This work was supported by NIH grants ARRA RO3 AI078145 and 1SC3GM113743 to RZ.

1.细胞提取物制剂<ol><li>生长的利什曼原虫细胞与气密的瓶盖密封的无菌塑料瓶在26ºC在制成1×M199的培养基中补充有20毫米的HEPES pH7.4的，100U / ml青霉素，100微克/ ml链霉素，5微克/毫升血红素0.35克/升NACO 2小时 ，0.1毫米腺嘌呤，和2微克/毫升生物蝶呤，无晃动。收获细胞，离心在1,500rpm离心5分钟，4ºC当它们到达的1-2×10 7 / ml的细胞密度。 </li><li>丢弃用吸管将上清液和重悬细胞沉淀用血清吸管在半冷的磷酸盐缓冲盐水的培养的体积（137毫摩尔NaCl，2.7毫米氯化钾洗细胞，10毫的 Na 2 HPO 4，1.8毫KH 2 PO 4，pH7.4）中。根据BL2安全规定进行处置细胞上清液。 </li><li>再次离心细胞在1,500rpm离心5分钟，在4ºC。丢弃用吸管将上清液。继续牛逼Ó下一步骤或卡扣冷冻细胞沉淀在液氮中用于长期贮存于-80℃（长达三个月）。 </li><li>制备2×裂解缓冲液（0.5M蔗糖，0.1M TrisHCl，pH7.5的，2毫摩尔EDTA，和2x蛋白酶抑制剂混合物），并保持在4ºC冰上。 </li><li>重悬细胞沉淀中的2×裂解缓冲液等体积​​（新鲜或冷冻）。加入玻璃珠1倍量。涡旋剧烈4ºC10分钟。 </li><li>加入2倍体积的1X裂解液混匀。离心细胞提取物在1500×g离心在4ºC10分钟沉淀完整细胞和核。 </li><li>转移用吸管将上清液到新的凉爽离心管，并保持细胞提取物在冰上，直至实验结束。 </li></ol> 2.确定细胞提取物的蛋白浓度使用蛋白估计套件如二喹啉酸含量<ol><li>制备双金鸡宁酸（BCA）溶液（1ml /管）通过混合所述BCA和铜（II）■ulfate在49:1的比例（体积/体积）。 </li><li>制备的蛋白标准0，10，20，30，40，50，和60微克/毫升稀释10毫克/毫升牛血清白蛋白（BSA）原液入1ml等分该BCA溶液。 </li><li>加入2微升细胞提取物在1ml中重复BCA溶液。在一个预热的60℃水浴中孵育标准和蛋白质样品10分钟。 </li><li>转移样品到冰3分钟。测量的标准和蛋白质样品用分光光度计的吸光度的562纳米的波长。 </li><li>计算使用标准BSA作为参考在制造商的方案所述的细胞提取物的蛋白质浓度。稀释细胞提取物，以10毫克/毫升用1×裂解缓冲液的蛋白质浓度。 </li></ol> 3.酶测定每管200微升注意:携带在化学罩以下步骤。 <ol><li>测试一式两份每个样品在15ml锥形管中。准备20微升的1M TrisHCl pH为7.5，每管，并保持它在冰上。准备2 ml的氯仿/甲醇（1:1（体积/体积）;终止液）在RT每个管。 </li><li>吸取20微升的1M TrisHCl pH值7.5中的每个的15毫升锥形管在冰上。 </li><li>按照辐射安全指引从这里开始。添加0.06微米（0.2微居里）S中的等效- [甲基3 H〕腺苷-L-蛋氨酸和50μM的冷的SAM每管，共50.06微米的SAM。请将X微升冷水，其中x = 200-（20（缓冲）+ 20（为细胞提取物）+冷和放射性SAM的容量）每管。 </li><li>每个锥形管转移到一个预热的30°C以上水浴中。添加到每个管20微升细胞提取物（200微克蛋白质当量）以开始反应。孵育所需的时间（0到45分钟）。 </li><li>停止反应，加入2ml氯仿/甲醇（1:1;体积/体积;终止液），以每管。锥形管转移至RT（20-256; C）。 </li></ol> 4.脂质提取注意:携带在化学罩以下步骤。 <ol><li>加入700微升水含酶反应样品每管。涡大力30秒。离心机在1500×g离心5分钟，在室温至有机从水相分离。 </li><li>传送下层有机相到一个新的15毫升锥形管用吸管。加入1毫升水，以含有管和涡剧烈30秒每"低级阶段"。离心再次在1500×g离心5分钟以有机从水相分离。 </li><li>传送下层有机相到闪烁管用吸管。根据N 2流干样品。处置含有非掺入放射性SAM和放射性锥形管作为每个辐射准则的水相。 </li><li>加入2毫升/闪烁液管。测量结合的放射性用闪烁凑NTER根据制造商的协议和仪器的用途。 </li><li>使用下列一般方程计算以nmol / mg蛋白质的酶活性: 的cpm值×103×[总（放射性和冷）浓度的SAM（毫摩尔）]放射性的SAM（次/毫摩尔）x的×5比活性[放射性SAM的浓度（毫摩尔）] 注:上述协议可以改变一个SAM浓度依赖性或蛋白质依赖PEMT检测。对于SAM浓度依赖性酶测定，时间保持恒定（15分钟，这是在线性范围）和各种量的冷的SAM被加入到测定而对蛋白依赖性PEMT测定中，SAM浓度（我们选择0.05毫摩尔）和时间保持恒定（15分钟）。另外，缓冲液的pH可以根据需要，如果一些PEMT酶具有不同的最佳pH值来改变。 </li></ol>

<ol>
	<li>Keogh, M. R., Courtney, P. D., Kinney, A. J., Dewey, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+phospholipid+N-.methyltransferases+from+Arabidopsis+and+soybean.">Functional characterization of phospholipid N-.methyltransferases from Arabidopsis and soybean.</a> J Biol Chem. 284 (23), 15439-15447 (2009).</li><li>Geiger, O., Lopez-Lara, I. M., Sohlenkamp, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylcholine+biosynthesis+and+function+in+bacteria.">Phosphatidylcholine biosynthesis and function in bacteria.</a> Biochim Biophys Acta. 1831 (3), 503-513 (2013).</li><li>Gao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+lipogenesis+in+white+adipose+tissue+contributes+to+the+resistance+to+high+fat+diet-induced+obesity+in+phosphatidylethanolamine+N-.methyltransferase-deficient+mice.">Decreased lipogenesis in white adipose tissue contributes to the resistance to high fat diet-induced obesity in phosphatidylethanolamine N-.methyltransferase-deficient mice.</a> Biochim Biophys Acta. 1851 (2), 152-162 (2015).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+phosphatidylethanolamine+N-.methyltransferase+alters+plasma+VLDL+phospholipids+and+attenuates+atherosclerosis+in+mice.">Lack of phosphatidylethanolamine N-.methyltransferase alters plasma VLDL phospholipids and attenuates atherosclerosis in mice.</a> Arterioscler Thromb Vasc Biol. 29 (9), 1349-1355 (2009).</li><li>Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+methylation+in+mammals:+from+biochemistry+to+physiological+function.">Phospholipid methylation in mammals: from biochemistry to physiological function.</a> Biochim Biophys Acta. 1838 (6), 1477-1487 (2014).</li><li>Nishimaki-Mogami, T., Suzuki, K., Takahashi, 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+role+of+phosphatidylethanolamine+methylation+in+the+secretion+of+very+low+density+lipoproteins+by+cultured+rat+hepatocytes:+rapid+inhibition+of+phosphatidylethanolamine+methylation+by+bezafibrate+increases+the+density+of+apolipoprotein+B48-containing+lipoproteins.">The role of phosphatidylethanolamine methylation in the secretion of very low density lipoproteins by cultured rat hepatocytes: rapid inhibition of phosphatidylethanolamine methylation by bezafibrate increases the density of apolipoprotein B48-containing lipoproteins.</a> Biochim Biophys Acta. 1304 (1), 21-31 (1996).</li><li>Noga, A. A., Zhao, Y., Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unexpected+requirement+for+phosphatidylethanolamine+N-.methyltransferase+in+the+secretion+of+very+low+density+lipoproteins.">An unexpected requirement for phosphatidylethanolamine N-.methyltransferase in the secretion of very low density lipoproteins.</a> J Biol Chem. 277 (44), 42358-42365 (2002).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+repression+of+phosphatidylethanolamine+N-.methyltransferase+(PEMT)+in+BRCA1-mutated+breast+cancer.">Epigenetic repression of phosphatidylethanolamine N-.methyltransferase (PEMT) in BRCA1-mutated breast cancer.</a> Oncotarget. 5 (5), 1315-1325 (2014).</li><li>Cui, Z., Houweling, M., Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+rat+hepatoma+cell+growth+by+expression+of+phosphatidylethanolamine+N-.methyltransferase-2.">Suppression of rat hepatoma cell growth by expression of phosphatidylethanolamine N-.methyltransferase-2.</a> J Biol Chem. 269 (40), 24531-24533 (1994).</li><li>Cui, Z., Shen, Y. J., Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverse+correlation+between+expression+of+phosphatidylethanolamine.N-.methyltransferase-2+and+growth+rate+of+perinatal+rat+livers.">Inverse correlation between expression of phosphatidylethanolamine.N-.methyltransferase-2 and growth rate of perinatal rat livers.</a> Biochim Biophys Acta. 1346 (1), 10-16 (1997).</li><li>Minder, A. C., de Rudder, K. E., Narberhaus, F., Fischer, H. M., Hennecke, H., Geiger, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylcholine+levels+in.Bradyrhizobium+japonicum.+membranes+are+critical+for+an+efficient+symbiosis+with+the+soybean+host+plant.">Phosphatidylcholine levels in.Bradyrhizobium japonicum. membranes are critical for an efficient symbiosis with the soybean host plant.</a> Mol Microbiol. 39 (5), 1186-1198 (2001).</li><li>Kodaki, T., Yamashita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+phosphatidylethanolamine+methylation+pathway.+Cloning+and+characterization+of+two+distinct+methyltransferase+genes.">Yeast phosphatidylethanolamine methylation pathway. Cloning and characterization of two distinct methyltransferase genes.</a> J Biol Chem. 262 (32), 15428-15435 (1987).</li><li>Tanaka, Y., Amano, F., Maeda, M., Nishijima, M., Akamatsu, Y. <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+phosphatidyl-N-.monomethylethanolamine+N-.methyltransferase,+the+enzyme+catalyzing+the+second+and+the+third+steps+in+the+phosphatidylethanolamine+N-.methyltransferase+system,+from+mouse+liver+microsomes.">Purification and properties of phosphatidyl-N-.monomethylethanolamine N-.methyltransferase, the enzyme catalyzing the second and the third steps in the phosphatidylethanolamine N-.methyltransferase system, from mouse liver microsomes.</a> Jpn J Med Sci Biol. 43 (3), 59-73 (1990).</li><li>Bibis, S. S., Dahlstrom, K., Zhu, T., Zufferey, R. <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+Leishmania+major+phosphatidylethanolamine+methyltransferases+LmjPEM1+and+LmjPEM2+and+their+inhibition+by+choline+analogs.">Characterization of Leishmania major phosphatidylethanolamine methyltransferases LmjPEM1 and LmjPEM2 and their inhibition by choline analogs.</a> Mol Biochem Parasitol. 196 (2), 90-99 (2014).</li><li>deRudder, K. E., Thomas-Oates, J. E., Geiger, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhizobium+meliloti.+mutants+deficient+in+phospholipid+N-.methyltransferase+still+contain+phosphatidylcholine.">Rhizobium meliloti. mutants deficient in phospholipid N-.methyltransferase still contain phosphatidylcholine.</a> J Bacteriol. 179 (22), 6921-6928 (1997).</li><li>Aktas, M., Narberhaus, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+the+enzyme+properties+of+the+phospholipid+N-.methyltransferase+PmtA+from+Agrobacterium+tumefaciens.">In vitro characterization of the enzyme properties of the phospholipid N-.methyltransferase PmtA from Agrobacterium tumefaciens.</a> J Bacteriol. 191 (7), 2033-2041 (2009).</li><li>Ridgway, N. D., Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylethanolamine+N-.methyltransferase+from+rat+liver.">Phosphatidylethanolamine N-.methyltransferase from rat liver.</a> Methods Enzymol. 209, 366-374 (1992).</li><li>Gaynor, P. M., Carman, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylethanolamine+methyltransferase+and+phospholipid+methyltransferase+activities+from+Saccharomyces+cerevisiae.+Enzymological+and+kinetic+properties.">Phosphatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties.</a> Biochim Biophys Acta. 1045 (2), 156-163 (1990).</li><li>Arondel, V., Benning, C., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+expression+in+Escherichia+coli.+of+a+gene+encoding+phosphatidylethanolamine+methyltransferase+(EC+2.1.1.17)+from+Rhodobacter+sphaeroides.">Isolation and functional expression in Escherichia coli. of a gene encoding phosphatidylethanolamine methyltransferase (EC 2.1.1.17) from Rhodobacter sphaeroides.</a> J Biol Chem. 268 (21), 16002-16008 (1993).</li><li>Wessel, M., Klusener, S., Godeke, J., Fritz, C., Hacker, S., Narberhaus, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Agrobacterium+tumefaciens.+requires+phosphatidylcholine+in+the+bacterial+membrane.">Virulence of Agrobacterium tumefaciens. requires phosphatidylcholine in the bacterial membrane.</a> Mol Microbiol. 62 (3), 906-915 (2006).</li><li>Klusener, S., Aktas, M., Thormann, K. M., Wessel, M., Narberhaus, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+physiological+relevance+of+Agrobacterium+tumefaciens.+phosphatidylcholine+biosynthesis+genes.">Expression and physiological relevance of Agrobacterium tumefaciens. phosphatidylcholine biosynthesis genes.</a> J Bacteriol. 191 (1), 365-374 (2009).</li><li>Henderson, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+the+gene+encoding+Leishmania+donovani.S.-adenosylhomocysteine+hydrolase,+a+potential+target+for+antiparasitic+chemotherapy.">Cloning of the gene encoding Leishmania donovani.S.-adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy.</a> Mol Biochem Parasitol. 53 (1-2), 169-183 (1992).</li><li>Koszalka, G. W., Krenitsky, T. A., Nyhan, W. L., Thompson, L. F., Watts, R. W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5&#39;-Methylthioadenosine+(MTA)+phosphorylase+from+promastigote+of+Leishmania+donovani.">5&#39;-Methylthioadenosine (MTA) phosphorylase from promastigote of Leishmania donovani.</a> Purine and Pyrimidine Metabolism in Man V, Adv Exp Med Biol. 131, 559-563 (1986).</li><li>Biastoff, S., Teuber, M., Zhou, Z. S., Dr&#228;ger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+activity+measurement+of+a+recombinant+putrescine+N.-methyltransferase+from+Datura+stramonium.">Colorimetric activity measurement of a recombinant putrescine N.-methyltransferase from Datura stramonium.</a> Planta Med. 72 (12), 1136-1141 (2006).</li><li>Hendricks, C. L., Ross, J. R., Pichersky, E., Noel, J. P., Zhou, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-coupled+colorimetric+assay+for+S.-adenosylmethionine-dependent+methyltransferases.">An enzyme-coupled colorimetric assay for S.-adenosylmethionine-dependent methyltransferases.</a> Anal Biochem. 326 (1), 100-105 (2004).</li><li>Cannon, L. M., Butler, F. N., Wan, W., Zhou, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stereospecific+colorimetric+assay+for+(S.,S.)-adenosylmethionine+quantification+based+on+thiopurine+methyltransferase-catalyzed+thiol+methylation.">A stereospecific colorimetric assay for (S.,S.)-adenosylmethionine quantification based on thiopurine methyltransferase-catalyzed thiol methylation.</a> Anal Biochem. 308 (2), 358-363 (2002).</li><li>Hoffman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+analysis+of+the+chiral+and+covalent+instability+of+S.-adenosyl-L-methionine.">Chromatographic analysis of the chiral and covalent instability of S.-adenosyl-L-methionine.</a> Biochemistry. 25 (15), 4444-4449 (1986).</li><li>Wu, S. E., Huskey, W. P., Borchardt, R. T., Schowen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+instability+at+sulfur+of+S.-adenosylmethionine.">Chiral instability at sulfur of S.-adenosylmethionine.</a> Biochemistry. 22 (12), 2828-2832 (1983).</li><li>Dorgan, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-coupled+continuous+spectrophotometric+assay+for+S.-adenosylmethionine-dependent+methyltransferases.">An enzyme-coupled continuous spectrophotometric assay for S.-adenosylmethionine-dependent methyltransferases.</a> Anal Biochem. 350 (2), 249-255 (2006).</li></ol>

没有利益冲突的声明

此简单，快速PEMT测定允许PE而导致的从SAM放射性甲基的使用全细胞提取物作为蛋白质来源转移到PE的甲基化形式的量化。它具有快速，灵敏，重现性好，也适用于纯化的酶17。单甲基或二甲基PE可以被添加到测定如果感兴趣的甲基是特定于这些基板而不是体育12,13,18,19。如果纯化PEMT酶时，PE可以被添加到测定。此协议的一个限制是，该测定不能识别的反应产物（单甲基PE，二甲基PE，或PC）。然而，该反应产物（单甲基PE，二甲基PE，PC）的身份可以进一步通过一维薄层层析如20,21描述进行分析。此外，一些的SAM降解产物如 S -adenosylhomocysteine ​​和5&#39;-甲硫可能抑制PEMT b活动Ÿ反馈抑制。然而， 利什曼原虫具有一个 S -adenosylhomocysteine ​​水解酶22，其裂解小号 -adenosylhomocysteine ​​成腺嘌呤和 S -ribosylhomocysteine，和一个甲硫腺苷磷酸，产生腺嘌呤和methylthioribose -1-磷酸23。然而，它不知道小号 -adenosylhomocysteine ​​水解酶和甲硫腺苷磷酸活动是否高到足以有效地代谢小号 -adenosylhomocysteine ​​和甲硫分别使得没有抑制PEMT活性发生。在该实例š-adenosylhomocysteine ​​水解酶和/或5&#39;-甲基硫代谢酶不存在于所关注的细胞，除了各自的纯化的重组酶可被加入到测定，以减轻由SAM降解产物24,25反馈抑制26。 还有在这个协议中四个关键步骤:i）的蛋白酶抑制剂混合物的粉末具有临使用之前加入到裂解缓冲液（步骤1.1）; ii）所述全细胞提取物将要在以下小时内使用的制剂（步骤1.4之后后）; iii）在此脂质提取步骤（步骤4.2和4.4），小心需要施加到不能传输任何相间或水相，其中含有过量的氚标记的SAM的，和iv）对等分放射性的S - [甲基3 H〕腺苷-L-蛋氨酸和在接收冷试剂如冷冻的重复循环和解冻降解成5&#39;-甲基硫腺苷和丝氨酸内酯接着水解腺嘌呤和 S -pentosylmethionine 26,27,28，这可能说明缺乏衡量PEMT活动。坏全细胞提取物也可以是负责没有酶活性。在这种情况下，细胞提取物的质量，可以通过测量另一种已知的酶活性进行评估。最后，缺乏PEMT活动可能依靠PEMT恩ZYME是具体到单-PE或二-PE，而不是PE。加成单甲基PE和/或二甲基PE底物测定的可还原PEMT活性。 另一种选择，非放射性SAM依赖性甲基转移酶测定法已被开发出来，它依赖于高半胱氨酸的酶量化，SAM 29的 transmethylation产品。该协议已成功应用于衡量PE特定的甲基转移酶活性16。不幸的是，该测定是仅适用于纯化的酶，而不是对全细胞提取物，并没有揭示的反应产物的身份任。 本协议可能被应用到任何类型的细胞。另外，该测定可用于测试特定于感兴趣在测试新的抗微生物化合物对抗饮食诱导的动脉粥样硬化的细菌感染或新的治疗的上下文中，PEMT潜在药物，肥胖和胰岛素抵抗。

磷脂甲基（PEMT）酶催化的使用 S -adenosylmethionine（SAM）作为甲基供体上的PE，单甲基PE或二甲基PE，得到单甲基PE，二甲基PE和/或一个或多个甲基的共价连接磷脂酰胆碱（PC）。这些酶是在动物细胞和真菌几乎无处不在。它们也可以在一些植物1和细菌的约10％，尤其是那些与真核生物2交互找到。 PEMTs有关的细胞的生物学不仅通过，而且还通过实现其他重要的细胞功能有助于生产PC的，这是在动物细胞中的主要脂质类。在哺乳动物中，PEMTs主要表达在它们所需的极低密度脂蛋白的分泌正常肝脏和它们还有助于膳食诱导的肥胖3，动脉粥样硬化4，和胰岛素抵抗ANCE 5。此外，哺乳动物PEMT也表达在脂肪细胞，虽然到较低的水平，并且参与脂肪沉积6,7。在癌症的发展8，9凋亡和细胞生长10 PEMT作用也被证明。在细菌中，PEMT酶已被证明是对正常细胞生长2，毒力2，和共生与宿主植物11重要。 本议定书的目标和基本原理是测量从全细胞提取PEMT活动，而不需要净化酶。两个不同的协议已被开发来测量PEMT活性。第一个也是最常见的一种测量含氚甲基基团的放射性SAM转移到PE，这是本文的主题。此协议已被最初开发用于测量从酵母12和哺乳动物细胞（肝）13 PEMT活性以获得一个underst在这些细胞中的PC生物合成安定以及确定这些酶的特异性。以后，这一技术已被应用到其它类型的细胞，如细 ​​菌2（使用用于测定虽然15碱性pH值）和原生动物寄生虫14。这种技术可以用全细胞提取物以及纯化的酶被使用，并且可以潜在地适用于任何细胞提取物的系统。一种非放射性测定法也被设计依赖在 S -adenosylhomocysteine，SAM 16的 transmethylation产物的酶量化。后者测定可能更方便，因为它不涉及放射性但它只适用于纯化的酶。

<table><tbody><tr><td>S-[Methyl-&lt;sup&gt;3&lt;/sup&gt;H]adenosyl-L-methionine (specific activity of 5-15 Ci/mMole)</td><td>Perkin Elmer</td><td>NET155050UC</td><td>Aliquot the reagent and freeze at -20 &amp;deg;C; follow radiation safety guidelines while using this reagent</td></tr><tr><td>Protease inhibitor cocktail</td><td>Roche Life Sciences</td><td>11836170001</td><td>dilute it fresh&amp;nbsp;</td></tr><tr><td>Glass beads, acid washed, 425-600 mm</td><td>Sigma Aldrich</td><td>G8772</td><td></td></tr><tr><td>Bicinchoninic acid solution</td><td>Sigma Aldrich</td><td>B9643</td><td></td></tr><tr><td>Copper (II) sulfate</td><td>Sigma Aldrich</td><td>C2284</td><td></td></tr><tr><td>Scintillation counter MicroBeta2 with 1-detector</td><td>Perkin Elmer&amp;nbsp;</td><td>2450-0010</td><td></td></tr><tr><td>Spectrophotometer Biomate 3</td><td>Thermo Scientific</td><td>840208300</td><td></td></tr><tr><td>BSA stock solution (10 mg/ml)</td><td>New England Biolabs</td><td>B9001S</td><td></td></tr><tr><td>Scintillation liquid&amp;nbsp;</td><td>Research Product International Corp</td><td>111198</td><td></td></tr><tr><td>S-(5&#039;-Adenosyl)-L-methionine chloride (hydrochloride)&amp;nbsp;</td><td>Cayman Chemicals</td><td>13956</td><td>dilute the reagent in 20 mM HCl and freeze aliquots at -80 &amp;deg;C&amp;nbsp;</td></tr></tbody></table>

in vitro assay to measure phosphatidylethanolamine methyltransferase activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

图1示出了时间相关的PEMT测定法，其中，进行与利什曼原虫的全细胞提取物作为使用内源性的PE作为底物的酶的来源。放射性的在有机相中的量进行定量闪烁计数。被利用所得到的数字来计算转印到PE氚化的甲基的量。所述PEMT活性是线性大约20分钟。然后，它达到了一个平台，在大约30分钟，之后将其保持恒定再15分钟。正如所料，未检测PEMT活动时没有细胞提取物加入到测定（ 图 2）。而且，该活动被废除的100μM的十八烷基溴化物的存在，它是L的抑制剂各大厂家PE甲基化LMJ PEM1和LMJ PEM2 14。 PEMT活动还蛋白浓度dependen吨，而此活动成线性比例的蛋白质所申请的酶分析（图3）的量。最后，一个SAM浓度依赖性PEMT进行测定，其中SAM增加浓度进行了测试（图4）。 PEMT活动达到了高原的大约0.5mm的SAM浓度。总之，这四个实验证明PEMT活性是特异性，并且可以从全细胞提取物测量，而不需要净化酶。 <img alt="图1" src="/files/ftp_upload/53302/53302fig1.jpg" /> 图1.时间相关PEMT测定。进行的酶测定两次重复用0.2毫克的全利什曼原虫细胞提取物作为时间的函数。 PEMT活性表示为每蛋白质和p毫克转印到PE纳摩尔甲基呃小时。被加入到含有SAM中测定溶液之前:为时间"O"，细胞提取物先用2m​​l制成氯仿/甲醇（1，1体积）的终止液的混合。示标准偏差。 <img alt="图2" src="/files/ftp_upload/53302/53302fig2.jpg" /> 图2.特异性PEMT活性。的 PEMT测定进行两次重复15分钟在0.05毫米的SAM的存在。 1，0.2 mg蛋白提取物; 2，无细胞提取物; 3，0.2 mg蛋白和0.1mM十八烷基三溴化物。示标准偏差。 <img alt="图3" src="/files/ftp_upload/53302/53302fig3.jpg" /> 图3.蛋白依赖性PEMT测定。于进行两次的酶测定重复在不存在（点"O"）或不同量的利什曼原虫的蛋白的存在下具有0.05毫米的SAM 15分钟。示标准偏差。 <img alt="图4" src="/files/ftp_upload/53302/53302fig4.jpg" /> 图4. PEMT测定进行两次重复用0.2毫克的全利什曼原虫细胞提取物在不同浓度的SAM的存在下进行15分钟。标准偏差一起表示。

Watch this Scientific Journal Video about In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity at JoVE.com

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

体外试验来测量磷脂酰乙醇胺甲基转移酶活

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

in-vitro-assay-to-measure-phosphatidylethanolamine-methyltransferase

Nanyang Technological University

Research

JoVE Journal

Biology

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

9.8K 次观看.  St John's University. The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine. 

视频: In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Defining Substrate Specificities for Lipase and Phospholipase Candidates

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. Alternatively, other (phospho)lipases may be physically associated with the producing organism causing a turnover of intrinsic lipids and frequently giving rise to a remodeling of the cellular membranes. Although potential (phospho)lipases can be predicted with a number of algorithms when the gene/protein sequence is available, experimental proof of the enzyme activities, substrate specificities, and potential physiological functions has frequently not been obtained. This manuscript describes the optimization of assay conditions for prospective (phospho)lipases with unknown substrate specificities and how to employ these optimized conditions in the search for the natural substrate of a respective (phospho)lipase. Using artificial chromogenic substrates, such as p-nitrophenyl derivatives, may help to detect a minor enzymatic activity for a predicted (phospho)lipase under standard conditions. Having encountered such a minor enzymatic activity, the distinct parameters of an enzyme assay can be varied in order to obtain a more efficient hydrolysis of the artificial substrate. After having determined the conditions under which an enzyme works well, a variety of potential natural substrates should be assayed for their degradation, a process that can be followed employing distinct chromatographic methods. The definition of substrate specificities for new enzymes, often provides hypotheses for a potential physiological role of these enzymes, which then can be tested experimentally. Following these guidelines, we were able to identify a phospholipase C (SMc00171) that degrades phosphatidylcholine to phosphocholine and diacylglycerol, in a crucial step for the remodeling of membranes in the bacterium Sinorhizobium meliloti upon phosphorus-limiting conditions of growth. For two predicted patatin-like phospholipases (SMc00930 and SMc01003) of the same organism, we could redefine their substrate specificities and clarify that SMc01003 is a diacylglycerol lipase.

Many predicted (phospho)lipases are poorly characterized with regard to their substrate specificities and physiological functions. Here we provide a protocol to optimize enzyme activities, search for natural substrates, and propose physiological functions for these enzymes.

定义底物特异性的脂肪酶和磷脂酶考生

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

科研

生物化学

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. 1, 2 It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. 2 A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, 3 Dictyostelium discoideum 4,5 and Drosophila. 6 Our recent work has shown that Ehmeth methylates tRNAAsp, and this finding indicates that this enzyme has a dual DNA/tRNAAsp methyltransferase activity. 7 This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. 8 The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

This protocol describes the preparation of a synthetic tRNA substrate for the Entamoeba histolytica DNA/tRNA methyltransferase 2 (Dnmt2) homolog Ehmeth and the measure of its methyltransferase activity. This experimental approach can be used for investigating the activity of other Dnmt2 proteins.

在体外的tRNA与阿米巴 DNA和tRNA甲基Dnmt2（Ehmeth）酶甲基化含量

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

免疫与感染

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNA甲基转移酶活性分析的无抗体测定

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

遗传学

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

阿西尔-PEGyl交换凝胶移位测定，用于脑膜蛋白棕榈酸化定量测定

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

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 ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

用于蛋白质磷酸化定量的优化单分子下拉测定

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

艰难梭菌(p) pppp 合成酶的纯化及体外活性测定

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

测量<em&gt;体外</em&gt;的酶学性质ATP酶活性

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate ...

生物学

Phospholipase Activity Assay: A Gel Diffusion Assay to Determine Phospholipase Activity in Crude Venom Extract from Anthopleura dowii

Source: Ram&#237;rez-Carreto, S., et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/63630">Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays.</a> J. Vis. Exp. (2022)
In this video, we demonstrate an assay to establish the presence of phospholipase in the crude venom extract from Anthopleura dowii&#8212;a marine sea anemone. The assay utilizes an agarose gel diffusion technique, in which the enzyme phospholipase diffuses and breaks down phospholipids incorporated in the solid media in a radial manner.

磷脂酶活性测定:一种凝胶扩散测定法，用于测定 Anthopleura dowii 粗毒液提取物中的磷脂酶活性

Source: Ram&#237;rez-Carreto, S., et al., Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward ...