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

1. Realizar la reacción de hidrólisis de ATP con la proteína purificada <ol><li> Las existencias de preparar todos los reactivos necesarios para la incubación con proteína purificada. <ol><li> Preparar tampón 5x HEPES / NaCl / glicerol (HNG) que contiene 100 mM de HEPES pH 8,5, NaCl 65 mM, y 5% de glicerol (u otro tampón de ensayo según el caso). </li><li> Preparar 100 mM de MgCl 2 (u otro metal, si es de metal ATPasa dependiente) en agua. </li><li> Preparar fresca mM ATP 100 en 200 mM Tris Base (no ajuste de pH más) utilizando ATP alta pureza. Alícuota y almacenar el archivo de ATP a -20 ° C durante no más de unas pocas semanas, en forma de ATP se descompone con el tiempo, y se abstengan de congelación y descongelación de la población de ATP. </li><li> Premix MgCl 2 y ATP en una relación 1: 1 justo antes de la creación de la reacción de hidrólisis de ATP. </li></ol></li><li> Preparar y etiquetar tubos de 1,5 ml para la recogida de muestras a intervalos regulares a lo largo de la reacción. Preparar tubos para recoger muestras en el tiempo 0 y en el tiempo de 15, 30,45, y 60 min. NOTA: Como alternativa, recoger muestras solamente en el tiempo 0 y el punto final. <ol><li> Añadir 245 l de tampón 1x HNG a cada tubo con el fin de diluir las muestras recogidas de las reacciones de hidrólisis de ATP 1:50. </li></ol></li><li> Preparar un baño de hielo seco y etanol para congelar rápidamente muestras para detener la reacción. En un cubo de hielo de caucho o recipiente de otro seguro (no plástico), añade varios trozos de hielo seco y se vierte con cuidado suficiente etanol 70-100% para cubrir el hielo seco. </li><li> Diluir la proteína purificada en tampón HNG 1x, según sea apropiado (normalmente, un 5-10 mM), y mantener en hielo. </li><li> Configurar las reacciones de hidrólisis de ATP. <ol><li> En distintos tubos de 0,5 ml para cada muestra, añadir los siguientes reactivos (en orden): H 2 O (hasta un volumen final de 30 l), 6 l 5x HNG u otro tampón, 3 l mM MgCl 100 mezcla 2 -ATP, y 0,25-5 proteína M. </li><li> Incluir un único amortiguador-condición de control negativo en el que no se añade ninguna proteína.</li></ol></li><li> Retire 5 l de la reacción en el tiempo 0, se diluye 1:50 en el tubo de 1,5 ml preparado que contiene 245 l de tampón HNG, y congelar inmediatamente la muestra en el baño de hielo seco / etanol. </li><li> Incubar las reacciones a 37 ° C para permitir la hidrólisis de ATP que se produzca durante 1 hora. En cada intervalo (15, 30, 45, y 60 min), retirar 5 ml de alícuotas de la reacción y añadir a los tubos de muestra etiquetados que contienen tampón HNG como en el paso 1.6. </li><li> Al final de la reacción de hidrólisis de ATP, mover muestras diluidas a un congelador -80 ° C para el almacenamiento. Para asegurarse de que todas las muestras se congelaron por completo, espere al menos 10 minutos antes de continuar. </li></ol> 2. Las muestras que contienen Incubar libre pi con el reactivo de detección <ol><li> Descongelar las muestras diluidas que contienen partes alícuotas de la reacción de hidrólisis de ATP en cada punto de tiempo (obtenidos a partir de los pasos 1.6 y 1.7) a temperatura ambiente. </li><li> Configurar una placa de 96 pocillos que contienen las muestras y patrones de fosfato. <ol><li> En una ma 0,5 mlser, diluir el estándar de fosfato (proporcionado con el reactivo de detección de Pi) de 800 mM a 40 mM mediante la adición de 5,5 l de tampón de la HNG 800 M estándar a 104,5 l. Mezclar bien y añadir 100 l de esta norma Pi 40 M a pocillo A1 de una placa de 96 pocillos. </li><li> Añadir tampón HNG 50 l a los pocillos B1-H1 para 1: 1 diluciones en serie de la norma Pi. </li><li> Retire 50 l de 40 mM Pi desde el pocillo A1 y añadir al tampón de ensayo en 50 l B1 así, mezclar y remover 50 l de bien B1 y C1 añadir a bien, diluciones continuas a través de G1 también. Desechar 50 l de G1 bien después de la mezcla para asegurar cada pocillo tiene el mismo volumen. Así H1 debe contener solamente tampón para crear un estándar a PI 40 a 0 M. NOTA: Si un factor adicional (tal como un inhibidor) se ha añadido a las muestras, crear una curva estándar que contiene ese factor en el control de cambios en la liberación de fosfato o absorbancia bajo esas condiciones. </li><li> Añadir 50 l de cada sampLe por duplicado a la placa. Añadir muestras del mismo punto de tiempo en las columnas verticalmente (muestra 1 hora 0 = A2, A3; muestra 2 tiempo 0 = B2, B3) y diferentes puntos de tiempo horizontalmente. Esto permite hasta 8 muestras y 5 puntos de tiempo por placa. </li></ol></li><li> Usar una pipeta estéril para eliminar suficiente reactivo de detección de verde malaquita / molibdato de Pi añadir 100 l a cada uno de los pocillos que contienen las muestras y los estándares (reactivo necesario (ml) = 0,1 x número de muestras y los estándares) y añadir a un plato para facilitar el pipeteado utilizando una pipeta multicanal. No vierta el reactivo de detección directamente en la cápsula, ya que es probable que se produzca contaminación Pi. </li><li> Con una pipeta multicanal, se añaden 100 l de reactivo de detección Pi a cada pocillo y mezclar cuidadosamente con la pipeta hacia arriba y abajo un número considerable de veces sin la introducción de burbujas, preferentemente en el orden de los últimos puntos de tiempo a los primeros momentos de la hora. </li><li> Incubar la placa durante 25 min a temperatura ambiente, o de acuerdo con la madirecciones de nufacturer. </li></ol> 3. cuantificar Resultados El uso de un lector de microplacas <ol><li> Leer la absorbancia de las muestras a 650 nm utilizando un lector de microplaca de absorbancia. </li><li> Hacer una curva estándar Pi. El uso de un software de gráficos, representar gráficamente los valores de absorbancia de las muestras estándar Pi frente a la concentración con el fin de encontrar una ecuación usada para resolver la cantidad de fosfato en cada muestra. </li><li> Calcular la actividad ATPasa para cada muestra mediante la calibración con el estándar de fosfato. Fosfato liberado = (OD 650 - Intersección) / pendiente. <ol><li> Promediar el Pi total a partir de duplicados de cada muestra. Restar lectura de la absorbancia del control de tampón de sólo de este número. Multiplique esto por el factor de dilución (50 en nuestro ejemplo). </li><li> Determinar el Pi liberado por nmol de proteína mol. Representación gráfica de estos valores para cada punto de tiempo en un ensayo de cinética debe producir ajustes lineales de al menos R = 0,99 (Figura 1); Si nOT, el ensayo puede ajustarse con más o menos proteína o un tiempo de incubación más largo o más corto. </li></ol></li><li> Representan los resultados como nmol Pi / mol de proteína / min (Figuras 2, 3), o como proteína nmol Pi / mg / min si se desea. </li></ol>

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E., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vacuolar-type+H+-ATPase+at+a+glance+-+more+than+a+proton+pump.">The vacuolar-type H+-ATPase at a glance - more than a proton pump.</a> J Cell Sci. 127 (23), 4987-4993 (2014).</li><li>Planet, P. J., Kachlany, S. C., DeSalle, R., Figurski, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+of+genes+for+secretion+NTPases:+Identification+of+the+widespread+tadA+subfamily+and+development+of+a+diagnostic+key+for+gene+classification.">Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification.</a> Proc Natl Acad Sci U S A. 98 (5), 2503-2508 (2001).</li><li>Robien, M. A., Krumm, B. E., Sandkvist, M., Hol, W. G. 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L., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+the+Vibrio+cholerae+type+II+secretion+ATPase+EpsE.">Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE.</a> J Bacteriol. 187 (1), 249-256 (2005).</li><li>Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+II+secretion+and+pathogenesis.">Type II secretion and pathogenesis.</a> Infect Immun. 69 (6), 3523-3535 (2001).</li><li>Brune, M., Hunter, J. L., Corrie, J. E. T., Webb, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct,+Real-time+measurement+of+rapid+inorganic+phosphate+release+using+a+novel+fluorescent+probe+and+its+application+to+actomyosin+subfragment+1+ATPase.">Direct, Real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.</a> Biochemistry. 33 (27), 8262-8271 (1994).</li><li>Carter, S. G., Karl, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inorganic+phosphate+assay+with+malachite+green:+An+improvement+and+evaluation.">Inorganic phosphate assay with malachite green: An improvement and evaluation.</a> J Biochem Biophys Methods. 7 (1), 7-13 (1982).</li><li>Henkel, R. D., VandeBerg, J. L., Walsh, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microassay+for+ATPase.">A microassay for ATPase.</a> Anal Biochem. 169 (2), 312-318 (1988).</li><li>Harder, K. W., Owen, P., Wong, L. K. H., Aebersold, R., Clark-Lewis, I., Jirik, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+kinetic+analysis+of+the+intracellular+domain+of+human+protein+tyrosine+phosphatase+&#946;+(HPTP+&#946;)+using+synthetic+phosphopeptides.">Characterization and kinetic analysis of the intracellular domain of human protein tyrosine phosphatase &#946; (HPTP &#946;) using synthetic phosphopeptides.</a> Biochem J. 298 (2), 395-401 (1994).</li><li>Camberg, J. L., Johnson, T. L., Patrick, M., Abendroth, J., Hol, W. 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The authors have nothing to disclose.

Este es un protocolo general para la medición de la actividad ATPasa in vitro de proteínas purificadas para la caracterización bioquímica. Este método se optimiza fácilmente; por ejemplo, el ajuste de la cantidad de proteína, tampón y composiciones de sal, temperatura, y la variación de la longitud de ensayo y los intervalos (incluyendo el aumento del número total de intervalos) puede mejorar la actividad de la cuantificación. Comercialmente disponibles malaquita reactivos basados ​​en v...

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

enzimas trifosfato-hidrólisis de la adenosina, o ATPasas, juegan un papel crítico en una diversa gama de funciones celulares. Estas proteínas dinámicas pueden generar energía para el trabajo mecánico, como el tráfico de proteínas y la degradación, transporte de solutos, y los movimientos celulares. El protocolo descrito aquí es un ensayo básico para la medición de la actividad in vitro de ATPasas purificadas para la caracterización funcional. Las proteínas se hidrolizan ATP en una reacción que resulta en la liberación de fosfato inorgánico, y la cantidad de fosfato liberado a continuación, se cuantifica mediante un ensayo colorimétrico. Este protocolo altamente adaptable se puede ajustar para medir la actividad de ATPasa en ensayos cinéticos o de punto final. Se proporciona aquí un protocolo representativo basado en la actividad y requisitos de EPSE, la AAA + ATPasa implicada en la secreción de Tipo II en la bacteria Vibrio cholerae. La cantidad de proteína purificada necesaria para medir la actividad, la duración del ensayo y la sincronización y la cantidad de saintervalos mpling, tampón y composición de sal, temperatura, cofactores, estimulantes (si lo hay), etc., pueden variar de los descritos aquí, y por lo tanto una optimización pueden ser necesarios. Este protocolo proporciona un marco básico para ATPasas que caracterizan y se puede realizar rápida y fácilmente ajustarse según sea necesario.

La actividad in vitro de la ATPasa T2S EPSE puede ser estimulada por copurification de EPSE con el dominio citoplásmico de EPSL (EPSE-cytoEpsL) y la adición de la cardiolipina fosfolípido ácido 12. También es posible determinar el papel de los residuos particulares EPSE en la hidrólisis de ATP mediante la comparación de la actividad de tipo salvaje (WT) a formas variantes de la proteína utilizando este ensayo. Aquí, el efecto de sustituir dos residuos de lisi...

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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.

Medición<em&gt; In Vitro</em&gt; Actividad ATPasa para enzimática Caracterización

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

measuring-in-vitro-atpase-activity-for-enzymatic-characterization

Research

JoVE Journal

Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

18.0K vistas.  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.

Video: Measuring In Vitro ATPase Activity for Enzymatic Characterization

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.

Ensayo de la actividad de proteína quinasa con ATP radiomarcado

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 ...

Investigación

Bioquímica

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.

Caracterización a nivel molecular utilizando enfoques bioquímicos robustos de una nueva proteína kinasa

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.

Una adaptación de semi-alto rendimiento del ensayo ATPase acoplado a NADH para la detección de inhibidores de moléculas pequeñas

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.

<meta charset="utf8"></head><body>Ensayo de nucleósidos trifosfato hidrolasas en Toxoplasma gondii y Neospora caninum para el cribado de alto rendimiento utilizando un brazo robótico

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.

Ensayo de nucleósidos trifosfato hidrolasas en Toxoplasma gondii y Neospora caninum para el cribado de alto rendimiento utilizando un brazo robótico

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.

La detección directa de la actividad de acetato de formación de la enzima cinasa de acetato

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

Biología

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.

Una purificación y análisis de actividad In Vitro de una (p) ppGpp sintetasa de Clostridium difficile

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

Inmunología e Infección

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.

Evaluación de las funciones mitocondriales y la viabilidad celular renal de células que sobreexpresan la proteína quinasa C Isoenzimas

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.

Los ensayos bioquímicos para Actividades Analizando de ATP-dependientes remodelación de la cromatina enzimas

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.

Medición

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