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 Preparation
<ol>
	<li>Grow the Leishmania cells in a sterile plastic bottle sealed with air tight cap at 26 &#186;C in a medium made of 1x M199 supplemented with 20 mM HEPES pH7.4, 100 U/ml penicillin, 100 &#181;g/ml streptomycin, 5 &#181;g/ml heme, 0.35 g/L NaCO2H, 0.1 mM adenine, and 2 &#181;g/ml biopterin without shaking. Harvest the cells by centrifugation at 1,500 g for 5 min at 4 &#186;C when they reach a cell density of 1-2 x 107/ml.</li>
	<li>Discard the supernatan...

<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. M., Sohlenkamp, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylcholine+biosynthesis+and+function+in+bacteria.">Phosphatidylcholine biosynthesis and function in bacteria.</a> Biochim Biophys Acta. 1831 (3), 503-513 (2013).</li><li>Gao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+lipogenesis+in+white+adipose+tissue+contributes+to+the+resistance+to+high+fat+diet-induced+obesity+in+phosphatidylethanolamine+N-.methyltransferase-deficient+mice.">Decreased lipogenesis in white adipose tissue contributes to the resistance to high fat diet-induced obesity in phosphatidylethanolamine N-.methyltransferase-deficient mice.</a> Biochim Biophys Acta. 1851 (2), 152-162 (2015).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+phosphatidylethanolamine+N-.methyltransferase+alters+plasma+VLDL+phospholipids+and+attenuates+atherosclerosis+in+mice.">Lack of phosphatidylethanolamine N-.methyltransferase alters plasma VLDL phospholipids and attenuates atherosclerosis in mice.</a> Arterioscler Thromb Vasc Biol. 29 (9), 1349-1355 (2009).</li><li>Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+methylation+in+mammals:+from+biochemistry+to+physiological+function.">Phospholipid methylation in mammals: from biochemistry to physiological function.</a> Biochim Biophys Acta. 1838 (6), 1477-1487 (2014).</li><li>Nishimaki-Mogami, T., Suzuki, K., Takahashi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+phosphatidylethanolamine+methylation+in+the+secretion+of+very+low+density+lipoproteins+by+cultured+rat+hepatocytes:+rapid+inhibition+of+phosphatidylethanolamine+methylation+by+bezafibrate+increases+the+density+of+apolipoprotein+B48-containing+lipoproteins.">The role of phosphatidylethanolamine methylation in the secretion of very low density lipoproteins by cultured rat hepatocytes: rapid inhibition of phosphatidylethanolamine methylation by bezafibrate increases the density of apolipoprotein B48-containing lipoproteins.</a> Biochim Biophys Acta. 1304 (1), 21-31 (1996).</li><li>Noga, A. A., Zhao, Y., Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unexpected+requirement+for+phosphatidylethanolamine+N-.methyltransferase+in+the+secretion+of+very+low+density+lipoproteins.">An unexpected requirement for phosphatidylethanolamine N-.methyltransferase in the secretion of very low density lipoproteins.</a> J Biol Chem. 277 (44), 42358-42365 (2002).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+repression+of+phosphatidylethanolamine+N-.methyltransferase+(PEMT)+in+BRCA1-mutated+breast+cancer.">Epigenetic repression of phosphatidylethanolamine N-.methyltransferase (PEMT) in BRCA1-mutated breast cancer.</a> Oncotarget. 5 (5), 1315-1325 (2014).</li><li>Cui, Z., Houweling, M., Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+rat+hepatoma+cell+growth+by+expression+of+phosphatidylethanolamine+N-.methyltransferase-2.">Suppression of rat hepatoma cell growth by expression of phosphatidylethanolamine N-.methyltransferase-2.</a> J Biol Chem. 269 (40), 24531-24533 (1994).</li><li>Cui, Z., Shen, Y. J., Vance, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverse+correlation+between+expression+of+phosphatidylethanolamine.N-.methyltransferase-2+and+growth+rate+of+perinatal+rat+livers.">Inverse correlation between expression of phosphatidylethanolamine.N-.methyltransferase-2 and growth rate of perinatal rat livers.</a> Biochim Biophys Acta. 1346 (1), 10-16 (1997).</li><li>Minder, A. C., de Rudder, K. E., Narberhaus, F., Fischer, H. M., Hennecke, H., Geiger, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylcholine+levels+in.Bradyrhizobium+japonicum.+membranes+are+critical+for+an+efficient+symbiosis+with+the+soybean+host+plant.">Phosphatidylcholine levels in.Bradyrhizobium japonicum. membranes are critical for an efficient symbiosis with the soybean host plant.</a> Mol Microbiol. 39 (5), 1186-1198 (2001).</li><li>Kodaki, T., Yamashita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+phosphatidylethanolamine+methylation+pathway.+Cloning+and+characterization+of+two+distinct+methyltransferase+genes.">Yeast phosphatidylethanolamine methylation pathway. 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. E., Geiger, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhizobium+meliloti.+mutants+deficient+in+phospholipid+N-.methyltransferase+still+contain+phosphatidylcholine.">Rhizobium meliloti. mutants deficient in phospholipid N-.methyltransferase still contain phosphatidylcholine.</a> J Bacteriol. 179 (22), 6921-6928 (1997).</li><li>Aktas, M., Narberhaus, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+the+enzyme+properties+of+the+phospholipid+N-.methyltransferase+PmtA+from+Agrobacterium+tumefaciens.">In vitro characterization of the enzyme properties of the phospholipid N-.methyltransferase PmtA from Agrobacterium tumefaciens.</a> J Bacteriol. 191 (7), 2033-2041 (2009).</li><li>Ridgway, N. D., Vance, D. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+expression+in+Escherichia+coli.+of+a+gene+encoding+phosphatidylethanolamine+methyltransferase+(EC+2.1.1.17)+from+Rhodobacter+sphaeroides.">Isolation and functional expression in Escherichia coli. of a gene encoding phosphatidylethanolamine methyltransferase (EC 2.1.1.17) from Rhodobacter sphaeroides.</a> J Biol Chem. 268 (21), 16002-16008 (1993).</li><li>Wessel, M., Klusener, S., Godeke, J., Fritz, C., Hacker, S., Narberhaus, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Agrobacterium+tumefaciens.+requires+phosphatidylcholine+in+the+bacterial+membrane.">Virulence of Agrobacterium tumefaciens. requires phosphatidylcholine in the bacterial membrane.</a> Mol Microbiol. 62 (3), 906-915 (2006).</li><li>Klusener, S., Aktas, M., Thormann, K. M., Wessel, M., Narberhaus, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+physiological+relevance+of+Agrobacterium+tumefaciens.+phosphatidylcholine+biosynthesis+genes.">Expression and physiological relevance of Agrobacterium tumefaciens. phosphatidylcholine biosynthesis genes.</a> J Bacteriol. 191 (1), 365-374 (2009).</li><li>Henderson, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+the+gene+encoding+Leishmania+donovani.S.-adenosylhomocysteine+hydrolase,+a+potential+target+for+antiparasitic+chemotherapy.">Cloning of the gene encoding Leishmania donovani.S.-adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy.</a> Mol Biochem Parasitol. 53 (1-2), 169-183 (1992).</li><li>Koszalka, G. W., Krenitsky, T. A., Nyhan, W. L., Thompson, L. F., Watts, R. W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5&#39;-Methylthioadenosine+(MTA)+phosphorylase+from+promastigote+of+Leishmania+donovani.">5&#39;-Methylthioadenosine (MTA) phosphorylase from promastigote of Leishmania donovani.</a> Purine and Pyrimidine Metabolism in Man V, Adv Exp Med Biol. 131, 559-563 (1986).</li><li>Biastoff, S., Teuber, M., Zhou, Z. S., Dr&#228;ger, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+activity+measurement+of+a+recombinant+putrescine+N.-methyltransferase+from+Datura+stramonium.">Colorimetric activity measurement of a recombinant putrescine N.-methyltransferase from Datura stramonium.</a> Planta Med. 72 (12), 1136-1141 (2006).</li><li>Hendricks, C. L., Ross, J. R., Pichersky, E., Noel, J. P., Zhou, Z. 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This simple, quick PEMT assay allows the quantification of methylated forms of PE that results from the transfer of radioactive methyl groups from SAM onto PE using whole cell extract as a protein source. It is fast, sensitive, reproducible, and also suitable for purified enzymes 17. Monomethyl- or dimethyl-PE can be added to the assay if the methyltransferase of interest is specific to these substrates rather than to PE 12,13,18,19. If purified PEMT enzyme is used, PE can be added to the assay. A l...

Phosphatidylethanolamine methyltransferase (PEMT) enzymes catalyze the covalent attachment of one or more methyl groups using S-adenosylmethionine (SAM) as the methyl group donor onto PE, monomethyl-PE or dimethyl-PE to give monomethyl-PE, dimethyl-PE and/or phosphatidylcholine (PC). These enzymes are almost ubiquitous in animal cells and fungi. They can also be found in some plants 1 and approximately 10% of bacteria, particularly those that interact with eukaryotes 2.
PEMTs are relevant to the biology of the cell not only by contributing to the production of PC, which is the main lipid class in animal cells, but also by fulfilling other important cellular functions. In mammals, PEMTs are mainly expressed in the liver where they are required for normal secretion of very low-density lipoprotein and they also contribute to diet-induced obesity 3, atherosclerosis 4, and insulin resistance 5. Additionally, mammalian PEMT are also expressed in adipocytes, although to lower levels, and participate in fat deposition 6,7. PEMT role in cancer development 8, apoptosis 9, and cell growth 10 have also been demonstrated. In bacteria, PEMT enzymes have been shown to be important for normal cell growth 2, virulence 2, and symbiosis with the host plant 11.
The goal and rationale of the present protocol is to measure PEMT activity from whole cell extracts without the need to purify the enzyme. Two distinct protocols have been developed to measure PEMT activity. The first and most common one measures the transfer of tritiated methyl group from radioactive SAM onto PE, which is the topic of this article. This protocol has been originally developed to measure PEMT activity from yeast 12 and mammalian cells (liver) 13 to gain an understanding of PC biosynthesis in these cells as well as to determine the specificity of these enzymes. Later, this technique has been applied to other cell types such as bacteria 2 (using a basic pH value for the assay though 15) and protozoan parasites 14. This technique can be used with whole cell extracts as well as purified enzyme, and can potentially be applied to any cell extract system. A non radioactive assay has also been designed that relies on the enzymatic quantification of S-adenosylhomocysteine, the transmethylation product of SAM 16. The latter assay may be more convenient as it does not involve radioactivity but it is only suitable for purified enzymes.

<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.

Figure 1 shows a time dependant 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. The resulting numbers were utilized to calculate the amount of tritiated methyl groups transferred onto PE. The PEMT activity was linear for approximately 20 min. It then reached a plateau at around 30 min, after which ...

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 Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

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

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

An In vitro FluoroBlok Tumor Invasion Assay

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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

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

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

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

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

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

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

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