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

1. Preparação Extracto celular <ol><li> Crescer as células de Leishmania em um frasco de plástico estéril, selado com tampa estanque ao ar 26 ° C num meio feito de 1x M199 suplementado com HEPES 20 mM, pH 7,4, 100 U / ml de penicilina, 100 ug / ml de estreptomicina, 5 ug / ml de heme , 0,35 g / L NACo 2H, adenina 0,1 mM, e 2 ug / ml de biopterina sem agitação. Colher as células por centrifugação a 1500 g durante 5 min a 4 ° C quando atingem uma densidade celular de 1-2 x 10 7 / ml. </li><li> Descartar o sobrenadante com uma pipeta e lava-se as células por ressuspensão do sedimento de células com uma pipeta serológica em metade do volume da cultura de solução salina de fosfato tamponada (NaCl 137 mM, KCl 2,7 mM, 10 mM de 2 HPO 4, 1,8 mM de KH 2 PO 4, pH 7,4). Descarte sobrenadantes de células de acordo com as diretrizes de segurança BL2. </li><li> Centrifugar células novamente em 1500 g durante 5 min a 4 ºC. Descartar o sobrenadante com uma pipeta. Prossiga tO passo seguinte ou encaixe congelar o sedimento celular em azoto líquido durante a armazenagem a longo prazo a -80 ° C (até três meses). </li><li> Preparar tampão de lise 2x (0,5 M de sacarose, 0,1 M Tris-HCl, pH 7,5, EDTA 2 mM, e 2x cocktail inibidor de protease) e mantê-lo a 4 ° C em gelo. </li><li> Ressuspender o sedimento de células (frescas ou congeladas) em igual volume de tampão de lise 2x. Adicionar o volume 1x de contas de vidro. Vortex vigorosamente a 4 ºC durante 10 minutos. </li><li> Adicionar 2 volumes de tampão de lise 1x e misturar. Extratos de células centrifugar a 1500 xg a 4 ºC durante 10 minutos para sedimentar as células intactas e núcleos. </li><li> Transferir o sobrenadante com uma pipeta para um tubo de centrífuga de fresco e manter fresco extractos de células em gelo até à conclusão da experiência. </li></ol> 2. Determinar a concentração de proteína do extracto celular utilizando o kit de Estimativa proteína tal como ácido bicinconínico Ensaio <ol><li> Prepara-se o ácido bicinconínico solução (BCA) (1 ml / tubo) por mistura do BCA e cobre (II) sulfate numa proporção de 49: 1 (v / v). </li><li> Prepare os padrões de proteína de 0, 10, 20, 30, 40, 50, e 60 ug / ml por diluição de um ml de solução concentrada a 10 mg / albumina de soro bovino (BSA) em aliquotas de 1 mL da solução de BCA. </li><li> Adicionar 2 mL de extractos de células em 1 ml de solução de BCA em duplicado. Incubar as amostras e os padrões de proteína durante 10 minutos num banho de água a 60 ° C pré-aquecido. </li><li> Amostras de transferência de gelo por 3 min. Medir a absorvância dos padrões e das amostras de proteína com um espectrofotómetro a um comprimento de onda de 562 nm. </li><li> Calcula-se a concentração de proteína dos extractos celulares, usando o padrão de BSA, como uma referência, tal como descrito no protocolo do fabricante. Dilui-se extractos de células para uma concentração de proteína de 10 mg / ml com tampão de lise 1x. </li></ol> 3. Ensaio enzimático em 200 ul por tubo NOTA: Siga os seguintes passos em uma capa química. <ol><li> Teste cada amostra em duplicado, a 15 mltubo cónico. Prepare 20 mL 1 M TrisHCl pH 7,5 por tubo e mantê-lo em gelo. Preparar 2 ml de clorofórmio / metanol (1: 1 (v / v); Solução de paragem) à temperatura ambiente para cada um dos tubos. </li><li> Pipetar 20 ul de Tris-HCl 1 M pH 7,5 em cada tubo de 15 ml em gelo. </li><li> Siga as orientações de segurança de radiação a partir daqui. Adicionar o equivalente a 0,06 uM (0,2 uCi) S - [metil-3H] adenosil-L-metionina e 50 uM frio SAM por tubo para um total de 50,06 uM de SAM. Adicionar x ul de água fria, onde x = 200- (20 (para buffer) + 20 (para extratos de células) + volume de frio e radioativo SAM) por tubo. </li><li> Transferir cada tubo cónico para um banho de água a 30 ° C pré-aquecido. Adicionar a cada tubo 20 ul de extractos de células (equivalente a 200 ug de proteína) para iniciar a reacção. Incubar durante o tempo desejado (0 a 45 min). </li><li> Parar a reacção por adição de 2 ml de clorofórmio / metanol (1: 1; v / v; Solução de paragem) para cada tubo. Transferir o tubo cónico para a TA (20-256; C). </li></ol> 4. Lipid Extração NOTA: Siga os seguintes passos em uma capa química. <ol><li> Adicionar 700 uL de água a cada tubo contendo a amostra de reacção enzimática. Vortex vigorosamente por 30 segundos. Centrifugar a 1.500 xg durante 5 min à temperatura ambiente para separar a fase orgânica da fase aquosa. </li><li> Transferir a fase orgânica inferior para um novo tubo cónico de 15 ml com uma pipeta. Adicionar 1 ml de água para cada &quot;fase inferior&quot; contendo tubo e agitar vigorosamente em vórtice durante 30 seg. Centrifugar novamente a 1500 xg durante 5 minutos para separar a fase orgânica da fase aquosa. </li><li> Transferir a fase orgânica inferior para um tubo de cintilação com uma pipeta. As amostras secas sob uma corrente de N2. Elimine as fases de água contendo o SAM radioativo não-incorporados e os tubos cônicos radioativos conforme as diretrizes de radiação. </li><li> Adicione 2 ml / tubo de líquido de cintilação. Medir a radioactividade incorporada com um par de cintilaçãonter acordo com o protocolo dos fabricantes e utilização do instrumento. </li><li> Calcula-se a actividade enzimática em nmol / mg de proteína usando a seguinte equação geral: valor cpm x 10 x 3 [total (radioativo e frio) concentração de SAM (mM)] x 5 atividade específica de SAM radioativo (Ci / mmol) x [concentração de substâncias radioativas SAM (mM)] NOTA: o protocolo acima pode ser alterada para uma concentração SAM dependente ou um ensaio PEMT dependente proteína. Para a concentração de SAM ensaio enzimático dependente, o tempo é mantido constante (15 minutos, o que está na gama linear) e várias quantidades de frio SAM são adicionados ao ensaio enquanto que para o ensaio PEMT dependente de proteína, concentração de SAM (escolheu 0,05 mM) e a hora são mantida constante (15 minutos). Além disso, o pH do tampão pode ser alterado conforme necessário, se algumas enzimas PEMT tem um valor de pH óptimo diferente. </li></ol>

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Este ensaio PEMT simples, rápida permite a quantificação das formas metiladas de PE que resulta da transferência de grupos metilo radioactivas de SAM na PE utilizando o extracto de célula completa como uma fonte de proteína. É rápido, sensível, reprodutível e também adequado para enzimas purificadas 17. Monometil ou dimetil-PE pode ser adicionado ao ensaio se a metiltransferase de interesse é específico para estes substratos em vez de PE 12,13,18,19. Se PEMT enzima purificado é usado,...

Fosfatidiletanolamina metiltransferase (PEMT) As enzimas catalisam a ligação covalente de um ou mais grupos de metilo utilizando S -adenosylmethionine (SAM) como o doador grupo metilo em PE, PE-monometil ou dimetil-PE para dar monometil-PE, dimetil-PE e / ou fosfatidilcolina (PC). Estas enzimas são quase ubíquo em células animais e de fungos. Eles também podem ser encontrados em algumas plantas 1 e cerca de 10% das bactérias, particularmente aqueles que interagem com 2 eucariotas. PEMTs são relevantes para a biologia da célula não só por contribuir para a produção de PC, que é a principal classe de lípidos em células animais, mas também por cumprir outras funções celulares importantes. Nos mamíferos, são principalmente PEMTs expressa no fígado, onde eles são necessários para a secreção normal de colesterol de lipoproteína de muito baixa densidade e que também contribuem para a obesidade induzida por dieta 3, 4 aterosclerose, e resistir à insulinaance 5. Além disso, PEMT de mamífero também são expressos em adipócitos, embora a níveis mais baixos, e participar em deposição de gordura 6, 7. PEMT papel no desenvolvimento do cancro 8, 9 a apoptose, e o crescimento celular 10 também foram demonstradas. Em bactérias, as enzimas PEMT têm sido mostrados como sendo importantes para o crescimento normal de células 2, 2 virulência, e a simbiose com a planta hospedeira 11. O objetivo e razão de ser do presente protocolo é o de medir a atividade PEMT a partir de extratos de células inteiras sem a necessidade de purificar a enzima. Dois protocolos distintos foram desenvolvidos para medir a actividade PEMT. A primeira e mais comum mede-se a transferência do grupo metil tritiada de radioativo SAM em PE, que é o tema deste artigo. Este protocolo foi originalmente desenvolvido para medir a actividade de levedura PEMT 12 e células de mamíferos (fígado) 13 para obter uma understAND de biossíntese PC nestas células, bem como para determinar a especificidade destas enzimas. Mais tarde, esta técnica tem sido aplicada a outros tipos de células tais como bactérias 2 (utilizando um valor de pH de base para o ensaio embora 15) e parasitas protozoários 14. Esta técnica pode ser utilizada com extractos de células completas, bem como de enzima purificada, e, potencialmente, pode ser aplicado a qualquer sistema de extracto celular. Um ensaio não radioactivo também foi projetado que se baseia na quantificação enzimática de S -adenosylhomocysteine, o produto transmetilação de SAM 16. O último ensaio pode ser mais conveniente, uma vez que não envolvem radioactividade, mas é somente apropriado para enzimas purificadas.

<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.

A Figura 1 mostra um ensaio PEMT dependente do tempo, o qual foi realizado com Leishmania extracto de célula completa como uma fonte de enzima utilizando PE endógeno como um substrato. A quantidade de radioactividade na fase orgânica foi quantificada por contagem de cintilação. Os números resultantes foram utilizados para calcular a quantidade de grupos metil tritiados transferidos para PE. A actividade PEMT foi linear durante, aproximadamente, 20 min. Em ...

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.

Ensaio in vitro para medir Fosfatidiletanolamina Metiltransferase Atividade

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 visualizações.  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. 

Vídeo: 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.

Definindo substrato Especificidades para a lipase e fosfolipase candidatos

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

Bioquímica

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.

Ensaio in vitro de metilação tRNA com a Entamoeba histolytica DNA e Dnmt2 tRNA metiltransferase (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 ...

Imunologia e Infecção

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.

Teste anticorpo-livre para a análise da atividade do methyltransferase do RNA

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

Genética

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.

Ensaio de mudança de gel de troca acila-PEGyl para determinação quantitativa de palmitoilação de proteínas de membrana cerebral

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.

Ensaio fluorométrico baseado em química de cliques para apolipoproteína N-aciltransferase da caracterização enzimática à triagem de alto rendimento

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;

Ensaio de metilação de DNA acoplado à endonuclease à base de fluorescência contínua para triagem de inibidores da DNA metiltransferase

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.

Um ensaio de recuo de molécula única otimizado para quantificação da fosforilação de proteínas

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.

Uma purificação e ensaio de atividade In Vitro para um ppGpp (p) sintetase de 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.

Medindo<em&gt; In Vitro</em&gt; Actividade ATPase para enzimática Caracterização

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

Biologia

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.

Ensaio de Atividade de Fosfolipase: Um Ensaio de Difusão em Gel para Determinar a Atividade da Fosfolipase em Extrato Bruto de Veneno de Anthopleura dowii

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