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. 세포 추출물 준비 <ol><li> 20 mM의 HEPES pH 7.4로, 100 U는 / ㎖ 페니실린, / ㎖ 스트렙토 마이신, 5 μg의 / ㎖ 헴 (heme) 100 μg의 보충 1X M199 만든 배지에서 26 ºC에서 기밀 캡으로 밀봉 멸균 플라스틱 병에 슈만 편모충 세포를 성장 흔들림없이, 0.35 G / L 나코 2 H, 0.1 mM의 아데닌, 2 μg의 / ㎖ biopterin. 그들은 1-2 X 107 / ml의 세포 밀도에 도달했을 때 4 ºC에서 5 분간 1,500g에서 원심 분리하여 세포를 수확. </li><li> 피펫으로 뜨는을 취소하고 차가운 인산염 완충 식염수의 문화의 볼륨 (137 mM의 염화나트륨, 2.7 밀리미터의 KCl의 절반에 혈청 학적 피펫으로 세포 펠렛을 재현 탁하여 세포를 씻어, 10 mM의 나 2 HPO 4, 1.8 mM의 KH 2 PO 4, pH 7.4로). BL2 안전 지침에 따라 세포의 상층 액을 폐기하십시오. </li><li> 원심 분리기 세포를 다시 4 ºC에서 5 분 1,500g에서. 피펫으로 상층 액을 버린다. T를 진행또는 다음 공정 o를하면 -80 ºC (최대 세 개월)에 장기 저장을 위해 액체 질소에서 세포 펠렛을 동결 스냅. </li><li> 배 용해 버퍼 (0.5 M 자당, 0.1 M TrisHCl, pH7.5, 2 mM의 EDTA 및 배 프로테아제 억제제 칵테일)를 준비하고 얼음에 4 ºC에서 보관하십시오. </li><li> 배속 용해 완충액의 동일 부피 (신선하거나 냉동) 세포 펠릿을 재현 탁. 유리 구슬의 1X 볼륨을 추가합니다. 소용돌이 적극적으로 10 분 동안 4 ºC에서. </li><li> 1X 용해 버퍼의 2 볼륨을 추가하고 혼합한다. 10 분 동안 4 ºC에서 1,500 XG에 원심 분리기 세포 추출물은 끊어지지 세포와 핵 펠렛합니다. </li><li> 신선한 시원한 원심 분리기 튜브에 피펫으로 뜨는을 전송하고 실험이 완료 될 때까지 얼음에 세포 추출물을 유지합니다. </li></ol> 2. 이러한 Bicinchoninic 산 분석으로 단백질 평가 키트를 사용하여 세포 추출물의 단백질 농도를 결정 <ol><li> bicinchoninic 산을 준비 (BCA) 솔루션 (BCA)과 구리 (II)를 혼합하여 (1 ㎖ / 튜브)의(49)의 비율 ulfate : 1 (V / V). </li><li> 단백질 표준을 제조 0, 10, 20, 30, 40, 50, 및 BCA 용액 1 ml의 분취 량으로 10 ㎎ / ㎖ 소 혈청 알부민 (BSA) 원액을 희석하여 60 ㎍ / mL로. </li><li> 중복에 BCA 용액 1 ml의 세포 추출물의 2 μl를 추가합니다. 미리 예열 60 ℃ 수조에서 10 분 동안 표준 및 단백질 시료를 배양한다. </li><li> 전송 샘플은 3 분 동안 얼음. 562 nm의 파장에서 분광 광도계 표준 및 단백질 샘플의 흡광도를 측정한다. </li><li> 제조사의 프로토콜에 기술 된 바와 같이 참조로서 BSA 표준을 사용하여 세포 추출물의 단백질 농도를 계산한다. 1X 용해 완충 용액과 10 밀리그램 / 단백질의 농도로 세포 추출물을 희석. </li></ol> 튜브에 200 μL 3. 효소 분석 참고 : 화학 후드에서 다음 단계를 수행하십시오. <ol><li> 15 ml의 중복 각 샘플을 테스트원뿔 튜브. 튜브 당 20 μL (1) M TrisHCl pH가 7.5을 준비하고 얼음에 보관하십시오. 각각의 튜브에 대한 실온에서; (솔루션을 정지 1 (V / V) 1) 클로로포름 / 메탄올 2 ㎖를 준비합니다. </li><li> 피펫 얼음에 각 15 ML 원뿔 튜브에 1 M TrisHCl의 pH 7.5의 20 μL. </li><li> 여기에에서 방사선 안전 지침을 따르십시오. SAM의 50.06 μm의 총 튜브 당 [메틸 3 H] 아데노-L 메티오닌 50 μm의 차가운 SAM - 0.06 μM (0.2 μCi를) S에 해당하는 추가. 찬물 μL 추가 x 여기서 x = 200 - ((20) (+ 세포 추출물 20 () + 추위와 방사성 SAM의 볼륨) 버퍼) 튜브 당. </li><li> 미리 예열 30 ºC 물을 욕조에 각 원뿔 튜브를 전송합니다. 반응을 시작하기 위해 각 튜브에 세포 추출물 (단백질 200 μg의 동일)의 20 μl를 추가합니다. 원하는 시간 (0-45 분)에 대한 품어. </li><li> 클로로포름 / 메탄올 2 ㎖ 첨가하여 반응을 정지 (1 : 1, V / V, 정지 용액) 각각의 튜브를. (실온 20 ~ 25을 원뿔 튜브로 이동6; C). </li></ol> 4. 지질 추출 참고 : 화학 후드에서 다음 단계를 수행하십시오. <ol><li> 효소 반응 샘플을 포함하는 각 튜브에 물을 700 μl를 추가합니다. 적극적으로 30 초 동안 소용돌이. RT에서 5 분 동안 1,500 XG 원심 분리기는 물, 유기 상으로부터 분리한다. </li><li> 피펫 새로운 15 ML 원뿔 튜브에 낮은 유기 상을 전송합니다. 30 초 동안 적극적으로 튜브와 소용돌이를 포함하는 각각의 &quot;낮은 단계&quot;에 물 1 ML을 추가합니다. 다시 원심 분리기에서 5 분 동안 1,500 XG에서 수상으로부터 유기물을 분리. </li><li> 피펫 섬광 관에 낮은 유기 상을 전송합니다. N 2의 스트림에서 건조 샘플. 비 방사성 혼입 SAM 방사선 지침에 따라 방사성 원추형 튜브를 함유하는 물 상을 폐기. </li><li> 2 ㎖ / 섬광 액체의 튜브를 추가합니다. 섬광 COU와 통합 된 방사능을 측정nter 제조 업체의 프로토콜과 기기의 사용에 따라. </li><li> 하기 일반 식을 이용을 nmol / mg의 단백질의 효소 활성을 계산한다 : CPM 값 × 10 3 × [SAM (MM)의 총 (방사성 차가운) 농도] 방사성 SAM (Cl / mmol)의 X의 X 5 특정 활동 [방사성 SAM의 농도 (MM)] 참고 : 위의 프로토콜에 의존 SAM 농도 또는 단백질에 의존 PEMT 분석을 위해 변경 될 수 있습니다. SAM 농도 의존 효소 분석을 위해, 시간 동안 단백질에 의존 PEMT 분석, SAM의 농도 분석에 추가됩니다 상수 (선형 범위에서 15 분) 차가운 SAM의 다양한 양을 유지 (우리는 mM의 0.05를 선택) 및 시간 상수 (15 분) 유지됩니다. 또한 완충액의 pH는 약 PEMT 효소는 다른 최적의 pH 값을 가질 경우, 필요에 따라 변경 될 수있다. </li></ol>

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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>

관심 없음 충돌 선언하지 않습니다

이 단순한 빠른 PEMT 분석은 단백질 원으로 전체 세포 추출액을 이용하여 PE 상 SAM에서 방사성 메틸기의 전달로 인한 PE의 메틸화 된 형태의 정량을 허용한다. 그것은 빠르고, 민감한, 재현성, 정제 효소 17도 적합하다. 관심 메틸화 된 오히려 PE 12,13,18,19보다 이들 기판에 특정한 경우 Monomethyl- 또는 디메틸 PE는 상기 분석에 첨가 될 수있다. 정제 PEMT 효소가 사용되는 경우, PE는 분석에 ...

포스파티딜 에탄올 아민의 메틸화 된은 (PEMT) 효소 모노-PE, 디메틸 PE 및 / 또는 제공하기 위해 PE, 모노 - PE 또는 디메틸 PE 상 메틸기 공여체로서 S의 -adenosylmethionine (SAM)을 사용하여 하나 이상의 메틸 그룹의 공유 결합을 촉진 포스파티딜콜린 (PC). 이 효소는 동물 세포와 곰팡이에 거의 어디에나 있습니다. 이들은 또한 몇몇 식물 1 세균의 약 10 %, 진핵 2와 상호 작용하는, 특히에서 찾을 수있다. PEMTs이뿐만 아니라, 다른 중요한 세포 기능을 다함으로써 동물 세포에서 주요 지질 클래스 PC의 생산에 기여함으로써 세포 생물학에 적합하다. 포유류에서 PEMTs 주로 그들은 매우 저밀도 지질 단백질의 정상적인 분비에 필요한 간에서 발현되며, 또한식이 유도 비만 (3), 동맥 경화증 (4)에 기여하고, 인슐린 저항ANCE 5. 또한, 포유류 PEMT은 비록 낮은 수준으로, 지방 세포에서 발현, 지방 침착 (6, 7)에 참여하고 있습니다. 암 발생 8, 9 사멸 및 세포 증식 10 PEMT 역할도 입증되었다. 박테리아, PEMT 효소는 숙주 식물 (11)와 정상 세포 성장 2, 2 병독성과 공생 중요한 것으로 밝혀졌다. 본 프로토콜의 목적 및 근거는 효소를 정제 할 필요없이 전체 세포 추출물로부터 PEMT 활성을 측정하는 방법이다. 두 가지 프로토콜 PEMT 활성을 측정하기 위해 개발되었다. 첫 번째 가장 일반적인 하나는이 문서의 주제 PE, 상 방사성 SAM에서 삼중 메틸 그룹의 전송을 측정한다. 이 프로토콜은 원래 underst을 얻기 위해 효모 및 포유 동물 세포 (12) (간) (13)로부터 PEMT 활성을 측정하기 위해 개발되어왔다이들 세포에서 PC 생합성 AND 작업뿐만 아니라 이들 효소의 특이성을 결정한다. 나중에,이 기술 및 원생 동물 기생충 14 (15 비록 분석 용 염기성 pH 값을 이용하여) (2) 박테리아와 같은 다른 세포 유형에 적용되고있다. 이 기술은 전체 세포 추출물뿐만 아니라, 정제 된 효소로 사용할 수 있고, 잠재적으로 모든 세포 추출물 시스템에 적용 할 수있다. 비 방사성 분석도의 S -adenosylhomocysteine, SAM (16)의 transmethylation 생성물의 정량 효소에 의존하는 설계되었다. 이 방사능을 포함하지 않는 한 후자의 분석은 더 편리 할 수​​ 있지만 정제 효소에만 적합합니다.

<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.

도 1은 기판으로서 PE를 사용하여 내인성 효소 원으로서 Leishmania의 전체 세포 추출물로 행하여, 시간 종속 PEMT 분석을 보여준다. 유기 상 중의 방사능의 양을 섬광 계수에 의해 정량화 하였다. 얻어진 번호는 PE 삼중 전사 메틸기의 양을 계산하는데 이용되었다. PEMT 활성은 약 20 분 동안 선형이었다. 그런 다음, 또 다른 15 분 동안 일정한 머문 후 약 30 분에...

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.

시험 관내 분석에서 포스파티딜 메틸화 된 역가를 측정하는 방법

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

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

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JoVE Journal

Biology

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

9.8K 조회수.  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. 

비디오: 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.

리파제 및 포스 후보자에 대한 기질 특이성을 정의

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

연구

생화학

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 DNA와 tRNA Methyltransferase Dnmt2 (Ehmeth) 효소를 체외 tRNA의 Methylation 분석에서

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

면역학 및 감염병학

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 메틸 트랜스퍼라제 활성 분석을 위한 항체 무첨가 분석

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

유전학

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.

아실-PEGyl 교환 젤 시프트 분석은 뇌막 단백질의 팔미토일화의 정량적 측정을 위한 것입니다.

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.

효소 특성화에서 고처리량 스크리닝까지 아포지단백질 N-아실트랜스퍼라제에 대한 클릭 화학 기반 형광 분석

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 메틸트랜스퍼라제 억제제를 스크리닝하기 위한 연속 형광 기반 엔도뉴클레아제 결합 DNA 메틸화 분석

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.

단백질 인산화의 정량화를 위한 최적화된 단일 분자 풀다운 분석

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.

정화 및 체 외에서 활동 분석 결과 (p) ppGpp Clostridium 남과 어울리지 않 는에서 합성에 대 한

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.

자질<em&gt; 체외</em효소 특성에 대한&gt; ATP 아제 활동

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

생물학

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.

Phospholipase Activity Assay: Anthopleura dowii 의 조독 추출물에서 Phospholipase 활성을 결정하기 위한 겔 확산 분석

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