Name: in vitro assay to measure phosphatidylethanolamine methyltransferase activity
Uploaded: 2016-01-05T13:00:01.000+00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity at JoVE.com

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

1. Hücre Özüt Hazırlama <ol><li> 20 mM HEPES pH7.4, 100 U / ml penisilin / ml streptomisin, 5 ug / ml heme 100 ug ile takviye edilmiş M199 1 x yapılmış bir ortam içinde 26 ° C &#39;de hava geçirmez kapak ile sızdırmaz steril plastik şişe Leishmania hücreleri büyümek çalkalamadan, 0.35 g / L NACO 2H, 0.1 mM adenin, ve 2 ug / ml biopterin. Bunlar 1-2 x 10 7 / ml&#39;lik bir hücre yoğunluğuna ulaştığında 4 ° C &#39;de 5 dakika süre ile 1500 g&#39;de santrifüje edilerek hücreler hasat. </li><li> Bir pipet ile süpernatant atılır ve soğuk fosfat tamponlu serum fizyolojik kültürün hacmi (137 mM NaCI, 2.7 mM KCI yarısı bir serolojik pipet hücre topağın tekrar askıda bırakılmasıyla yıkama hücreleri, 10 mM Na-2 HPO 4, 1.8 mM KH 2 PO 4, pH 7.4). BL2 güvenlik kurallarına göre hücre süpernatantlar bertaraf edin. </li><li> Hücrelerin tekrar santrifüj 4 ° C&#39;de 5 dakika boyunca 1500 g&#39;de. Bir pipet ile süpernatant atın. T Devamya da bir sonraki adıma o -80 ºC (üç ay) uzun süreli saklama için sıvı nitrojen içinde hücre pelletini dondurmak oturtun. </li><li> 2x lizis tamponu (0.5 M sukroz, 0.1 M TrisHCl, pH 7.5, 2 mM EDTA ve 2 x proteaz inhibitör kokteyli) hazırlayın ve buz üzerinde 4 ° C&#39;de tutun. </li><li> 2x lizis tamponu eşit hacimde (taze veya dondurulmuş) hücre pelletini. Cam boncuklar 1x hacmi ekleyin. Vorteks kuvvetli bir şekilde 10 dakika boyunca 4 ° C&#39;de. </li><li> 1x liziz tamponu 2 hacim ekleyin ve karıştırın. 10 dakika boyunca 4 ° C&#39;de 1500 x g&#39;de santrifüjleyin hücre ekstreleri kesintisiz hücre ve çekirdeklerin: pelet. </li><li> Bir taze soğuk bir santrifüj tüpü içine pipetle aktarın ve süpernatant deney tamamlanana kadar buz üzerinde hücre ekstreleri tutun. </li></ol> 2. Bu tür Bikinkoninik Asit Testi Protein Tahmin Kiti Kullanımı Cep Özü Protein Konsantrasyon belirleyin <ol><li> Bisinkoninik asit hazırlanması (BCA) çözeltisi BCA ve bakır (II) karıştırılması ile (1 mL / tüp) s49 oranında ulfate: 1 (v / v). </li><li> Protein standartlarını hazırlayın 0, 10, 20, 30, 40, 50 ve BCA solüsyonun 1 ml&#39;lik alikolar içinde bir 10 mg / ml bovin serum albümini (BSA) stok solüsyonu seyreltilerek 60 ug / ml olmuştur. </li><li> Tekrarlardaki BCA çözeltisi 1 ml hücre ekstrelerinin 2 ul ekleyin. Önceden ısıtılmış 60 ° C su banyosu içinde 10 dakika için standartlar ve protein örnekleri inkübe edin. </li><li> Aktarım numuneler 3 dakika boyunca buz. 562 nm bir dalga boyunda bir spektrofotometre ile standart ve protein örnekleri absorbansı ölçülür. </li><li> Üreticinin protokolünde tarif edildiği gibi, bir referans olarak BSA standardı kullanılarak hücre ekstrelerinin protein konsantrasyonu hesaplanır. 1x lizis tamponu ile mi, 10 mg / bir protein konsantrasyonuna hücre ekstreleri seyreltilir. </li></ol> Tüp başına 200ul 3. Enzimatik Deneyi NOT: Bir kimyasal kaputu aşağıdaki adımları uygulayın. <ol><li> 15 ml nüsha halinde her bir numune test edinkonik tüp. Tüp başına 20 ul 1 M TrisHCl pH 7.5 hazırlayın ve buz üzerinde tutmak. Her bir tüp, oda sıcaklığında, (çözelti durdurma 1 (h / h) 1) kloroform / metanol 2 ml hazırlayın. </li><li> Pipet, buz üzerinde, her 15 ml konik bir tüp içinde 1 M TrisHCl pH 7.5, 20 ul. </li><li> Burada radyasyon güvenlik kurallarına uyun. SAM 50,06 uM toplam tüp başına [metil- 3 H] adenosil-L-metionin ve 50 uM soğuk SAM - 0.06 uM (0.2 uCi), S eşdeğer ekleyin. Soğuk su ul x ekle x = 200- (20 (+ hücre ekstreleri için 20 () + soğuk ve radyoaktif SAM hacmi) tamponu için) tüp başına. </li><li> Önceden ısıtılmış 30 ºC su banyosunda her konik tüp aktarın. Reaksiyonu başlatmak için her bir tüpe, hücre ekstreleri (proteinin 200 ug eşdeğer) 20 ul ekle. İstediğiniz zaman (0-45 dakika) inkübe edin. </li><li> Kloroform / metanol 2 ml ilave ederek reaksiyonu durdurun (1: 1; v / v; durdurma çözeltisi) her tüpe. (Oda sıcaklığına 20-25 konik tüp transfer6 ° C). </li></ol> 4. Lipid Ekstraksiyon NOT: Bir kimyasal kaputu aşağıdaki adımları uygulayın. <ol><li> Enzimatik reaksiyon örneğini içeren her tüpe su 700 ul ekle. Kuvvetlice 30 saniye boyunca girdap. Oda sıcaklığında 5 dakika boyunca 1500 x g&#39;de santrifüjleyin su fazının organik ayırmak için. </li><li> Bir pipet yardımıyla yeni bir 15 ml konik tüp içine alt organik fazı aktarın. 30 saniye boyunca kuvvetli bir şekilde boru ve vorteks içeren her &quot;düşük değerli faz&quot; su 1 ml ilave edilir. Tekrar santrifüj 5 dakika boyunca 1500 x g&#39;de su fazının organik ayırmak için. </li><li> Bir pipet, bir sintilasyon tüpüne alt organik faz transfer edin. N2 akışı altında kuru örnekleri. Olmayan anonim radyoaktif SAM ve radyasyon kurallarına göre radyoaktif konik tüpler içeren su fazları bertaraf edin. </li><li> 2 ml / sintilasyon sıvısı tüp ekleyin. Bir sintilasyon cou ile radyoaktiviteyi ölçünnter üreticilerin protokol ve enstrümanın kullanıma göre. </li><li> Aşağıdaki genel denklemi kullanarak nmol / mg protein enzimatik aktivite hesaplanır cpm x değeri 10 3 x [SAM (mM) &#39;nin toplam (radyoaktif ve soğuk) konsantrasyonu], radyoaktif SAM (Ci / mmol) x x 5 spesifik aktivitesi [radyoaktif SAM konsantrasyonu (mM)] Not: Yukarıdaki protokole bağlı SAM konsantrasyonu ya da bir protein bağlı PEMT tahlili için değiştirilebilir. SAM, konsantrasyona bağlı enzimatik deney için, zaman ise, protein bağlı PEMT tahlilinde, SAM konsantrasyonu için tahlil eklenir sabit (lineer aralığında 15 dakika) ve soğuk SAM çeşitli miktarlarda tutulur (biz mM 0.05 seçtik) ve zaman sabiti (15 dakika) tutulur. Ayrıca tampon çözeltisinin pH değeri, bazı PEMT enzimler farklı optimal pH değerine sahip olması gerektiği gibi değiştirilebilir. </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>

Herhangi bir çıkar çatışması ilan

Bu basit, pratik PEMT tahlil, bir protein kaynağı olarak bütün hücre ekstresi kullanarak PE üzerine SAM radyoaktif metil gruplarının transferi sonucu PE metillenmiş formlarının ölçümü sağlar. Bu, hızlı, hassas ve tekrarlanabilir ve saflaştırılmış enzimler 17 için de uygundur. Ilgi metiltransferaz yerine PE 12,13,18,19 daha Bu yüzeylerde özgü olup olmadığını monometil veya dimetil-PE tahlilinde eklenebilir. Saflaştınldı PEMT enzim kullanılırsa, PE tahlilinde eklen...

Fosfatidiletanolamin metiltransferaz (PEMT) PEMT enzimler monometil-PE, dimetil-PE ve / veya vermek için PE, monometil-PE veya dimetil-PE üzerine metil grubu donörü S -adenosylmethionine (SAM) ile bir veya daha fazla metil grubu kovalent bağlanmasını katalize eden bir fosfatidilkolin (PC). Bu enzimler, hayvan hücreleri ve mantarlar hemen hemen her zaman görülmektedir. Ayrıca, bazı bitkilerin 1 ve bakterilerin yaklaşık% 10, ökaryotlarda 2 ile etkileşim özellikle bulunabilir. PEMTs değil, aynı zamanda diğer önemli hücresel fonksiyonları yerine getirerek hayvan hücrelerinde ana lipid sınıf PC, üretimine katkıda bulunarak hücre biyolojisine alakalı. Memelilerde, PEMTs esas olarak çok düşük yoğunluklu lipoprotein normalin üzerinde bir salgılama için gerekli olan karaciğer olarak ifade edilmiştir ve ayrıca diyet indükte obeziteye 3, ateroskleroz 4 katkıda bulunur ve insülin karşıance 5. Buna ek olarak, bir memeli PEMT da her ne kadar düşük seviyelere, adipositler ile ifade edilmiştir ve yağ depolanması 6, 7 katılırlar. Kanser gelişimi 8, 9 apoptoz ve hücre büyümesi 10&#39;da PEMT rolü de ortaya konmuştur. Bakterilerde PEMT enzimler ana bitki 11, normal hücre çoğalması 2, virülans 2 ve simbiyoz için önemli olduğu gösterilmiştir. Mevcut protokolün amacı ve gerekçesi enzimi arındırmak gerek kalmadan bütün hücre ekstrelerinden PEMT aktivitesini ölçmektir. İki farklı protokoller PEMT aktivitesini ölçmek için, geliştirilmiştir. İlk ve en yaygın olanı, bu makalenin konusu olan PE, üzerine radyoaktif SAM trityumlu metil grubunun transferini ölçer. Bu protokol, ilk olarak bir underst elde etmek için maya 12 ve memeli hücrelerinde (karaciğer) 13 den PEMT aktivitesini ölçmek için, geliştirilmiştirBu hücrelerde PC biyosentezinin anding yanı sıra, bu enzim özgüllüğünü belirlemek için. Daha sonra, bu teknik ve tek hücreli parazitlerin 14 (15 olsa deneyi için bir bazik pH değeri kullanılarak) bu tür bakteri 2 gibi diğer hücre tiplerinde uygulanmıştır. Bu teknik, bütün hücre hülasaları hem de saflaştırılmış enzim ile birlikte kullanılabilir ve potansiyel olarak herhangi bir hücre ekstresi sistemine uygulanabilir. Olmayan bir radyoaktif deneyi de S -adenosylhomocysteine, SAM 16 metillenmenin ürünün enzimatik miktarının dayandığı dizayn edilmiştir. Bu radyoaktivite içermeyen olarak ikinci deney daha uygun olabilir ancak saflaştınldı enzimler için uygundur.

<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.

Şekil 1, bir alt-tabaka olarak endojen PE kullanılarak, bir enzim kaynağı olarak Leishmania bütün hücre ekstresi ile gerçekleştirildiği zamana bağlı PEMT deneyi göstermektedir. Organik faz içinde radyoaktivite miktarı, sintilasyon sayımı ile nicelendirildi. Elde edilen PE numaraları üzerine transfer tritiye metil gruplarının miktarını hesaplamak için kullanıldı. PEMT aktivitesi yaklaşık 20 dakika boyunca doğrusal olmuştur. Daha sonr...

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.

Vitro Tahlil Fosfatidiletanolamin Metiltransferaz Aktivite Tedbir

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 Görüntüleme.  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.

Lipaz ve Fosfolipaz Adayları için substrat özellikleri tanımlama

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

Biyokimya

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.

Entamoeba histolytica DNA ve tRNA Metiltransferaz Dnmt2 (Ehmeth) Enzim ile in vitro tRNA Metilasyon Testi

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

İmmünoloji ve Enfeksiyon

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

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

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

RNA metiltransferaz aktivite analizi için Antibody Içermeyen tahlil

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

Genetik

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.

Beyin Zarı Proteinlerinin Palmitoylarasyonunun Kantitatif Tayini için Acyl-PEGyl Exchange Jel Shift Tayini

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.

Enzim Karakterizasyonundan Yüksek Verimli Taramaya Kadar Apolipoprotein N-asiltransferaz için Click-Kimya Tabanlı Florometrik Analiz

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;

DNA Metiltransferaz İnhibitörlerini Taramak için Sürekli Floresan Bazlı Endonükleaz Bağlantılı DNA Metilasyon Testi

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.

Protein Fosforilasyonunun Miktarının Belirlenmesi için Optimize Edilmiş Tek Moleküllü Bir Pull-Down Testi

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.

Bir arınma ve In Vitro etkinliği tahlil için (p) ppGpp sentetaz Clostridium difficile üzerinden

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.

ölçme<em&gt; İn Vitro</emEnzimatik Karakterizasyonu için&gt; ATPaz Aktivitesi

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

Biyoloji

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.

Fosfolipaz Aktivite Testi: Anthopleura dowii'den Ham Venom Ekstraktında Fosfolipaz Aktivitesini Belirlemek İçin Bir Jel Difüzyon Testi

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