Name: in vitro assay to measure phosphatidylethanolamine methyltransferase activity
Uploaded: 2016-01-05T13:00:01
Description: Watch this Scientific Journal Video about In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity at JoVE.com

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

1. Preparación de la célula Extracto <ol><li> Cultivar las células de Leishmania en una botella de plástico estéril sellado con tapa de aire ajustado a 26 ºC en un medio hecho de 1x M199 suplementado con 20 mM HEPES pH 7,4, 100 U / ml de penicilina, 100 mg / ml de estreptomicina, 5 mg / ml heme , 0,35 g / L NaCO 2 H, adenina 0,1 mM y 2 g / ml biopterina sin agitación. Recoger las células por centrifugación a 1500 g durante 5 min a 4 ºC cuando alcanzan una densidad celular de 1-2 x 10 <sup...

<ol>
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Este ensayo PEMA simple, rápida permite la cuantificación de las formas metiladas de PE que resulta de la transferencia de grupos metilo radiactivos de SAM en PE utilizando extracto de células enteras como fuente de proteína. Es rápido, sensible, reproducible, y también es adecuado para las enzimas purificadas 17. Monometil- o dimetil-PE se pueden añadir a el ensayo si el metiltransferasa de interés es específica para estos sustratos en lugar de PE 12,13,18,19. Si se utiliza la enzima puri...

Metiltransferasa fosfatidiletanolamina (PEMA) enzimas catalizan la unión covalente de uno o más grupos metilo utilizando S -adenosylmethionine (SAM) como donante de metilo en PE grupo, monometil o dimetil-PE-PE para dar monometil-PE, dimetil-PE y / o fosfatidilcolina (PC). Estas enzimas son casi omnipresentes en las células animales y hongos. También se pueden encontrar en algunas plantas 1 y aproximadamente el 10% de las bacterias, particularmente aquellos que interactúan con 2 eucariotas. PEMTs son relevantes para la biología de la célula no sólo contribuyendo a la producción de PC, que es la principal clase de lípidos en las células animales, sino también mediante el cumplimiento de otras funciones celulares importantes. En los mamíferos, PEMTs se expresan principalmente en el hígado donde son requeridas para la secreción normal de la lipoproteína de muy baja densidad y también contribuyen a la obesidad inducida por la dieta 3, 4 aterosclerosis, y la insulina se resistenANCE 5. Además, PEMA mamíferos también se expresan en los adipocitos, aunque a niveles más bajos, y participar en la deposición de grasa 6, 7. PEMA papel en el desarrollo del cáncer 8, 9 apoptosis, y 10 el crecimiento celular también se ha demostrado. En las bacterias, las enzimas PEMA han demostrado ser importantes para el crecimiento normal de las células 2, la virulencia 2, y la simbiosis con la planta huésped 11. El objetivo y razón de ser del presente protocolo es para medir la actividad PEMA a partir de extractos de células enteras, sin la necesidad de purificar la enzima. Dos protocolos distintos se han desarrollado para medir la actividad PEMA. La primera y más común se mide la transferencia de un grupo metilo tritiada de SAM radiactivo en PE, que es el tema de este artículo. Este protocolo ha sido desarrollado originalmente para medir la actividad de la levadura PEMA 12 y células de mamíferos (hígado) 13 para obtener una understanding de la biosíntesis de PC en estas células, así como para determinar la especificidad de estas enzimas. Más tarde, esta técnica se ha aplicado a otros tipos de células tales como bacterias 2 (utilizando un valor de pH básico para el ensayo aunque 15) y parásitos protozoarios 14. Esta técnica se puede utilizar con extractos de células enteras, así como enzima purificada, y potencialmente se puede aplicar a cualquier sistema de extracto celular. Un ensayo no radiactivo también se ha diseñado que se basa en la cuantificación enzimática de S -adenosylhomocysteine, el producto de transmetilación de SAM 16. El último ensayo puede ser más conveniente, ya que no implica la radiactividad pero sólo es adecuado para las enzimas purificadas.

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">S-[Methyl-3H]adenosyl-L-methionine (specific activity of 5-15 Ci/mMole)</td>
			<td style="width:107px;">Perkin Elmer</td>
			<td style="width:119px;">NET155050UC</td>
			<td style="width:188px;">Aliquot the reagent and freeze at -20 &#176;C; follow radiation safety guidelines while using this reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Roche Life Sciences</td>
			<td style="width:119px;">11836170001</td>
			<td>dilute it fresh&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass beads, acid washed, 425-600 mm</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bicinchoninic acid solution</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">B9643</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Copper (II) sulfate</td>
			<td style="width:107px;">Sigma Aldrich</td>
			<td style="width:119px;">C2284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scintillation counter MicroBeta2 with 1-detector</td>
			<td style="width:107px;">Perkin Elmer&#160;</td>
			<td style="width:119px;">2450-0010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectrophotometer Biomate 3</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td>840208300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA stock solution (10 mg/ml)</td>
			<td style="width:107px;">New England Biolabs</td>
			<td style="width:119px;">B9001S</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Scintillation liquid&#160;</td>
			<td style="width:107px;">Research Product International Corp</td>
			<td style="width:119px;">111198</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">S-(5&#39;-Adenosyl)-L-methionine chloride (hydrochloride)&#160;</td>
			<td style="width:107px;">Cayman Chemicals</td>
			<td>13956</td>
			<td style="width:188px;">dilute the reagent in 20 mM HCl and freeze aliquots at -80 &#176;C&#160;</td>
		</tr>
	</tbody>
</table>

in vitro assay to measure phosphatidylethanolamine methyltransferase activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

La Figura 1 muestra un ensayo de PEMA dependiente del tiempo, que se llevó a cabo con extracto de células enteras Leishmania como una fuente de enzima usando PE endógena como sustrato. La cantidad de radiactividad en la fase orgánica se cuantificó por recuento de centelleo. Los números resultantes se utilizan para calcular la cantidad de grupos metilo tritiados transferidos a PE. La actividad PEMA fue lineal durante aproximadamente 20 min. A continuación,...

Watch this Scientific Journal Video about In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity at JoVE.com

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

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A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

A Matrigel basado en tubo de ensayo de formación para evaluar la actividad de las células tumorales vasculogénica

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Assaying the Kinase Activity of LRRK2 in vitro

Assaying the Kinase Activity of LRRK2 in vitro

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

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

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 ...

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro Methylation Assay to Study Protein Arginine Methylation

In vitro metilación de ensayo para estudiar la proteína arginina metilación

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Cuantitativa inmunofluorescencia Ensayo para medir la variación en los niveles de proteína en centrosomas

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Ensayo Cometa como una medida indirecta de estrés oxidativo sistémico

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

El uso del ensayo de Ramsay medir la secreción de fluidos y de iones Flux tarifas en el<em&gt; Drosophila melanogaster</em&gt; Túbulo de Malpighi

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...