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

1. Zellextrakt Vorbereitung <ol><li> Wachsen die Leishmania-Zellen in einem sterilen Plastikflasche mit luftdichten Deckel verschlossen bei 26 ºC in einem Medium mit M199 1x machte mit 20 mM HEPES pH 7,4, 100 U / ml Penicillin, 100 ug / ml Streptomycin, 5 ug / ml Häm ergänzt 0,35 g / L NaCO 2 H, 0,1 mM Adenin, und 2 &amp; mgr; g / ml biopterin ohne Schütteln. Ernte der Zellen durch Zentrifugation bei 1.500 g für 5 min bei 4 ºC, wenn sie eine Zelldichte von 1-2 x 10 7 / ml zu erreichen. </li><li> Verwerfen Sie den Überstand mit einer Pipette und waschen Sie die Zellen durch Resuspendieren des Zellpellets mit einer serologischen Pipette in die Hälfte der Kultur Volumen von kaltem phosphatgepufferter Kochsalzlösung (137 mM NaCl, 2,7 mM KCl, 10 mM Na 2 HPO 4, 1,8 mM KH 2 PO 4, pH 7,4). Entsorgen Zellüberständen nach BL2 Sicherheitsrichtlinien. </li><li> Zentrifuge Zellen erneut bei 1500 g für 5 min bei 4 ºC. Überstand verwerfen mit einer Pipette. Gehen to mit dem nächsten Schritt oder Schnapp einfrieren das Zellpellet in flüssigem Stickstoff zur Langzeitlagerung bei -80 ° C (bis zu drei Monaten). </li><li> Bereiten 2x Lysepuffer (0,5 M Sucrose, 0,1 M Tris-HCl, pH 7,5, 2 mM EDTA, und 2x-Protease-Inhibitor-Cocktail) und halten Sie sie bei 4 ° C auf Eis. </li><li> Zellpellet (frisch oder gefroren) in gleichen Volumen 2x Lysepuffer. Hinzufügen 1x Volumen von Glasperlen. Vortex kräftig bei 4 ° C für 10 min. </li><li> In 2 Volumen 1x Lysepuffer und mischen. Centrifuge Zellextrakte bei 1.500 × g bei 4 ° C für 10 min pelletiert ungebrochen Zellen und Kerne. </li><li> Den Überstand mit einer Pipette in ein frisches kühles Zentrifugenröhrchen und bewahren Zellextrakten auf Eis bis zum Abschluss des Experiments. </li></ol> 2. Bestimmen Sie die Proteinkonzentration der Zellextrakt unter Verwendung von Protein Estimation Kit Wie Bicinchoninsäure-Assay <ol><li> Bereiten Sie die Bicinchoninsäure (BCA) (1 ml / Röhrchen) durch Mischen des BCA und Kupfer (II) sulfate in einem Verhältnis von 49: 1 (v / v). </li><li> Vorbereitung der Proteinstandards von 0, 10, 20, 30, 40, 50 und 60 ug / ml durch Verdünnen einer 10 mg / ml Rinderserumalbumin (BSA) Stammlösung in 1 ml-Aliquots der BCA-Lösung. </li><li> Add 2 ul Zellen Extrakte in 1 ml BCA-Lösung in Duplikaten. Standards und Proteinproben inkubieren für 10 Minuten in einem vorgewärmten 60 ° C Wasserbad. </li><li> Transfer Proben für 3 min Eis. Die Extinktion der Standards und der Proteinproben mit einem Spektrophotometer bei einer Wellenlänge von 562 nm. </li><li> Berechnung der Proteinkonzentration der Zellextrakte unter Verwendung der BSA-Standard als Referenz, wie in dem Protokoll des Herstellers beschrieben. Verdünnte Zellextrakten zu einer Proteinkonzentration von 10 mg / ml mit 1x Lysepuffer ml. </li></ol> 3. enzymatischen Assay in 200 ul pro Rohr HINWEIS: Führen Sie die folgenden Schritte in einer chemischen Abzugshaube. <ol><li> Testen Sie jede Probe in zweifacher Ausfertigung in einem 15 mlkonischen Rohr. Es werden 20 &amp; mgr; l 1 M Tris-HCl pH 7,5 pro Röhrchen und bewahren Sie es auf Eis. Vorbereitung wurden 2 ml Chloroform / Methanol (1: 1 (v / v); Stopp-Lösung) bei Raumtemperatur für jedes Röhrchen. </li><li> Pipettieren Sie 20 ul 1 M Tris-HCl pH 7,5, in jeder 15 ml konischen Röhrchen auf Eis. </li><li> Folgen Strahlenschutzrichtlinien von hier an. Füge das Äquivalent von 0,06 um (0,2 uCi) S - [Methyl- 3 H] Adenosyl-L-Methionin und 50 &amp; mgr; M kaltes SAM pro Röhrchen für insgesamt 50,06 uM SAM. Add x ul kaltem Wasser mit x = 200- (20 (für Puffer) + 20 (für die Zellextrakte) + Volumen von kaltem und radioaktivem SAM) pro Röhrchen. </li><li> Übertragen Sie jede konische Röhrchen in eine vorgewärmte 30 ºC Wasserbad. Hinzufügen zu jedem Röhrchen 20 ul Zellextrakte (Äquivalent von 200 ug Protein), um die Reaktion zu starten. Inkubation für die gewünschte Zeit (0-45 min). </li><li> Stoppen der Reaktion durch Zugabe von 2 ml Chloroform / Methanol (1: 1; v / v; Stopplösung) in jedes Röhrchen. Übertragen Sie die konische Röhrchen auf Raumtemperatur (20-256; C). </li></ol> 4. Lipidextraktion HINWEIS: Führen Sie die folgenden Schritte in einer chemischen Abzugshaube. <ol><li> Fügen Sie 700 ul Wasser zu jedem Röhrchen die enzymatische Reaktion enthaltenden Probe. Vortex kräftig für 30 Sekunden. Zentrifuge bei 1500 × g für 5 min bei RT, die organische von der Wasserphase trennen. </li><li> Übertragen die untere organische Phase in ein neues 15 ml konischen Röhrchen mit einer Pipette. 1 ml Wasser in jedes &quot;Unterphase&quot; Rohr und Wirbel enthält kräftig für 30 Sekunden. Zentrifuge wieder bei 1.500 xg für 5 min, um die organische von der Wasserphase zu trennen. </li><li> Übertragen die untere organische Phase wird in einem Scintillationsröhrchen mit einer Pipette. Trockenproben unter einem Strom von N 2. Entsorgen Sie die Wasserphasen, die das nicht-radioaktive eingebaut SAM und die radioaktiven konische Röhrchen nach Strahlungs Richtlinien. </li><li> 2 ml / Röhrchen Szintillationsflüssigkeit. Messen Sie die eingebaute Radioaktivität mit einem Scintillations counter gemäß dem Protokoll des Herstellers und die Verwendung Instruments. </li><li> Berechnung der enzymatischen Aktivität in nmol / mg Protein unter Verwendung der folgenden allgemeinen Gleichung: cpm Wert x 10 3 x [Gesamt (radioaktive und kalt) Konzentration von SAM (mM)] x 5 spezifische Aktivität des radioaktiven SAM (Ci / mmol) x [Konzentration von radioaktivem SAM (mM)] HINWEIS: der obige Protokoll kann für eine SAM-Konzentration abhängige oder einem Protein abhängig PEMT Test geändert werden. SAM konzentrationsabhängigen enzymatischen Assay wird die Zeitkonstante (15 min, die in dem linearen Bereich ist) und verschiedenen Mengen von kalter SAM gehalten, während für Protein abhängigen PEMT Assay SAM-Konzentration werden dem Assay hinzugefügt (wählten wir 0,05 mM) und Zeit konstant (15 min) gehalten wird. Auch der pH-Wert des Puffers kann wie erforderlich, wenn einige PEMT Enzyme haben eine unterschiedliche optimale pH-Wert verändert werden. </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>

Kein Interessenkonflikt erklärt

Diese einfache, schnelle PEMT Assay ermöglicht die Quantifizierung von methylierten Formen von PE, die aus der Übertragung von radioaktiver Methylgruppen von SAM auf PE mit Gesamtzellextrakt als Proteinquelle führt. Es ist schnell, empfindlich, reproduzierbar, und auch für die gereinigten Enzyme 17 geeignet. Monomethyl- oder Dimethyl-PE zu dem Assay zugegeben werden, wenn die Methyltransferase von Interesse spezifisch auf diesen Substraten anstatt PE 12,13,18,19. Wenn gereinigt PEMT Enzym verwe...

Phosphatidylethanolamin methyltransferase (PEMT) Enzyme katalysieren die kovalente Bindung von einem oder mehreren Methylgruppen mit S -adenosylmethionine (SAM) als Methylgruppendonator auf PE, PE oder Monomethyl-dimethyl-PE zu Monomethyl-PE, Dimethyl-PE und / oder geben Phosphatidylcholin (PC). Diese Enzyme sind fast allgegenwärtig in Tierzellen und Pilze. Sie können auch in einigen Anlagen 1 und etwa 10% der Bakterien, insbesondere solche, die mit Eukaryonten 2 interagieren gefunden werden. PEMTs sind nicht nur, indem sie zur Herstellung von PC, die Hauptlipidklasse in tierischen Zellen, aber auch von der Erfüllung anderer wichtigen zellulären Funktionen, die für die Biologie der Zelle. Bei Säugetieren sind PEMTs hauptsächlich in der Leber, wo sie für normale Sekretion von Very Low Density Lipoprotein erforderlich sind, ausgedrückt, und sie auch ernährungsbedingte Fettleibigkeit 3, Atherosklerose 4 beitragen und Insulin Resistance 5. Zusätzlich werden Säugetier PEMT auch in Adipozyten exprimiert, wenn auch in den unteren Ebenen, und beteiligen sich an Fettablagerung 6, 7. PEMT Rolle bei der Krebsentwicklung 8. Apoptose 9, und das Zellwachstum 10 wurden ebenfalls demonstriert. In Bakterien haben PEMT Enzyme gezeigt, wichtig für die normale Zellwachstum 2, Virulenz 2 und Symbiose mit der Wirtspflanze 11 zu sein. Das Ziel und Grundprinzip der vorliegenden Protokolls ist es, PEMU-Aktivität aus Gesamtzellextrakten zu messen, ohne die Notwendigkeit, um das Enzym zu reinigen. Zwei unterschiedliche Protokolle wurden entwickelt, um PEMT Aktivität zu messen. Die erste und häufigste misst die Übertragung von tritiiertem Methylgruppe von radioaktivem SAM auf PE, die das Thema dieses Artikels. Dieses Protokoll wurde ursprünglich entwickelt, um PEMU-Aktivität zu messen aus Hefe 12 und Säugerzellen (Leber) 13, eine underst gewinnenVerknüpfen der PC-Biosynthese in diesen Zellen als auch die Spezifität dieser Enzyme zu bestimmen. Später wurde diese Technik auf andere Zelltypen, wie beispielsweise Bakterien 2 (unter Verwendung eines basischen pH-Wertes für den Assay, obwohl 15) und Protozoenparasiten 14 angewendet. Diese Technik kann mit Gesamtzellextrakten sowie gereinigtes Enzym verwendet werden, und können möglicherweise zu jedem Zellextrakt System angewendet werden. Ein nichtradioaktiver Assay wurde ebenfalls entworfen, das auf die enzymatische Quantifizierung von S -adenosylhomocysteine, der Trans Produkt der SAM 16 stützt. Letztere Assay kann einfacher sein, da es sich nicht um die Radioaktivität, aber es ist nur gereinigte Enzyme geeignet.

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">S-[Methyl-3H]adenosyl-L-methionine (specific activity of 5-15 Ci/mMole)</td>
			<td style="width:107px;">Perkin Elmer</td>
			<td style="width:119px;">NET155050UC</td>
			<td style="width:188px;">Aliquot the reagent and freeze at -20 &#176;C; follow radiation safety guidelines while using this reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Roche Life Sciences</td>
			<td style="width:119px;">11836170001</td>
			<td>dilute it fresh&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed, 425-600 mm</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bicinchoninic acid solution</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">B9643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Copper (II) sulfate</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">C2284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scintillation counter MicroBeta2 with 1-detector</td>
			<td style="width:107px;">Perkin Elmer&#160;</td>
			<td style="width:119px;">2450-0010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectrophotometer Biomate 3</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td>840208300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA stock solution (10 mg/ml)</td>
			<td style="width:107px;">New England Biolabs</td>
			<td style="width:119px;">B9001S</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Scintillation liquid&#160;</td>
			<td style="width:107px;">Research Product International Corp</td>
			<td style="width:119px;">111198</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">S-(5&#39;-Adenosyl)-L-methionine chloride (hydrochloride)&#160;</td>
			<td style="width:107px;">Cayman Chemicals</td>
			<td>13956</td>
			<td style="width:188px;">dilute the reagent in 20 mM HCl and freeze aliquots at -80 &#176;C&#160;</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.

Figur 1 zeigt eine zeitabhängige PEMT Assay, der mit Leishmania Gesamtzellextrakt als Enzymquelle mit endogenen PE als Substrat durchgeführt wurde. Die Menge der Radioaktivität in der organischen Phase wurde durch Szintillationszählung quantifiziert. Die erhaltenen Zahlen wurden genutzt, um die Menge an tritiiertem Methylgruppen auf PE übertragen berechnen. Die PEMT Aktivität war linear für ca. 20 min. Es erreichte dann ein Plateau bei rund 30 min, wonach...

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-Test an Phosphatidylethanolamin Methyltransferase-Aktivität zu messen

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 Aufrufe.  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. 

Serie: In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

A Behavioral Assay, um die Reaktionsfähigkeit der Zebrafisch zu Änderungen in Lichtintensitäten messen

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Forschung

Biologie

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Ein In-vitro- FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Proteinmembran Overlay Assay: Ein Protokoll, die Interaktion zwischen löslichen und unlöslichen Proteinen testen In-vitro-</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Ein Matrigel-Based Rohr Formation Assay zur vaskulären Aktivität von Tumorzellen Beurteilen

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Untersuchen der Kinase-Aktivität von LRRK2 In-vitro-</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In-vitro-Test zur Methylierung Protein-Arginin-Methylierung Studieren

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Quantitative Immunfluoreszenz-Assay, um die Variation der Protein-Ebene an Zentrosomen Messen

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Comet Assay als ein indirektes Maß für Systemische Oxidativer Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

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.

Messung<em&gt; In Vitro</em&gt; ATPase Aktivität für die enzymatische Charakterisierung

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

In vitro-Test an Phosphatidylethanolamin Methyltransferase-Aktivität zu messen

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

A Behavioral Assay, um die Reaktionsfähigkeit der Zebrafisch zu Änderungen in Lichtintensitäten messen

Ein In-vitro- FluoroBlok Tumor Invasion Assay

Proteinmembran Overlay Assay: Ein Protokoll, die Interaktion zwischen löslichen und unlöslichen Proteinen testen In-vitro-

Ein Matrigel-Based Rohr Formation Assay zur vaskulären Aktivität von Tumorzellen Beurteilen

Untersuchen der Kinase-Aktivität von LRRK2 In-vitro-

Isolierung von Ribosom gebunden Nascent Polypeptide

In-vitro-Test zur Methylierung Protein-Arginin-Methylierung Studieren

Quantitative Immunfluoreszenz-Assay, um die Variation der Protein-Ebene an Zentrosomen Messen

Comet Assay als ein indirektes Maß für Systemische Oxidativer Stress

Messung