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

1. cella estratto Preparazione <ol><li> Crescere le cellule Leishmania in una bottiglia di plastica sterile, sigillato con tappo a tenuta d&#39;aria a 26 ° C in un mezzo fatto di 1x M199 supplementato con 20 mM HEPES pH7.4, 100 U / ml di penicillina, 100 mg / ml di streptomicina, 5 mg / ml eme , 0,35 g / L Naco 2 H, adenina mM 0.1, e 2 mg / ml biopterina senza agitare. Raccogliere le cellule per centrifugazione a 1.500 g per 5 minuti a 4 ° C quando raggiungono una densità cellulare di 1-2 x 10 7 / ml. </li><li> Eliminare il surnatante con una pipetta e lavare le cellule risospendendo il pellet cellulare con una pipetta sierologica a metà del volume del cultura del fosfato salino freddo tamponata (137 mM NaCl, 2.7 mM KCl, Na 10 mm 2 HPO 4, 1.8 KH mM 2 PO 4, pH7.4). Smaltire surnatanti cellulari secondo le linee guida di sicurezza BL2. </li><li> Centrifugare le cellule di nuovo a 1.500 g per 5 minuti a 4 ° C. Eliminare il surnatante con una pipetta. Procedere to il prossimo passo elastico, congelare il pellet di cellule in azoto liquido per la conservazione a lungo termine a -80 ° C (fino a tre mesi). </li><li> Preparare 2x tampone di lisi (0,5 M di saccarosio, 0,1 M TrisHCl, pH7.5, 2 mM EDTA, e inibitore della proteasi 2x cocktail) e tenerlo a 4 ° C su ghiaccio. </li><li> Risospendere il pellet cellulare (fresco o congelato) in volume uguale di tampone di lisi 2x. Aggiungere il volume 1x di perline di vetro. Vortex vigorosamente a 4 ° C per 10 min. </li><li> Aggiungere 2 volumi di tampone di lisi 1x e mescolare. Estratti cellulari centrifugare a 1500 xg a 4 ° C per 10 minuti a pellet cellule intatte e nuclei. </li><li> Trasferire il surnatante con una pipetta in una provetta da centrifuga fresca aperta e mantenerlo estratti cellulari in ghiaccio fino al completamento dell&#39;esperimento. </li></ol> 2. Determinare la concentrazione della proteina del estratto cellulare utilizzando proteine ​​Stima Kit Come bicinconinico Acid Assay <ol><li> Preparare la soluzione di acido bicinconinico (BCA) (1 ml / tubo) mescolando il BCA e rame (II) sulfate in un rapporto di 49: 1 (v / v). </li><li> Preparare gli standard proteici di 0, 10, 20, 30, 40, 50, e 60 ug / ml diluendo un / ml albumina di siero bovino (BSA) soluzione madre 10 mg in 1 ml aliquote della soluzione BCA. </li><li> Aggiungere 2 ml di cellule estratti in 1 ml di soluzione di BCA in duplicato. Incubare standard e campioni di proteine ​​per 10 minuti in un bagno d&#39;acqua a 60 ° C pre-riscaldato. </li><li> Campioni di trasferimento di ghiaccio per 3 min. Misurare l&#39;assorbanza degli standard e dei campioni di proteine ​​con uno spettrofotometro ad una lunghezza d&#39;onda di 562 nm. </li><li> Calcolare la concentrazione proteica degli estratti cellulari utilizzando lo standard BSA come riferimento, come descritto nel protocollo del produttore. Diluire estratti cellulari ad una concentrazione proteica di 10 mg / ml con tampone di lisi 1x. </li></ol> 3. enzimatica Assay in 200 pl per Tubo NOTA: Eseguire le seguenti operazioni in una cappa chimica. <ol><li> Testare ciascun campione in duplicato in 15 mltubo conico. Preparare 20 ml 1 M TrisHCl pH 7,5 per tubo e tenerlo in ghiaccio. Preparare 2 ml di cloroformio / metanolo (1: 1 (v / v); soluzione di stop) a temperatura ambiente per ogni provetta. </li><li> Pipettare 20 ml di 1 M pH 7,5 TrisHCl in ciascun tubo da 15 ml su ghiaccio. </li><li> Seguire le linee guida di sicurezza di radiazioni da qui in poi. Aggiungere l&#39;equivalente di 0,06 mM (0,2 pCi) S - [Methyl- 3 H] adenosil-L-metionina e 50 mM freddo SAM per provetta per un totale di 50,06 mM di SAM. Aggiungi x ml di acqua fredda dove x = 200- (20 (per il buffer) + 20 (per estratti cellulari) + volume di freddo e radioattivi SAM) per provetta. </li><li> Trasferire ogni tubo conico a 30 ° C bagno di acqua preriscaldata. Aggiungere a ciascuna provetta 20 l di estratti cellulari (pari a 200 mg di proteine) per attivare la reazione. Incubare per il tempo desiderato (da 0 a 45 min). </li><li> Arrestare la reazione aggiungendo 2 ml di cloroformio / metanolo (1: 1; v / v; soluzione di arresto) ad ogni provetta. Trasferire il tubo conico di RT (20-256; C). </li></ol> 4. lipidi Estrazione NOTA: Eseguire le seguenti operazioni in una cappa chimica. <ol><li> Aggiungere 700 ml di acqua a ciascuna provetta contenente il campione reazione enzimatica. Vortex energicamente per 30 sec. Centrifugare a 1500 xg per 5 minuti a RT per separare il organica dalla fase acquosa. </li><li> Trasferire la fase organica inferiore in un nuovo tubo da 15 ml con una pipetta. Aggiungere 1 ml di acqua per ogni &quot;fase inferiore&quot; contenente provetta e agitare vigorosamente per 30 secondi. Centrifugare nuovamente a 1.500 xg per 5 min per separare il organica dalla fase acquosa. </li><li> Trasferire la fase organica inferiore in un tubo scintillazione con una pipetta. Campioni secco sotto flusso di N 2. Smaltire le fasi d&#39;acqua contenenti la SAM radioattivo non incorporato e tubi coniche radioattive come da linee guida di radiazione. </li><li> Aggiungere 2 ml / tubo di scintillazione liquida. Misurare la radioattività incorporata con un paio di scintillazionenter secondo il protocollo dei costruttori e l&#39;uso dello strumento. </li><li> Calcolare l&#39;attività enzimatica in nmol / mg proteina usando la seguente equazione generale: valore cpm x 10 x 3 [totale (radioattivi e freddo) concentrazione di SAM (mM)] x 5 attività specifica di SAM radioattivi (Ci / mmol) x [concentrazione di SAM radioattivi (mM)] NOTA: il protocollo di cui sopra può essere modificato per una concentrazione SAM dipendente o una proteina dipendente test PEMT. Per concentrazione SAM saggio enzimatico dipendente, il tempo viene mantenuta costante (15 min, che è nel range lineare) e varie quantità di freddo SAM vengono aggiunti al dosaggio mentre per proteina dipendente dosaggio PEMT, concentrazione SAM (abbiamo scelto 0,05 mM) e tempo sono mantenuto costante (15 min). Anche il pH del tampone può essere cambiata come necessario se alcuni enzimi PEMT hanno un diverso valore ottimale del pH. </li></ol>

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	<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>

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Questa semplice, rapido test PEMT consente la quantificazione di forme metilate di PE che deriva dal trasferimento di gruppi metilici radioattive da SAM su PE utilizzando estratto cellulare tutto come fonte proteica. È veloce, sensibile, riproducibile e adatto anche per enzimi purificati 17. Monomethyl- o dimetil-PE possono essere aggiunti al test se il metiltransferasi di interesse specifico per questi substrati anziché PE 12,13,18,19. Se viene utilizzato purificato enzima PEMT, PE può essere a...

Fosfatidiletanolamina metiltransferasi (PEMT) enzimi catalizzano l&#39;attacco covalente di uno o più gruppi metilici utilizzando S -adenosylmethionine (SAM) come il gruppo metilico donatore sul PE, PE o monometil-dimetil-PE dare monometil-PE, dimetil-PE e / o fosfatidilcolina (PC). Questi enzimi sono quasi onnipresenti nelle cellule animali e funghi. Possono anche essere trovati in alcune piante 1 e circa il 10% dei batteri, in particolare quelli che interagiscono con eucarioti 2. PEMTs sono rilevanti alla biologia della cellula non solo contribuendo alla produzione di PC, che è la classe principale lipidi nelle cellule animali, ma anche compiendo altre importanti funzioni cellulari. Nei mammiferi, PEMTs sono espressi principalmente nel fegato dove sono richiesti per il normale secrezione di lipoproteine ​​a bassissima densità e contribuiscono anche all&#39;obesità indotta dalla dieta 3, aterosclerosi 4, e l&#39;insulina resistonoANCE 5. Inoltre, PEMT mammiferi sono espressi anche negli adipociti, anche se a livelli più bassi, e partecipare a deposizione di grasso 6, 7. Ruolo PEMT nello sviluppo del cancro 8, 9 apoptosi, e la crescita delle cellule 10 sono stati anche dimostrato. Nei batteri, enzimi PEMT hanno dimostrato di essere importante per la normale crescita delle cellule 2, la virulenza 2, e simbiosi con la pianta ospite 11. L&#39;obiettivo e le motivazioni del presente protocollo è quello di misurare l&#39;attività PEMT da estratti cellulari interi, senza la necessità di purificare l&#39;enzima. Due protocolli distinti sono stati sviluppati per misurare l&#39;attività PEMT. Il primo e più comune misura il trasferimento del gruppo metilico triziata da SAM radioattivo su PE, che è l&#39;argomento di questo articolo. Questo protocollo è stato originariamente sviluppato per misurare l&#39;attività PEMT dal lievito 12 e cellule di mammifero (fegato) 13 per ottenere un understANDing di biosintesi PC in queste cellule, nonché per determinare la specificità di questi enzimi. Successivamente, questa tecnica è stata applicata ad altri tipi di cellule, come i batteri 2 (utilizzando un valore pH basico per il test se 15) e protozoi parassiti 14. Questa tecnica può essere utilizzata con estratti cellulari interi nonché enzima purificato, e può potenzialmente essere applicato a qualsiasi sistema di estratto cellulare. Un test non radioattivo è stato progettato che si basa sulla quantificazione enzimatica di S -adenosylhomocysteine, il prodotto di transmetilazione SAM 16. Quest&#39;ultimo test può essere più conveniente in quanto non comporta radioattività ma è adatto solo per gli enzimi purificati.

<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.

La figura 1 mostra un tempo dipendente PEMT dosaggio, che è stata effettuata con Leishmania estratto cellula intera come fonte dell&#39;enzima utilizzando PE endogena come substrato. La quantità di radioattività nella fase organica è stata quantificata mediante conteggio a scintillazione. I numeri ottenuti sono stati utilizzati per calcolare la quantità di gruppi metilici triziata trasferiti verso PE. L&#39;attività PEMT era lineare per circa 20 min. È qu...

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.

In vitro misurare phosphatidylethanolamine metiltransferasi attività

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

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

Research

JoVE Journal

Biology

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

9.8K Views.  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. 

Video: 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.

La definizione del substrato Specificità per lipasi e fosfolipasi candidati

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

Ricerca

Biochimica

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.

Test in vitro Metilazione tRNA con l&#39;Entamoeba histolytica del DNA e Dnmt2 tRNA metiltransferasi (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 ...

Immunologia e infezioni

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.

Analisi anticorp-free per l'analisi dell'attività di RNA methyltransferase

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

Genetica

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.

Acyl-PEGyl Exchange Gel Shift Analisi per la Determinazione Quantitativa della Palmitoylation delle Proteine della Membrana Cerebrale

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.

Saggio fluorometrico basato sulla chimica dei clic per l'apolipoproteina N-aciltransferasi dalla caratterizzazione enzimatica allo screening ad alta produttività

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;

Saggio di metilazione del DNA accoppiato a endonucleasi a fluorescenza continua per lo screening degli inibitori della DNA metiltransferasi

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.

Un test di pull-down a singola molecola ottimizzato per la quantificazione della fosforilazione delle proteine

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.

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

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.

misurazione<em&gt; In Vitro</em&gt; Attività ATPasi per enzimatica Caratterizzazione

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

Biologia

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/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.

Saggio dell'attività della fosfolipasi: un test di diffusione su gel per determinare l'attività della fosfolipasi nell'estratto di veleno grezzo da Anthopleura dowii

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

In vitro misurare phosphatidylethanolamine metiltransferasi attività

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