Vitro Tahlil Fosfatidiletanolamin Metiltransferaz Aktivite Tedbir
Summary
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.
Abstract
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.
Introduction
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'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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grants ARRA RO3 AI078145 and 1SC3GM113743 to RZ.
Materials
| S-[Methyl-3H]adenosyl-L-methionine (specific activity of 5-15 Ci/mMole) | Perkin Elmer | NET155050UC | Aliquot the reagent and freeze at -20 °C; follow radiation safety guidelines while using this reagent |
| Protease inhibitor cocktail | Roche Life Sciences | 11836170001 | dilute it fresh |
| Glass beads, acid washed, 425-600 mm | Sigma Aldrich | G8772 | |
| Bicinchoninic acid solution | Sigma Aldrich | B9643 | |
| Copper (II) sulfate | Sigma Aldrich | C2284 | |
| Scintillation counter MicroBeta2 with 1-detector | Perkin Elmer | 2450-0010 | |
| Spectrophotometer Biomate 3 | Thermo Scientific | 840208300 | |
| BSA stock solution (10 mg/ml) | New England Biolabs | B9001S | |
| Scintillation liquid | Research Product International Corp | 111198 | |
| S-(5'-Adenosyl)-L-methionine chloride (hydrochloride) | Cayman Chemicals | 13956 | dilute the reagent in 20 mM HCl and freeze aliquots at -80 °C |
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