸8毫米，100毫米的钠2 - （N -吗啉）ethanesulphonic酸（MES），pH值6.5，0.2毫米核苷酸，核苷酸无论是：鸟苷二磷酸总值（GDP），三磷酸鸟苷（GTP），5&#39; -鸟苷二磷酸，3&#39; -二磷酸果糖（ppGpp），或5&#39; -鸟苷三磷酸，3&#39; -二磷酸（pppGpp），0.1毫米哆5&#39; -磷酸（PLP），1毫米β-巯基乙醇/（β- ME）。 1毫升溶液B，包括100?...

<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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赖氨酸脱羧酶试验，TNBS是L -赖氨酸和尸伯胺反应形成，TNP -赖氨酸和TNP -尸加合物（图1）。由于TNP -赖氨酸的羧酸基团的存在，这种加合物仍然易溶于水，而TNP -尸，缺乏羧酸 ，甲苯11分区。其他类型羧酸组的损失发生在反应过程中的氨基酸，可以更广泛地利用这类型的检测。这发生在L -鸟氨酸诱导鸟氨酸脱羧酶SPEF的脱羧形成多胺腐胺和L -精氨酸脱羧诱导精氨酸脱羧酶ADIA形成多胺胍丁...

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

大肠杆菌是肠道细菌能够较广泛的pH值（pH值5 - 9）增长1的，令人难以置信的，是能够生存的极端酸强调，通过哺乳动物的胃pH值下降，包括通过低作为pH值1 - 2 2。为了使这样一个广泛偏酸生存，E.大肠杆菌拥有四种不同的诱导氨基酸decarboxylases decarboxylate基板在质子依赖性氨基酸，从而提高内部的pH值。 decarboxylases包括谷氨酸decarboxylases嘎达和GadB 3，精氨酸脱羧酶ADIA 4，赖氨酸脱羧酶LdcI 5，6和7鸟氨酸脱羧酶SPEF。所有这些酶的利用吡哆醛-5 -磷酸作为共同因子8，函数内的膜基板产品逆向转运，删除脱羧产品在外部介质换取新鲜的基板2。在LdcI的情况下，赖氨酸尸逆向转运蛋白被称为农发行。最近，我们确定了X射线晶体结构的LdcI 2.0 A，我们发现了一个新的小分子绑定到LdcI严格的反应调节鸟苷5&#39; -二磷酸，3&#39; -二磷酸果糖（ppGpp）14 。严格的反应时，会发生细胞急剧增长的经验营养剥夺或其他9讲。因此，细胞产生ppGpp导致信号级联，最终从指数的增长转移到固定相增长10。我们已经证明，ppGpp是LdcI 14特异性抑制剂。在这里，我们描述赖氨酸脱羧酶检测，修改从藩等人开发的的实验。11，我们用于确定LdcI活动pppGpp / ppGpp该活动的影响。 LdcI脱羧反应，消除了L -赖氨酸的α-羧基组和产生的二氧化碳和尸胺（1,5 - diaminopentane） 5。 L -赖氨酸和尸，可在高pH值2,4,6 trinitrobenzensulfonic酸（TNBS）反应生成N，N - bistrinitrophenylcadaverine（TNP -尸）和N，N&#39; - bistrinitrophenyllysine（TNP -赖氨酸），分别11。 TNP -尸可以分开，前者是可溶于有机溶剂，如甲苯，而后者则没有（见图1），TNP -赖氨酸。检测的线性范围为使用纯化尸凭经验确定。

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

2,4,6 - trinitrobenzensulfonic酸反应的底物L -赖氨酸和产品尸形成加合物在甲苯的溶解度差，诱导赖氨酸脱羧酶的活动都受到监控。

用于测量的活性测定大肠埃希氏菌诱导赖氨酸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 ...

Research

JoVE Journal

Biology

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

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

科研

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

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对于电感受的制备快速协议<em&gt;大肠杆菌</em&gt;和<em&gt;霍乱弧菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

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协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

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检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

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膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

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蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

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非经典氨基酸的具体残留纳入到模型蛋白质使用<em&gt;大肠杆菌</em&gt;无细胞转录翻译系统

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

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