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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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 verde son muy sensibles y pueden detectar pequeñas cantidades de fosfato libre (~ 50 pmol de cada 100 l). Debido a la sensibilidad de este ensayo, es crucial usar artículos de plástico desechable, agua ultrapura, tampones y reactivos que carecen de contaminación de fosfato. Después de purificación de proteínas, se recomienda la exclusión de tamaño o cromatografía de intercambio iónico para mejorar la pureza de proteínas y eliminar los contaminantes. Para las proteínas que muestran débil de la actividad ATPasa in vitro, los estimulantes pueden ser añadidos a la reacción para mejorar enzymatic actividad. Muchos factores que estimulan la actividad de ATPasa se han caracterizado. Por ejemplo, cardiolipina y otros lípidos de membrana, proteínas cliente de chaperones tales como Hsp90, y otras proteínas que intervienen en el apoyo conformaciones o entornos particulares en las que funcionan ATPasas 13-15. Nuestro laboratorio caracteriza en primer lugar EPSE mediante la purificación de monómeros con la actividad ATPasa débil en comparación con las ATPasas homólogas 6. Más tarde descubrimos que cuando EPSE se copurified con el dominio citoplásmico de EPSL, una proteína transmembrana y pareja de unión de EPSE en el sistema T2S, la adición de fosfolípidos ácidos tales como cardiolipina a la mezcla de reacción aumentó en gran medida la actividad de la ATPasa de EPSE 12. Es probable que imita las condiciones experiencias EPSE en la membrana citoplasmática que puede promover la oligomerización. Muchas técnicas se han utilizado para cuantificar la actividad ATPasa in vitro de proteínas purificadas. γ- radiactivo <sup&gt; 32 P se ha aplicado con frecuencia y con éxito para cuantificar la hidrólisis de ATP 14,16, sin embargo, la seguridad es una preocupación y requiere la aprobación para el uso en laboratorio de radiactividad. Aunque muy sensible, la corta vida media de γ- 32 P es también una desventaja. Otros métodos de detección de fosfato comerciales están disponibles, tales como los que se basan en la formación de un producto fluorescente. Algunos de estos métodos también son muy sensibles, pero con frecuencia requieren la adición de otras enzimas para la reacción, dando como resultado los reactivos que son menos estables en el tiempo. Además, los kits están disponibles que detectan ADP liberada durante la hidrólisis de ATP usando un reactivo basado en luminiscencia estable, pero estos pueden ser menos ideales para ATPasas con bajos niveles de actividad 17. El ensayo descrito aquí consiste en un solo paso de medición liberación de fosfato, es muy sensible, y por lo general se puede realizar dentro de unos hr. También es posible prepare un sustrato que contiene verde de malaquita para evitar la compra de un kit de un proveedor comercial 6,9. Una consideración antes de emprender este ensayo es que la etapa entre añadir el reactivo de detección de fosfato y tomando lecturas de absorción es relativamente sensible al tiempo y debe estar entre 20-30 min. Este protocolo básico se puede utilizar para determinar el papel de los estimulantes (como se describe), antagonistas, subunidades, dominios y residuos específicos en la actividad de ATPasa 15,16,18,19. Este ensayo también se puede extender para medir la actividad de las fosfatasas u otras enzimas que liberan fosfato durante la catálisis. Además, este método se puede aplicar a cribado de alto rendimiento para los inhibidores de la ATPasa 20.

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 border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">HEPES buffer</td>
			<td style="width:128px;">Fisher</td>
			<td style="width:108px;">BP310-500</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine triphosphate (ATP)</td>
			<td>Fisher</td>
			<td>BP41325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well plates (clear, flat-bottom)</td>
			<td>VWR</td>
			<td>82050-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BIOMOL Green</td>
			<td>Enzo Life Sciences</td>
			<td>BML-AK111</td>
			<td>Preferred phosphate detection reagent. Caution: irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td></td>
			<td></td>
			<td>BioTek Synergy or comparable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism 5</td>
			<td colspan="2">GraphPad Software</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 lisina en el dominio de unión a zinc-EPSE se mide comparando la actividad de ATPasa de purificado WT EPSE-cytoEpsL a la variante EPSE K417AK419A-cytoEpsL. En la Figura 1, la liberación de fosfato procede linealmente en el transcurso de un ensayo cinético 1 hr, un requisito para la cuantificación precisa de la actividad ATPasa cinética. Figura 2 muestra los datos de las mismas muestras de proteínas purificadas como la Figura 1 que se ensayaron tres veces por duplicado y se cuantificaron en términos deLa tasa media de liberación de fosfato. Estos datos muestran que K417 y K419 parecen ser importantes para la actividad ATPasa EPSE. Sin embargo, la comparación de (no cardiolipina) las tasas de actividad ATPasa no estimuladas (Figura 3) muestra que K417 y K419 no contribuyen a la actividad basal de EPSE sino más bien a la capacidad de la proteína a ser estimulada por la cardiolipina. Las lisinas cargados positivamente en el dominio de unión a zinc-EPSE pueden interactuar directamente con los fosfolípidos cargados negativamente, lo que contribuye a la estimulación de fosfolípidos mediada por la actividad de ATPasa EPSE. <img alt="Figura 1" src="/files/ftp_upload/54305/54305fig1.jpg" /> Figura 1: fosfato es liberado linealmente en un ensayo cinético ATPasa Una hora cinética cardiolipina estimulada ensayo ATPasa comparación de la liberación de fosfato de 0,5 M WT EPSE-cytoEpsL a EPSE K417AK419A-cytoEpsL con albúmina de suero bovino (BSA) ensayó como.un control negativo. Datos [cantidad de fosfato liberado (eje y) frente al tiempo (eje x)] se representaron gráficamente y se sometieron a análisis de regresión lineal. La pendiente representa la velocidad de hidrólisis de ATP. Se muestra un gráfico representativo de las ecuaciones de regresión lineal para las tres proteínas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54305/54305fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54305/54305fig2.jpg" /> Figura 2:.. Doble lisina mutaciones en EPSE Zinc-dominio de unión Reducir estimulada ATPasa Actividad Los resultados del ensayo cardiolipina estimulada cinética 1 hr ATPasa con las mismas proteínas y condiciones que en la figura 1 los ensayos se realizaron tres veces separadas por duplicado técnica y la tasa de hidrólisis de ATP se calculó como fosfato nmol generado por minuto por M pr<html> Otein usando ecuaciones de regresión lineal. Los resultados de medias con el error estándar se muestran. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54305/54305fig2large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="figura 3" src="/files/ftp_upload/54305/54305fig3.jpg" /> Figura 3: Doble Lisina Las mutaciones en EPSE zinc-dominio de unión no interfieran con Unstimulated actividad ATPasa de una noche (16 h) punto final sin estimular (sin cardiolipina) Ensayo de ATPasa que compara la actividad de 5 M WT EPSE-cytoEpsL a EPSE K417AK419A-cytoEpsL con BSA. como control negativo. Los ensayos se realizaron en tres ocasiones separadas por duplicado técnica y resultados de medias con el error estándar se muestra. El nivel basal de actividad no estimulada EPSE ATPasa se ensayó durante un período de 16 horas es de ~ 1000 veces menor que 1 hr actividad cardiolipina estimulada cinética.e.com/files/ftp_upload/54305/54305fig2large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura.

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.

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

Nanyang Technological University

Research

JoVE Journal

Biology

Measuring In Vitro ATPase Activity for Enzymatic Characterization

17.9K 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

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un ensayo para medir la actividad de Escherichia coli Inducible lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Investigación

Biología

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Ensayo de la actividad quinasa de LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Medición de transporte de cationes por Na, K y H, K-ATPasa en<em&gt; Xenopus</em&gt; Los ovocitos mediante espectrofotometría de absorción atómica: una alternativa a ensayos de radioisótopos

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Medición inducida glutatión-Alimentación Respuesta en Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Reconstitución funcional y la actividad del canal Las mediciones de Purificada Wildtype y mutante CFTR Proteína

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

Método para medir la actividad de enzimas deubiquitinating en líneas celulares y muestras de tejido

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

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.

Ensayo in vitro para medir Fosfatidiletanolamina metiltransferasa actividad

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

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Medición de actividad enzimática de la lactasa en el laboratorio de enseñanza

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

In Vitro Ensayo de la sumoilación estudiar SUMO E3 ligasa actividad

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...