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. 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>

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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><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.

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

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.

Definieren von Substratspezifität für Lipase und Phospholipase-Kandidaten

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

Forschung

Biochemie

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.

In-vitro-tRNA Methylierungs-Assay mit der Entamoeba histolytica DNA und 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 ...

Immunologie und Infektion

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.

Antikörperfreier Assay für rna-Methyltransferase-Aktivitätsanalyse

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.

Acyl-PEGyl Exchange Gel Shift Assay zur quantitativen Bestimmung der Palmitoylierung von Gehirnmembranproteinen

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.

Klickchemischer fluorometrischer Assay für Apolipoprotein-N-Acyltransferase von der Enzymcharakterisierung bis zum Hochdurchsatz-Screening

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;

Kontinuierlicher fluoreszenzbasierter Endonuklease-gekoppelter DNA-Methylierungsassay zum Screening auf DNA-Methyltransferase-Inhibitoren

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.

Ein optimierter Einzelmolekül-Pulldown-Assay zur Quantifizierung der Proteinphosphorylierung

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.

Eine Reinigung und In vitro- Aktivität Assay für ein (p) PpGpp Synthestase von Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Measuring In Vitro ATPase Activity for Enzymatic Characterization

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization. Proteins hydrolyze ATP in a reaction that results in inorganic phosphate release, and the amount of phosphate liberated is then quantitated using a colorimetric assay. This highly adaptable protocol can be adjusted to measure ATPase activity in kinetic or endpoint assays. A representative protocol is provided here based on the activity and requirements of EpsE, the AAA+ ATPase involved in Type II Secretion in the bacterium Vibrio cholerae. The amount of purified protein needed to measure activity, length of the assay and the timing and number of sampling intervals, buffer and salt composition, temperature, co-factors, stimulants (if any), etc. may vary from those described here, and thus some optimization may be necessary. This protocol provides a basic framework for characterizing ATPases and can be performed quickly and easily adjusted as necessary.

Measuring In Vitro ATPase Activity for Enzymatic Characterization

We describe a basic protocol for quantitating in vitro ATPase activity. This protocol can be optimized based on the level of activity and requirements for a given purified ATPase.

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 ...

Biologie

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: Ein Geldiffusionsassay zur Bestimmung der Phospholipase Aktivität in Rohgiftextrakt aus Anthopleura dowii

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