Name: measuring in vitro atpase activity for enzymatic characterization
Uploaded: 2016-08-23T14:00:00.000+00:00
Duration: 7 min 38 s
Description: Watch this Scientific Journal Video about Measuring In Vitro ATPase Activity for Enzymatic Characterization at JoVE.com

The authors would like to acknowledge funding from a National Institutes of Health grant RO1AI049294 (to M. S.).

1. Effectuer ATP hydrolyse Réaction avec de la protéine purifiée <ol><li> Préparer les stocks de tous les réactifs nécessaires pour incubation avec de la protéine purifiée. <ol><li> Préparer 5x HEPES / NaCl / glycérol (HNG) contenant 100 mM de HEPES pH 8,5, 65 mM de NaCl et 5% de glycerol (ou un autre tampon d&#39;essai selon le cas). </li><li> Préparer 100 mM MgCl 2 (ou autre métal, si ATPase est en métal-dépendant) dans l&#39; eau. </li><li> Préparer frais mM ATP 100 dans 200 Tris base mM (ne pas ajuster le pH plus loin) en utilisant la haute ATP de pureté. Aliquoter et stocker le stock ATP à -20 ° C pendant plus de quelques semaines, que l&#39;ATP se décomposera au fil du temps, et de ne pas le gel et le dégel du stock ATP. </li><li> Premix MgCl2 et ATP à un rapport 1: 1 juste avant la mise en place de la réaction d&#39;hydrolyse de l&#39; ATP. </li></ol></li><li> Préparer et étiqueter tubes de 1,5 ml pour prélever des échantillons à intervalles réguliers tout au long de la réaction. Préparer des tubes pour recueillir des échantillons au temps 0 et au moment 15, 30,45 et 60 minutes. NOTE: Comme alternative, prélever des échantillons seulement au temps 0 et le point de terminaison. <ol><li> Ajouter tampon 1x HNG 245 pi à chaque tube afin de diluer les échantillons prélevés dans les réactions d&#39;hydrolyse de l&#39;ATP 1:50. </li></ol></li><li> Préparer un bain de glace sèche et d&#39;éthanol pour la congélation rapide des échantillons pour arrêter la réaction. Dans un seau à glace en caoutchouc ou un autre coffre-fort (non plastique) récipient, ajouter plusieurs morceaux de glace sèche et versez délicatement assez 70-100% d&#39;éthanol pour couvrir la glace sèche. </li><li> Diluer protéine purifiée dans un tampon HNG 1x, le cas échéant (typiquement 5-10 uM), et garder sur la glace. </li><li> Mettre en place les réactions d&#39;hydrolyse de l&#39;ATP. <ol><li> Dans 0,5 ml tubes séparés pour chaque échantillon, ajouter les réactifs suivants (dans l&#39; ordre): H 2 O (jusqu&#39;à un volume final de 30 pi), 6 pi 5x HNG ou un autre tampon, 3 ul mM MgCl 100 2 -ATP mélange, et 0,25 à 5 protéine iM. </li><li> Inclure une seule mémoire tampon condition de contrôle négatif dans lequel aucune protéine est ajoutée.</li></ol></li><li> Retirer 5 ul de la réaction au temps 0, diluer à 1:50 dans le tube de 1,5 ml préparé contenant 245 tampon HNG ul, et geler immédiatement l&#39;échantillon dans le bain de glace / éthanol sec. </li><li> Incuber les réactions à 37 ° C pour permettre l&#39;hydrolyse de l&#39;ATP de se produire pendant 1 heure. A chaque intervalle (15, 30, 45 et 60 min), enlever aliquotes de 5 ul de la réaction et ajouter à tubes d&#39;échantillons marqués contenant un tampon HNG comme à l&#39;étape 1.6. </li><li> A la fin de la réaction d&#39;hydrolyse de l&#39;ATP, de déplacer les échantillons dilués dans un congélateur C -80 ° C pendant le stockage. Pour assurer que tous les échantillons sont complètement gelés, attendre au moins 10 minutes avant de poursuivre. </li></ol> 2. Les échantillons Incuber contenant Pi gratuit avec le réactif de détection <ol><li> décongeler les échantillons contenant de l&#39;ATP dilué des aliquotes de la réaction d&#39;hydrolyse à chaque point de temps (obtenues à partir des étapes 1.6 et 1.7) à la température ambiante. </li><li> Mettre en place une plaque à 96 puits contenant des échantillons et des étalons de phosphate. <ol><li> Dans un ml tu 0,5soit, diluer le standard de phosphate (fourni avec le réactif de détection Pi) à partir de 800 uM à 40 uM en y ajoutant 5,5 ul du tampon HNG 800 pm à la norme 104,5 ul. Bien mélanger et ajouter 100 pi de cette 40 uM norme Pi bien A1 d&#39;une plaque de 96 puits. </li><li> Ajouter un tampon HNG 50 ul aux puits B1-H1 à 1: 1 des dilutions en série de la norme Pi. </li><li> Retirer 50 ul de 40 uM Pi du puits A1 et ajouter au tampon d&#39;essai de 50 pi dans le puits B1, mélanger et retirer 50 ul du puits B1 et ajouter ainsi C1, dilutions poursuivies par G1 bien. Jeter 50 pi de G1 bien après mélange pour assurer chaque puits a le même volume. Eh bien H1 ne doit contenir que tampon pour créer une norme Pi 40-0 uM. NOTE: Si un facteur supplémentaire (tel qu&#39;un inhibiteur) a été ajouté aux échantillons, créer une courbe standard contenant ce facteur pour contrôler les changements dans la libération de phosphate ou absorbance dans ces conditions. </li><li> Ajouter 50 ul de chaque sample en double exemplaire à la plaque. Ajouter des échantillons à partir du même point de temps dans les colonnes verticalement (échantillon de 1 heure 0 = A2, A3; échantillon 2 temps 0 = B2, B3) et différents points de temps horizontalement. Cela permet jusqu&#39;à 8 échantillons et 5 points de temps par plaque. </li></ol></li><li> Utilisez une pipette stérile pour éliminer assez molybdate réactif malachite verte / Pi détection d&#39;ajouter 100 pi à chacun des puits contenant des échantillons et des normes (réactif nécessaire (ml) = 0,1 x nombre d&#39;échantillons et des normes) et ajouter à un plat pour pipetage facile à l&#39;aide d&#39;une pipette multicanaux. Ne pas verser le réactif de détection directement dans le plat, car la contamination Pi est susceptible de se produire. </li><li> En utilisant une pipette multicanaux, ajouter 100 ul du réactif de détection Pi dans chaque puits et mélanger soigneusement pipetant un nombre constant de fois sans bulles d&#39;introduction, de préférence dans l&#39;ordre depuis les derniers points de temps pour les premiers points de temps. </li><li> Incuber la plaque pendant 25 min à température ambiante, ou selon la maLes directions de structeur. </li></ol> 3. Résultats quantifient en utilisant un lecteur de microplaques <ol><li> Lire l&#39;absorbance des échantillons à 650 nm en utilisant un lecteur de microplaques absorbance. </li><li> Faire une courbe standard Pi. L&#39;utilisation d&#39;un logiciel graphique, graphique les valeurs d&#39;absorbance pour les échantillons standards Pi par rapport à la concentration en vue de trouver une équation utilisée pour résoudre pour la quantité de phosphate dans chaque échantillon. </li><li> Calculer l&#39;activité de l&#39;ATPase pour chaque échantillon en calibrant avec le standard de phosphate. Phosphate libéré = (DO 650 - Y interception) / pente. <ol><li> La moyenne du total Pi de doubles de chaque échantillon. Soustraire la lecture d&#39;absorbance de la commande de mémoire tampon uniquement à partir de ce numéro. Multiplier par le facteur de dilution (50 dans notre exemple). </li><li> Déterminer la Pi nmol libérée par la protéine pmol. Graphes ces valeurs pour chaque point de temps dans un essai cinétique devrait donner des crises linéaires d&#39;au moins R = 0,99 (figure 1); Si not, le dosage peut être ajusté avec plus ou moins de protéines ou d&#39;un temps plus ou moins incubation. </li></ol></li><li> Représenter les résultats comme nmol Pi / pmol protéine / min (figures 2, 3), ou sous forme de protéine nmol Pi / pg / min si on le souhaite. </li></ol>

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The authors have nothing to disclose.

Ceci est un protocole général pour la mesure de l&#39;activité ATPase in vitro de protéines purifiées pour la caractérisation biochimique. Cette méthode est facilement optimisée; par exemple, en ajustant la quantité de protéines, un tampon et des compositions de sel, la température, et à faire varier la durée de l&#39;essai et les intervalles (y compris l&#39;augmentation du nombre total d&#39;intervalles) peut améliorer l&#39;activité de quantification. réactifs verts à base de malach...

ATPases are integral enzymes in many processes across all kingdoms of life. ATPases act as molecular motors that use the energy of ATP hydrolysis to power such diverse reactions as protein trafficking, unfolding, and assembly; replication and transcription; cellular metabolism; muscle movement; cell motility; and ion pumping1-3. Some ATPases are transmembrane proteins involved in transporting solutes across membranes, others are cytoplasmic and may be associated with a biological membrane such as the plasma membrane or those of organelles.
AAA+ ATPases (ATPases associated with various cellular activities) make up a large group of ATPases that share some sequence and structural conservation. These proteins contain conserved nucleotide binding motifs such as Walker-A and -B boxes and form oligomers (generally hexamers) in their active state1. Large conformational changes in these proteins upon nucleotide binding have been characterized among diverse members of the AAA+ family. EpsE is a AAA+ ATPase and member of the bacterial Type II/IV secretion subfamily of NTPases4-6. EpsE powers Type II Secretion (T2S) in Vibrio cholerae, the causative agent of cholera. The T2S system is responsible for the secretion of a wide variety of proteins, such as the virulence factor cholera toxin that causes profuse watery diarrhea when V. cholerae colonizes the human small intestine7. 
Techniques for quantitating in vitro ATPase activity are varied, but commonly measure phosphate release using colorimetric, fluorescent, or radioactive substrates8-11. We describe a basic method for determining in vitro ATPase activity of purified proteins using a colorimetric assay based on a commercially available malachite green-containing substrate that measures liberated inorganic phosphate (Pi). At low pH, malachite green molybdate forms a complex with Pi and the level of complex formation can be measured at 650 nm. This simple and sensitive assay may be used to functionally characterize new ATPases and to evaluate the roles of potential activators or inhibitors, to determine the importance of domains and/or specific residues, or to assess the effect of particular treatments on enzymatic activity.

<table><tbody><tr><td>HEPES buffer</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>BP358-212</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td></td></tr><tr><td>Adenosine triphosphate (ATP)</td><td>Fisher</td><td>BP41325</td><td></td></tr><tr><td>96-well plates (clear, flat-bottom)</td><td>VWR</td><td>82050-760</td><td></td></tr><tr><td>BIOMOL Green</td><td>Enzo Life Sciences</td><td>BML-AK111</td><td>Preferred phosphate detection reagent. Caution: irritant.</td></tr><tr><td>Microplate reader</td><td></td><td></td><td>BioTek Synergy or comparable</td></tr><tr><td>Prism 5</td><td>GraphPad Software</td><td></td><td></td></tr></tbody></table>

measuring in vitro atpase activity for enzymatic characterization

enzymes triphosphate-hydrolyser adénosine, ou ATPases, jouent un rôle essentiel dans un large éventail de fonctions cellulaires. Ces protéines dynamiques peuvent générer de l&#39;énergie pour le travail mécanique, comme le trafic de protéines et la dégradation, le transport de soluté, et les mouvements cellulaires. Le protocole décrit ici est un dosage de base pour mesurer l&#39;activité in vitro des ATPases purifiés pour la caractérisation fonctionnelle. Les protéines hydrolysent l&#39;ATP dans une réaction qui entraîne la libération de phosphate inorganique, et la quantité de phosphate libéré est ensuite quantifié en utilisant un dosage colorimétrique. Ce protocole hautement adaptable peut être ajustée pour mesurer l&#39;activité d&#39;ATPase dans les essais cinétiques ou le terminal. Un protocole représentatif est fourni ici sur la base de l&#39;activité et les exigences de Epse, l&#39;AAA + ATPase impliqué dans Type II Sécrétion dans la bactérie Vibrio cholerae. La quantité de protéine purifiée nécessaire pour mesurer l&#39;activité, la durée de l&#39;essai et le moment et le nombre de saintervalles mpling, tampon et de la composition de sel, température, co-facteurs, les stimulants ( le cas échéant), etc. peuvent varier de ceux décrits ici, et donc une certaine optimisation peuvent être nécessaires. Ce protocole fournit un cadre de base pour ATPases caractérisant et peut être effectuée rapidement et facilement ajustée si nécessaire.

L&#39;activité in vitro de la T2S ATPase Epse peut être stimulée par copurification de Epse avec le domaine cytoplasmique de EpsL (Epse-cytoEpsL) et ajout de la cardiolipine phospholipide acide 12. Il est également possible de déterminer le rôle de certains résidus Epse dans hydrolyse de l&#39;ATP en comparant l&#39;activité de type sauvage (WT) des variantes de formes de la protéine à l&#39;aide de ce test. Ici, l&#39;effet de substituer deux résidus de ly...

Watch this Scientific Journal Video about Measuring In Vitro ATPase Activity for Enzymatic Characterization at JoVE.com

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.

Mesure<em&gt; In vitro</em&gt; Activité ATPase pour Enzymatic Caractérisation

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

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Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

18.0K vues.  University of Michigan. 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.

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Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Dosage de l&#39;activité des protéines kinase avec ATP radiomarqué

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

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Biochimie

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Caractérisation au niveau moléculaire à l'aide d'approches biochimiques robustes d'une nouvelle protéine Kinase

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

Adaptation semi-haute débit de l'analyse ATPase couplée au NADH pour le dépistage des petits inhibiteurs des molécules

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Détection directe de l&#39;activité Acétate de formation de l&#39;acétate kinase Enzyme

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Biologie

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.

Une Purification et analyse d’activité In Vitro pour un (p) ppGpp synthétase de Clostridium difficile

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

Immunologie et infection

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Évaluation des fonctions mitochondriales et la viabilité cellulaire dans les cellules rénales surexprimant la protéine kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.

Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner.

Les dosages biochimiques pour l&#39;analyse des activités de l&#39;ATP-dépendant remodelage de la chromatine enzymes

Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and ...

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.

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.

Essai in vitro pour mesurer Phosphatidyléthanolamine Methyltransferase activité

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

Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

Source: Pokhrel, A., et al. A <a href="https://jove.unicartagenaproxy.elogim.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018).
This video demonstrates a thin-layer chromatography-based enzyme assay to determine pyrophosphokinase activity using radiolabeled nucleotides. The method provides insights into nucleotide synthesis and phosphotransfer reactions.

Dosage de l’activité enzymatique basé sur la chromatographie sur couche mince : une technique pour déterminer l’activité de la pyrophosphokinase in vitro à l’aide de la chromatographie sur couche mince

Source: Pokhrel, A., et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018).
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Chapitres dans cette vidéo

Dosage de l'activité des protéines kinase avec ATP radiomarqué

Caractérisation au niveau moléculaire à l'aide d'approches biochimiques robustes d'une nouvelle protéine Kinase

Adaptation semi-haute débit de l'analyse ATPase couplée au NADH pour le dépistage des petits inhibiteurs des molécules

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Détection directe de l'activité Acétate de formation de l'acétate kinase Enzyme

Une Purification et analyse d’activité In Vitro pour un (p) ppGpp synthétase de Clostridium difficile

Évaluation des fonctions mitochondriales et la viabilité cellulaire dans les cellules rénales surexprimant la protéine kinase C Isozymes

Les dosages biochimiques pour l'analyse des activités de l'ATP-dépendant remodelage de la chromatine enzymes

Essai in vitro pour mesurer Phosphatidyléthanolamine Methyltransferase activité

Dosage de l’activité enzymatique basé sur la chromatographie sur couche mince : une technique pour déterminer l’activité de la pyrophosphokinase in vitro à l’aide de la chromatographie sur couche mince