这项工作是由国家科学基金会奖＃0920274和南卡罗来纳州试验站项目（SC - 1700340）幼稚园资助计划的支持。本文是技术贡献的克莱姆森大学实验站5929号。

此协议的总体方案概述图1。 1。标准曲线和检测解决方案的准备工作<ol><li>准备100毫升2 mol / L的羟胺盐酸溶液。称取13.9克盐酸羟胺（兆瓦69.49克/摩尔），溶于约50毫升蒸馏水，去离子水（DDH 2 O）的。使用氢氧化钾颗粒或浓缩液，调整pH至7.0。带来的最终体积至100 mL。该解决方案可在常温下保存长达30天，或在4 ° C下90天。 </li><li>准备一个三氯化铁/盐酸溶液100毫升。称取13.5克氯化铁（兆瓦270.32克/摩尔），溶于约50毫升DDH 2 O添加41.3毫升的浓盐酸（12.1 mol / L的），使终体积为100 mL。氯化铁的终浓度0.5 mol / L的终浓度的盐酸，将5 MOL/ L。解决的办法是在常温下保存。 </li><li>准备100毫升trichoroacetic酸了1.84 mol / L的解决方案。称取30.0克的三氯乙酸（兆瓦163.39克/摩尔），溶于DDH 2 O，使最终体积至100 mL。解决的办法是在常温下保存。 </li><li>准备0.1 mol / L的乙酰，磷酸盐溶液5毫升。称取0.092克乙酰磷酸（兆瓦184.06克/摩尔），溶于DDH 2 O，使最终体积至5 mL。分装的解决方案，离心管（0.25毫升每管），并在-20℃冻结，直到使用。解冻的解决方案应放在冰，可用于约1小时。乙酰磷酸解决方案已经解冻的，不应该再冷冻和再利用。 </li><li>准备5毫升0.1 mol / L的ADP的解决方案。称取0.24克（兆瓦472.5克/摩尔）的ADP，溶于DDH 2 O，并把最终体积至5 mL。分装在离心管（每管0.5毫升）的解决方案，并冻结在-20 ° C直到使用。保持解冻冰上直至使用的解决方案。 </li><li>准备50毫升的1 mol / L的三解决方案。称取6.06克三（兆瓦121.14克/摩尔），溶于40毫升DDH 2 O溶液的pH值调整到7.0用盐酸和带来最终容至50 ml。在室温下的存储解决方案。 </li><li>准备1 mol / L的氯化镁溶液10毫升。称取2.03克氯化镁（兆瓦203.31克/摩尔），溶于DDH 2 O，并把最后容至10 mL。储存在室温下的解决方案。 </li><li>准备好三氯化铁/盐酸溶液和三氯乙酸溶液等体积混合25毫升的开发解决方案。开发解决方案，应准备新鲜的每一天，在室温下储存。 </li></ol> 2。乙酰磷酸标准曲线的制备<ol><li>乙酰磷酸标准曲线生成乙酰磷酸已知金额。标准曲线的6至eighT分是合适的。该标准的范围应该每300μL，这与0.1〜3 mmol / L的终浓度从0.03μmoles到0.9μmoles稀乙酰磷酸原液100毫米三在终体积为300μL的解决方案，在适当的浓度。还应该准备一个没有控制乙酰磷酸样本。 </li><li>每个标准控制在37 ° C的热块1分钟。 </li><li>添加50μL盐酸羟胺溶液到每一个标准和晃动三次，控制和混合。放入60 ° C的热块的标准，并孵育5分钟。 </li><li>加入100μL的开发解决方案的标准和控制，通过摇动三次拌匀，并让颜色在室温下至少一分钟的发展。 </li><li>离心机中离心1分钟18000 XG的所有标准和控制。 </li><li>分光光度计一个测量样品吨540 nm处使用1毫升塑料试管，用分光空白的控制。 </li><li>使用适当的数据分析和绘图软件（如Kaleidagraph，协同软件）生成一个标准的曲线。 R 2的值大于或等于0.99是可取的。数据绘制与乙酰磷酸目前μmoles，在540 nm处的吸光度。 </li></ol> 3。醋酸激酶的活性含量为<ol><li>使用所述的解决方案，准备一个反应混合物中包含终浓度为0.1 mol / L的Tris缓冲液，10 mmol / L的氯化镁，5 mmol / L的ADP，和2 mmol / L的乙酰磷酸。在10 mL反应混合物将需要8.2毫升DDH 2 O，1.0毫升三的解决方案，为0.1 mL氯化镁溶液，0.5毫升ADP的解决方案，0.2毫升乙酰磷酸盐溶液。分布在300μL分装离心管的反应混合。动力学参数和底物浓度的优化的测定，一个基板可以多样和其他在恒定浓度的反应举行。 </li><li>预1分钟的反应在37 ° C的热块。 </li><li>添加酶反应所需的量，并立即返回到37℃的热块管。还包括一个控制反应没有酶添加。如果进行几个反应管，在特定的时间间隔，添加酶。一定要跟踪时间。 （注:酶的使用量将需要优化，以确保乙酰磷酸或ADP基板都不会枯竭 - 见讨论部分） </li><li> 5分钟后，加入盐酸羟胺溶液50μL，每个反应和控制，混合，放置在60 ° C的热块管。如果正在执行的一些反应，这应该在相同的时间间隔添加酶。 </li><li> 5分钟的反应和控制孵育60 ° C。 </li><li>新增100＆万亩; L的开发解决方案，每个反应和控制，混合，在实验室工作台上的试管架。允许发展至少1分钟的颜色。 </li><li>离心机中离心1分钟18000 XG管。 </li><li>分光光度计在540 nm，使用1毫升的塑料试管和乙酰磷酸标准曲线进行比较，以确定乙酰磷酸目前量的测量样品。 </li><li>乙酰磷酸耗尽基板量可以由乙酰磷酸减去从量控制缺乏酶的乙酰磷酸酶反应后的剩余金额。使用适当的数据和图形软件（例如Kaleidagraph，协同软件）进行数据分析。 R 2的值比0.97更大的是可取的。 </li></ol> 4。代表性的成果: 这个实验的目的是直接测量醋酸激酶活性离子的醋酸形成。这是通过测量乙酰磷酸基板的消费，因为是不容易直接测量，醋酸生产的。乙酰磷酸酶反应过程中给定的时间后剩余是转化为乙酰氧肟，并在其后铁氧肟复杂，其中有一个颜色偏红（图2），可在540 nm处测定。标准曲线的绘制吸光度与μmoles标准的乙酰磷酸目前用于确定每个反应中剩余的乙酰磷酸量。标准曲线应通过零点的线性。一个有代表性的标准曲线如图3所示的1.2332％μmole乙酰磷酸目前和 R 2值0.99吸光度单位，表明该数据符合线性方程的斜率。标准曲线数据的线性拟合方程用于计算乙酰磷酸反应中剩余的数量。该μmoles乙酰消耗磷酸是在目前缺乏酶和乙酰磷酸酶反应后剩余的μmoles控制反应的乙酰磷酸μmoles之间的差异决定的。 纯化，重组M。嗜热醋酸激酶检测使用所描述的协议。如图4所示的实验，ADP浓度保持不变在5 mmol / L的浓度变化的反应乙酰磷酸乙酰磷酸，以确定的 K m。正如所料，这个实验表明，这种酶如下每个基板标准米氏般的动力学，并产生了双曲饱和曲线在策划活动与乙酰磷酸盐浓度。明显的 K m值乙酰磷酸为0.27 ± 0.01 mmol / L时相比，毫不逊色出版的 K m值0.47 ± 0.01 mmol / L的使用加上作为说13。 直接分析，也可以用来衡量利用衬底比ATP，这是不可能使用标准的耦合检测的醋酸激酶的活动。纯化的重组大肠杆菌阿米巴醋酸激酶16，PP 我 ，而不是ATP的产生在醋酸形成的方向，受到这个实验中，使用ADP的地方磷酸钠。反应磷酸钠的浓度保持不变，在0.2 mol / L的乙酰磷酸盐浓度是不同的。至于的M.嗜热醋酸激酶， 大肠杆菌阿米巴醋酸激酶遵循米氏般的动力学和乙酰磷酸双曲饱和曲线（图5）。明显的 K m值乙酰磷酸为0.50 ± 0.006毫摩尔/升。 Reeves和格思里15如何确定的 K m值的0.06毫摩尔/大号原酶的乙酰磷酸;<html>以往，他们的酶只是部分纯化和检测涉及四个耦合的酶，也只有部分纯化。因此，它是很难直接比较这些值。 协议计划<ol><li>生成乙酰磷酸标准曲线</li><li>稀释到所需浓度的酶</li><li>准备反应混合物和分装300μL离心管</li><li> Preinc​​ubate反应在37 ° C为1分钟</li><li>启动通过添加酶在特定的时间间隔反应</li><li>添加羟胺在相同的时间间隔，停止反应，并培育在60℃，5分钟</li><li>添加氯化铁/三氯乙酸溶液显色</li><li>离心管18,000 1分钟XG </li><li>在540 nm处测量吸光度</li><li>乙酰消耗磷矿量确定标准曲线比较</li></ol>tp_upload/3474/3474fig1.jpg"/&gt; 图1。议定书"的计划 <img alt="图2" src="/files/ftp_upload/3474/3474fig2.jpg" /> 图2。乙酰磷酸标准 。乙酰磷酸浓度增加的反应，羟胺和颜色所述。缺乏乙酰磷酸的控制是亮黄色，并含有对乙酰磷酸越来越多的反应都先后在颜色较深。这种颜色的变化是在540 nm处测量。 <img alt="图3" src="/files/ftp_upload/3474/3474fig3.jpg" /> 图3。乙酰磷酸样品标准曲线。与羟胺反应的乙酰磷酸数额不等，颜色开发解决方案和吸光度在540 nm处测定。与μmoles乙酰磷酸在540 nm的吸光度，绘制线性拟合的数据应用。/ P&gt; <img alt="图4" src="/files/ftp_upload/3474/3474fig4.jpg" /> 图4 ATP生产M.乙酰磷酸的利用。 嗜热醋酸激酶 。酶活性测定醋酸形成中存在的5 mmol / L的ADP与不同浓度的乙酰磷酸的方向。乙酰磷酸消耗金额被认定为起点金额乙酰磷酸反应和乙酰磷酸酶反应后剩余的金额之间的差额。检测显示，错误的酒吧一式三份。 <img alt="图5" src="/files/ftp_upload/3474/3474fig5.jpg" /> 图5。E.利用由PP 我生产的乙酰磷酸阿米巴醋酸激酶 。在醋酸形成的方向酶活性测定在0的存在。2 mol / L的不同浓度的乙酰磷酸磷酸钠。乙酰磷酸消耗金额被认定为起点金额乙酰磷酸反应和乙酰磷酸酶反应后剩余的金额之间的差额。检测显示，错误的酒吧一式三份。

<ol>
	<li>Bork, P., Sander, C., Valencia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ATPase+domain+common+to+prokaryotic+cell+cycle+proteins,+sugar+kinases,+actin,+and+hsp70+heat+shock+proteins.">An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 7290-7294 (1992).</li><li>Bork, P., Sander, C., Valencia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+evolution+of+similar+enzymatic+function+on+different+protein+folds:+the+hexokinase,+ribokinase,+and+galactokinase+families+of+sugar+kinases.">Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.</a> Protein Sci. 2, 31-40 (1993).</li><li>Buss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urkinase:+structure+of+acetate+kinase,+a+member+of+the+ASKHA+superfamily+of+phosphotransferases.">Urkinase: structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases.</a> J. Bacteriol. 183, 680-686 (2001).</li><li>Hurley, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sugar+kinase/heat+shock+protein+70/actin+superfamily:+implications+of+conserved+structure+for+mechanism.">The sugar kinase/heat shock protein 70/actin superfamily: implications of conserved structure for mechanism.</a> Annu. Rev. Biophys. Biomol. Struct. 25, 137-162 (1996).</li><li>Holmes, K. C., Sander, C., Valencia, 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+new+ATP-binding+fold+in+actin,+hexokinase+and+Hsc70.">A new ATP-binding fold in actin, hexokinase and Hsc70.</a> Trends. Cell. Biol. 3, 53-59 (1993).</li><li>Ingram-Smith, C., Martin, S. R., Smith, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetate+kinase:+not+just+a+bacterial+enzyme.">Acetate kinase: not just a bacterial enzyme.</a> Trends. Microbiol. 14, 249-253 (2006).</li><li>Aceti, D. J., Ferry, J. G. <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+characterization+of+acetate+kinase+from+acetate-grown+Methanosarcina+thermophila.+Evidence+for+regulation+of+synthesis.">Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis.</a> J. Biol. Chem. 263, 15444-15448 (1988).</li><li>Latimer, M. T., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning,+sequence+analysis,+and+hyperexpression+of+the+genes+encoding+phosphotransacetylase+and+acetate+kinase+from+Methanosarcina+thermophila.">Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.</a> J. Bacteriol. 175, 6822-6829 (1993).</li><li>Ingram-Smith, C., Barber, R. D., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+histidines+in+the+acetate+kinase+from+Methanosarcina+thermophila.">The role of histidines in the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 275, 33765-33770 (2000).</li><li>Miles, R. D., Iyer, P. P., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-directed+mutational+analysis+of+active+site+residues+in+the+acetate+kinase+from+Methanosarcina+thermophila.">Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 276, 45059-45064 (2001).</li><li>Miles, R. D., Gorrell, A., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+transition+state+analog,+MgADP-aluminum+fluoride-acetate,+in+acetate+kinase+from+Methanosarcina+thermophila.">Evidence for a transition state analog, MgADP-aluminum fluoride-acetate, in acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 277, 22547-22552 (2002).</li><li>Ingram-Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+acetate+binding+pocket+in+the+Methanosarcina+thermophila+acetate+kinase.">Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase.</a> J. Bacteriol. 187, 2386-2394 (2005).</li><li>Gorrell, A., Lawrence, S. H., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+kinetic+analyses+of+arginine+residues+in+the+active+site+of+the+acetate+kinase+from+Methanosarcina+thermophila.">Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila.</a> J. Biol. Chem. 280, 10731-10742 (2005).</li><li>Gorrell, A., Ferry, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+Methanosarcina+thermophila+acetate+kinase+mechanism+by+fluorescence+quenching.">Investigation of the Methanosarcina thermophila acetate kinase mechanism by fluorescence quenching.</a> Biochemistry. 46, 14170-14176 (2007).</li><li>Reeves, R. E., Guthrie, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetate+kinase+(pyrophosphate).+A+fourth+pyrophosphate-dependent+kinase+from+Entamoeba+histolytica.">Acetate kinase (pyrophosphate). A fourth pyrophosphate-dependent kinase from Entamoeba histolytica.</a> Biochem. Biophys. Res. Commun. 66, 1389-1395 (1975).</li><li>Fowler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+and+structural+characterization+of+the+novel+PPi-dependent+acetate+kinase+from+the+parasite+Entamoeba+histolytica.">Kinetic and structural characterization of the novel PPi-dependent acetate kinase from the parasite Entamoeba histolytica.</a> , (2011).</li><li>Lipmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+synthesis+of+acetyl+phosphate.">Enzymatic synthesis of acetyl phosphate.</a> J. Biol. Chem. 155, 55-70 (1944).</li><li>Lipmann, F., Tuttle, L. 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+specific+micromethod+for+determination+of+acyl+phosphates.">A specific micromethod for determination of acyl phosphates.</a> J. Biol. Chem. 159, 21-28 (1945).</li><li>Lipmann, F., Jones, M. E., Black, S., Flynn, R. 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+mechanism+of+the+ATP-CoA-acetate+reaction.">The mechanism of the ATP-CoA-acetate reaction.</a> J. Cell. Physiol. Suppl. 41, 109-112 (1953).</li><li>Rose, I. A., Grunberg-Manago, M., Korey, S. F., Ochoa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+phosphorylation+of+acetate.">Enzymatic phosphorylation of acetate.</a> J. Biol. Chem. 211, 737-756 (1954).</li><li>Allen, S. H., Kellermeyer, R. W., Stjernholm, R. L., Wood, H. G. <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+properties+of+enzymes+involved+in+the+proponic+acid+fermentation.">Purification and properties of enzymes involved in the proponic acid fermentation.</a> Journal of Bacteriology. 87, 171-187 (1964).</li><li>Clarke, P. M., Payton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+assay+for+acetate+in+spent+bacterial+culture+supernatants.">An enzymatic assay for acetate in spent bacterial culture supernatants.</a> Anal. Biochem. 130, 402-405 (1983).</li><li>Mukhopadhyay, S., Hasson, M. S., Sanders, D. 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+continuous+assay+of+acetate+kinase+activity:+measurement+of+inorganic+phosphate+release+generated+by+hydroxylaminolysis+of+acetyl+phosphate.">A continuous assay of acetate kinase activity: measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate.</a> Bioorg. Chem. 36, 65-69 (2008).</li><li>Nakajima, H., Suzuki, K., Imahori, K. <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+properties+of+acetate+kinase+from+Bacillus+stearothermophilus.">Purification and properties of acetate kinase from Bacillus stearothermophilus.</a> J. Biochem. 84, 193-203 (1978).</li></ol>

没有利益冲突的声明。

在这个实验中的乙酰磷酸的检测是根据足够浓度的盐酸羟胺和氯化铁溶液的浓度和酸度依赖。更改的检测量将需要重新考虑这两个组件。酶促反应这里描述的是37 °与5分钟，在60 ° C时，羟胺终止。这较高的温度是至关重要的，以便其余乙酰磷酸迅速转化为乙酰氧肟。颜色一步发展和吸光度的测量时间是不太重要的，羟胺溶液此外，可以在短短的5分钟至15分钟进行。样品应该离心读取吸光度之前，...

<table border="1"><tr><td width="160"> 试剂名称 </td><td width="108"> 公司 </td><td width="134"> 目录编号 </td><td width="103"> 评论 </td></tr><tr><td>乙酰磷酸</td><td>西格玛爱秩序</td><td> 01409 </td><td>锂盐（97％） </td></tr><tr><td>钠磷酸二氢（脱水） </td><td> ThermoFisher </td><td> S381 </td><td></td></tr><tr><td>钠磷酸氢二钠（无水） </td><td> ThermoFisher </td><td> S374 </td><td></td></tr><tr><td>氯化镁（六水） </td><td> ThermoFisher </td><td> M33 </td><td></td></tr><tr><td> Tris碱</td><td> ThermoFisher </td><td> B152 </td><td></td></tr><tr><td>三氯化铁（六水） </td><td> ThermoFisher </td><td> I88 </td><td></td></tr><tr><td>三氯乙酸</td><td> ThermoFisher </td><td> A324 </td><td></td></tr><tr><td>盐酸羟胺</td><td> ThermoFisher </td><td> H330 </td><td></td></tr><tr><td>腺苷5&#39; -二磷酸钠盐</td><td>西格玛爱秩序</td><td> A2754 </td><td></td></tr><tr><td> Biomate三分光光度计</td><td> ThermoFisher </td><td> 142982082 </td><td>标准紫外/可见分光光度计</td></tr></table>

direct detection of the acetate-forming activity  of the enzyme acetate kinase

醋酸激酶，醋酸盐和糖激酶- HSP70 -肌动蛋白（ASKHA）1-5酶超家族成员，负责对醋酸的可逆磷酸化乙酰磷酸盐作为基质利用ATP 。醋酸激酶是无处不在的细菌 ，发现一个古属的，而且也存在于 真核生物 6微生物。最特征的醋酸激酶是由产甲烷古菌甲烷嗜 热 7-14 。 阿米巴 ，阿米巴痢疾的病原体已被隔离醋酸激酶只能利用聚丙烯 ，但不乙酰磷酸形成的方向ATP，并迄今只发现该属15,16。 在乙酰磷酸形成的方向，醋酸激酶活性通常是测量使用的氧肟检测，首先由李普曼描述17-20日 ，耦合检测的ATP转化为ADP耦合NADH氧化为NAD +的酶丙酮酸激酶和乳酸脱氢酶21,22，或乙酰磷酸盐产品的反应后释放的无机磷的实验测量羟胺23。醋酸形成方向相反的活动，是衡量耦合ATP的形成，从ADP 减少NADP +，NADPH酶己糖激酶和葡萄糖-6 - 磷酸脱氢酶24。 在这里，我们描述了醋酸醋酸形成，不需要耦合酶的方向激酶活性检测的方法，而是被直接测定乙酰磷酸消费。酶反应后，剩余的乙酰磷酸转化为羟肟检测，可以测量分光光度计的一个铁氧肟复杂。因此，不像STandard加上这个方向是依赖于从ADP ATP的生产检测，这种直接的检测可用于醋酸激酶产生ATP或PP 我 。

Watch this Scientific Journal Video about Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase at JoVE.com

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

醋酸激酶活性测定方法的描述。本实验利用一个直接反应在醋酸形成的方向确定不同磷受体酶的活性和乙酸激酶动力学。此外，这种方法可以用于化验等乙酰磷酸盐或乙酰CoA利用酶。

醋酸形成醋酸酶激酶活性的直接检测

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

direct-detection-acetate-forming-activity-enzyme-acetate

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

23.0K 次观看.  Clemson University. 醋酸激酶活性测定方法的描述。本实验利用一个直接反应在醋酸形成的方向确定不同磷受体酶的活性和乙酸激酶动力学。此外，这种方法可以用于化验等乙酰磷酸盐或乙酰CoA利用酶。

视频: Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme&#39;s active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

科研

生物化学

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

用放射性标记的ATP测定蛋白激酶活性

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

利用多功能生化方法鉴定新型 ck2 激酶基材

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. 
Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). 
Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

<meta charset="utf8"></head><body>评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

In this paper, standardized ecotoxicological methods for the evaluation of biomarkers in neotropical anuran species are presented. Specifically, this paper details several methodologies at different scales of ecotoxicological evaluation, such as the genetic, cellular-histological, biochemical, morphological, and individual levels.

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that ...

环境科学

开发基于点击的检测方法，用于筛选 HAT1 酶活性

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

作者聚焦:开发用于 HAT1 抑制剂筛选的乙酰点击检测

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

醋酸形成醋酸酶激酶活性的直接检测

摘要

探索更多视频

此视频中的章节

制备及

用放射性标记的ATP测定蛋白激酶活性

利用多功能生化方法鉴定新型 ck2 激酶基材

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

评估新热带 Anurans 不同尺度生物标志物的生态毒理学方法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

在大肠杆菌中生成特异的乙酰蛋白的简易协议