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. Cell Extract Voorbereiding <ol><li> Groeien de Leishmania cellen in een steriele plastic fles afgesloten met luchtdichte deksel bij 26 ° C in een medium van 1x M199 aangevuld met 20 mM HEPES pH 7,4, 100 U / ml penicilline, 100 ug / ml streptomycine, 5 ug / ml heem , 0,35 g / L NACO 2 H, 0,1 mM adenine en 2 ug / ml biopterine zonder schudden. Oogst de cellen door centrifugeren bij 1500 g gedurende 5 minuten bij 4 ° C wanneer zij een celdichtheid van 1-2 x 10 7 / ml bereikt. </li><li>...

<ol>
	<li>Keogh, M. R., Courtney, P. D., Kinney, A. J., Dewey, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+phospholipid+N-.methyltransferases+from+Arabidopsis+and+soybean.">Functional characterization of phospholipid N-.methyltransferases from Arabidopsis and soybean.</a> J Biol Chem. 284 (23), 15439-15447 (2009).</li><li>Geiger, O., Lopez-Lara, I. 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Cloning and characterization of two distinct methyltransferase genes.</a> J Biol Chem. 262 (32), 15428-15435 (1987).</li><li>Tanaka, Y., Amano, F., Maeda, M., Nishijima, M., Akamatsu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+properties+of+phosphatidyl-N-.monomethylethanolamine+N-.methyltransferase,+the+enzyme+catalyzing+the+second+and+the+third+steps+in+the+phosphatidylethanolamine+N-.methyltransferase+system,+from+mouse+liver+microsomes.">Purification and properties of phosphatidyl-N-.monomethylethanolamine N-.methyltransferase, the enzyme catalyzing the second and the third steps in the phosphatidylethanolamine N-.methyltransferase system, from mouse liver microsomes.</a> Jpn J Med Sci Biol. 43 (3), 59-73 (1990).</li><li>Bibis, S. S., Dahlstrom, K., Zhu, T., Zufferey, 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+of+Leishmania+major+phosphatidylethanolamine+methyltransferases+LmjPEM1+and+LmjPEM2+and+their+inhibition+by+choline+analogs.">Characterization of Leishmania major phosphatidylethanolamine methyltransferases LmjPEM1 and LmjPEM2 and their inhibition by choline analogs.</a> Mol Biochem Parasitol. 196 (2), 90-99 (2014).</li><li>deRudder, K. E., Thomas-Oates, J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylethanolamine+N-.methyltransferase+from+rat+liver.">Phosphatidylethanolamine N-.methyltransferase from rat liver.</a> Methods Enzymol. 209, 366-374 (1992).</li><li>Gaynor, P. M., Carman, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylethanolamine+methyltransferase+and+phospholipid+methyltransferase+activities+from+Saccharomyces+cerevisiae.+Enzymological+and+kinetic+properties.">Phosphatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties.</a> Biochim Biophys Acta. 1045 (2), 156-163 (1990).</li><li>Arondel, V., Benning, C., Somerville, C. 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Deze eenvoudige, snelle PEMT test maakt de kwantificering van gemethyleerde vormen van PE als gevolg van de overdracht van radioactieve methylgroepen van SAM op PE middels gehele celextract als eiwitbron. Het is snel, gevoelig en reproduceerbaar, en ook geschikt voor gezuiverde enzymen 17. Monomethyl- of dimethyl-PE kan worden toegevoegd aan de test indien het methyltransferase van belang is specifiek voor deze substraten plaats van PE 12,13,18,19. Als PEMT gezuiverde enzym wordt gebruikt, kan PE w...

Fosfatidylethanolamine methyltransferase (PEMT) enzymen katalyseren de covalente aanhechting van één of meer methylgroepen met S -adenosylmethionine (SAM) als methylgroepdonor op PE, monomethyl-dimethyl-PE of PE naar monomethyl-PE, dimethyl-PE en / of geeft fosfatidylcholine (PC). Deze enzymen zijn vrijwel overal in dierlijke cellen en schimmels. Ze kunnen ook in sommige planten 1 en ongeveer 10% van de bacteriën, met name die interageren met eukaryoten 2. PEMTs de biologie van de cel betrokken zijn niet alleen bijdragen aan de productie van PC, die de belangrijkste lipideklasse in dierlijke cellen, maar ook door te voldoen aan andere belangrijke cellulaire functies. In zoogdieren worden PEMTs voornamelijk tot expressie in de lever, waar zij nodig zijn voor normale afscheiding van zeer lage dichtheid lipoproteïne en ze dragen ook bij aan dieet-geïnduceerde obesitas 3, atherosclerose 4 en insuline weerstandkomstig 5. Daarnaast zijn zoogdieren PEMT eveneens uitgedrukt in adipocyten, hoewel lagere niveaus, en deelnemen vetafzetting 6, 7. PEMT rol in kanker ontwikkeling 8, 9 apoptose en celgroei 10 zijn ook aangetoond. In bacteriën zijn PEMT enzymen getoond belangrijk voor normale celgroei 2, virulentie 2 en symbiose met de gastplant 11 zijn. Het doel en de reden van het huidige protocol is het PEMT activiteit van volledige celextracten te meten zonder de noodzaak om het enzym te zuiveren. Twee verschillende protocollen ontwikkeld om PEMT te meten. De eerste en meest voorkomende meet de overdracht van getritieerd methylgroep van radioactieve SAM op PE, dat is het onderwerp van dit artikel. Dit protocol werd oorspronkelijk ontwikkeld om PEMT activiteit meet van gist 12 en zoogdiercellen (lever) 13 om een underst winnenanding PC biosynthese in deze cellen en de specificiteit van deze enzymen te bepalen. Later is deze techniek toegepast op andere celtypen zoals bacteriën 2 (met een basische pH-waarde voor de test hoewel 15) en protozoa parasieten 14. Deze techniek kan worden gebruikt met gehele celextracten en gezuiverd enzym, en kan mogelijk worden toegepast op elk celextract systeem. Een niet radioactieve assay is ook ontworpen dat steunt op de enzymatische kwantificering van S -adenosylhomocysteine ​​het transmethylatie product van SAM 16. Laatstgenoemde bepaling kan handiger omdat het geen betrekking radioactiviteit maar het is alleen geschikt voor gezuiverde enzymen.

<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.

Figuur 1 toont een tijdsafhankelijke PEMT test, die met Leishmania gehele celextract werd uitgevoerd als een enzym bron met endogene PE als substraat. De hoeveelheid radioactiviteit in de organische fase werd gekwantificeerd door scintillatietelling. De verkregen getallen werden gebruikt om de hoeveelheid getritieerd methylgroepen overgebracht naar PE te berekenen. De PEMT activiteit was lineair gedurende ongeveer 20 min. Vervolgens bereikte een plateau bij onge...

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.

In vitro test te meten Fosfatidylethanolamine methyltransferase activiteit

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

The overall goal of this methodology is to quantify phosphatidylethanolamine methyltransferase or PEMT activity from whole cell extracts. In this method we are allowed to answer key questions into the lipid metabolism field such as the importance of the phosphatidylethanolamine methyltransferases in the biosynthesis of phosphatidylcholine. The main advantage of this protocol is that it can be measured from whole cell extract and does not require purification of the enzyme.So this protocol can give some insights into how the parasites synthesizes its most abundant lipid phosphatidylcholine. This protocol can also be applied to other cell types such as mammal cells and bacteria. Growth of Leishmania cells in a sterile plastic bottle sealed with an airtight cap at 26 degree Celsius in supplemented M199 medium without shaking.Harvest the cells by centrifugation at 1500G for five minutes at four degree Celsius. When they reach a cell density of 10 to 20 million cells per milliliter. After removing the supernatant with a pipette wash the cells by resuspending the cell pellet with a serological pipette in half of the culture's volume of cold phosphate buffered saline.Centrifuge the cells again at 1550G for five minutes at four degree Celsius. Discard the supernatant with a pipette. Proceed to the next step or snap freeze the cell pellet in liquid nitrogen for long-term storage at minus 80 degree Celsius.Next, re-suspend the cell pellet in an equal volume 2X lysis buffer. Add 1X volume of glass beads and vortex vigorously at four degree Celsius for 10 minutes. Add two volumes of 1X lysis buffer and mix.Then centrifuge the cell extracts at 1500G and four degree Celsius for 10 minutes to pellet unbroken cells and nuclei. Transfer the supernatant with a pipette into a fresh cool centrifuge tube and keep the cell extracts on ice until the completion of the experiment. Add two microliters of cell extracts to one milliliter of bicinchoninic acid solution and duplicate.Incubate the standards and protein samples for 10 minutes in a pre-warmed 60 degree Celsius water bath. Transfer the samples to ice for three minutes. Measure the absorbents at the standards and the protein samples with a spectrophotometer at a wavelength of 562 nanometers.Calculate the protein concentration of the cell extracts by using the bovine serum albumin standard as a reference as described in the manufacturer's protocol. Dilute the cell extracts to a protein concentration of 10 milligrams per milliliter with 1X lysis buffer. In a chemical hood, test each sample and duplicate in a 15 milliliter conical tube.Prepare 20 microliters of one molar TrisHCL PH 7.5 per tube and keep it on ice. Also prepare two milliliters of chloroform methanol stopping solution at room temperature for each tube. Pipette 20 microliters of the one molar TrisHCL into each 15 milliliter conical tube on ice.Then following radiation safety guidelines, at the equivalent of zero point zero six micromolar tritiated SAM and 50 micromolar cold SAM per tube for a total of 50 point zero six micromolar SAM. At this point, add a volume of cold water per tube that has been calculated according to the text protocol. Transfer each conical tube to a pre-warmed 30 degree Celsius water bath.Add 20 microliters of cell extracts to each tube to start the reaction. Incubate the samples for the desired time. Stop the reaction by adding two milliliters of chloroform methanol to each tube.Transfer the conical tubes to room temperature. Working in a chemical hood, add 700 microliters of water to each tube containing the enzymatic reaction sample. Vortex vigorously for 30 seconds.Then centrifuge at 1500g for five minutes at room temperature to separate the organic from the water phase. It is critical while transferring the lower phase to not take any of the water phase as it will bias the calculation of the assay. Use a pipette to transfer the lower organic phase into a new 15 milliliter conical tube.Add one milliliter of water to each lower phase containing tube and vortex vigorously for 30 seconds. Then, centrifuge again at 1500G for five minutes to separate the organic phase from the water phase. Transfer the lower organic phase into a scintillation tube with a pipette.Dry the samples under a stream of nitrogen gas. Dispose of the water phases containing the non-incorporated radioactive SAM and the radioactive conical tubes as per radiation guidelines. Next, add two milliliters of scintillation liquid per tube.Measure the incorporated radioactivity with a scintillation counter according to the manufacturer's protocol, and instruments used. Calculate the enzymatic activity in nanomoles per milligram of protein using the equation listed in the text protocol. Shown here is a time dependent PEMT assay which was carried out with Leishmania whole cell extract as an enzyme source using endogenous PE as a substrate.The amount of radioactivity in the organic phase was quantified by scintillation counting and utilized to calculate the amount of tritiated methyl groups transferred on to PE.The PEMT activity was linear for approximately 20 minutes. It then reached a plateau at around 30 minutes, after which it stayed constant for another 15 minutes. As expected, PEMT activity was not detected when no cell extracts were added and was detected when cell extracts were added to the assay.Further, this activity was abolished in the presence of 100 micromolar octadecyltrimethylammonium bromide which is a specific inhibitor of leishmania major phosphatidylethanolamine methyltransferases LMJ PEM One and LMJ PEM two. PEMT activity was also protein concentration dependent and this activity was linearly proportional to the amount of protein applied for the enzymatic assay. Lastly, a SAM concentration dependent PEMT assay was carried out, in which increasing concentrations of SAM were tested.PEMT activity reached a plateau at a SAM concentration of approximately zero point five millimolar. One masters this protocol can be carried out in three to five hours depending on the number of samples. While attempting this protocol, it is very important to use recently purchased SAM as this compound is unstable.Don't forget that while working with pathogens and radioactivity can be extremely hazardous. Therefore, it is important to follow the safety guidelines and to wear proper laboratory equipment.

Research

JoVE Journal

Biology

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

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