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.
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.
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.
1. Cell Extract Preparation
2. Determine the Protein Concentration of the Cell Extract Using Protein Estimation Kit Such as Bicinchoninic Acid Assay
3. Enzymatic Assay in 200 μl per Tube
NOTE: Carry the following steps in a chemical hood.
4. Lipid Extraction
NOTE: Carry the following steps in a chemical hood.
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 it stayed constant for another 15 min. As expected, PEMT activity was not detected when no cell extracts were added to the assay (Figure 2). Further, this activity was abolished in the presence of 100 μM octadecyltrimethylammonium bromide, which is an inhibitor of L. major PE methyltransferases LmjPEM1 and LmjPEM2 14. PEMT activity was also protein concentration dependent, and this activity was linearly proportional to the amount of protein applied for the enzymatic assay (Figure 3). Lastly, a SAM concentration dependent PEMT assay was carried out, in which increasing concentrations of SAM were tested (Figure 4). PEMT activity reached a plateau at SAM concentration of approximately 0.5 mM. All together, these four assays demonstrate that PEMT activity is specific and can be measured from whole cell extracts without the need to purify the enzyme(s).
Figure 1. Time dependent PEMT assay. The enzymatic assay was performed twice in duplicate with 0.2 mg of whole Leishmania cell extracts as a function of time. PEMT activity is represented as nmol methyl groups transferred onto PE per mg of protein and per hr. For time "O", cell extracts were first mixed with 2 ml of stopping solution made of chloroform/methanol (1:1, by volume) before being added to the assay solution containing SAM. Standard deviations are shown.
Figure 2. Specificity of PEMT activity. PEMT assay was carried out twice in duplicate for 15 min in the presence of 0.05 mM SAM. 1, 0.2 mg protein extract; 2, no cell extract; 3, 0.2 mg protein and 0.1 mM octadecyltrimethylammonium bromide. Standard deviations are shown.
Figure 3. Protein dependent PEMT assay. The enzymatic assay was performed twice in duplicate in the absence (point 'O') or presence of various amounts of Leishmania proteins with 0.05 mM SAM for 15 min. Standard deviations are shown.
Figure 4. The PEMT assay was carried out twice in duplicate with 0.2 mg of whole Leishmania cell extracts in the presence of various concentrations of SAM for 15 min. Standard deviations are shown.
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 limitation of this protocol is that the assay does not identify the reactions products (monomethyl-PE, dimethyl-PE, or PC). However, the identity of the reaction products (monomethyl-PE, dimethyl-PE, PC) can further be analyzed by one dimensional thin layer chromatography as described in 20,21. Further, some of the SAM degradation products such as S-adenosylhomocysteine and 5'-methylthioadenosine may inhibit PEMT activity by feedback inhibition. However, Leishmania possesses an S-adenosylhomocysteine hydrolase 22, which cleaves S-adenosylhomocysteine into adenine and S-ribosylhomocysteine, and a methylthioadenosine phosphorylase, which produces adenine and methylthioribose-1-phosphate 23. However, it is not known whether S-adenosylhomocysteine hydrolase and methylthioadenosine phosphorylase activities are high enough to efficiently metabolize S-adenosylhomocysteine and methylthioadenosine, respectively, so that no inhibition of PEMT activity occurs. In the instance S-adenosylhomocysteine hydrolase and/or 5'-methylthioadenosine metabolic enzyme are absent in the cell of interest, addition of the respective purified, recombinant enzymes can be added to the assay to relieve feedback inhibition by SAM degradation products 24,25,26.
There are four critical steps in this protocol: i) the protease inhibitor cocktail powder has to be added to the lysis buffer just before use (step 1.1); ii) the whole cell extracts are to be utilized within the following hours after preparation (after step 1.4); iii) during the lipid extraction steps (steps 4.2 and 4.4), caution needs to be applied to not transfer any of the interphase or water phase, which contains the excess tritiated SAM, and iv) to aliquot the radioactive S-[Methyl-3H]adenosyl-L-methionine and cold reagent upon receipt as repeated cycles of freeze and thaw degrade it into 5'-methylthioadenosine and homoserine lactone followed by hydrolysis to adenine and S-pentosylmethionine 26,27,28, which may account for lack of measurable PEMT activity. Bad whole cell extracts may also be responsible for no enzymatic activity. In this case, the quality of the cell extract can be assessed by measuring another known enzymatic activity. Last, lack of PEMT activity may rely on the PEMT enzyme being specific to monomethyl-PE or dimethyl-PE rather than PE. Addition of monomethyl-PE and/or dimethyl-PE substrates to the assay may restore PEMT activity.
An alternative, non-radioactive SAM dependent methyltransferase assay has been developed, that relies on the enzymatic quantification of homocysteine, the transmethylation products of SAM 29. This protocol has been successfully applied to measure PE specific methyltransferase activity 16. Unfortunately, this assay is only suitable for purified enzyme and not for whole cell extract, and does not reveal the identity of the reaction products either.
The present protocol can possibly be applied to any cell type. Additionally, this assay can be used to test potential drugs specific to a PEMT of interest in the context of testing new anti-microbial compounds to fight bacterial infections or novel therapeutics against diet-induced atherosclerosis, obesity, and insulin resistance.
The authors have nothing to disclose.
This work was supported by NIH grants ARRA RO3 AI078145 and 1SC3GM113743 to RZ.
S-[Methyl-3H]adenosyl-L-methionine (specific activity of 5-15 Ci/mMole) | Perkin Elmer | NET155050UC | Aliquot the reagent and freeze at -20 °C; follow radiation safety guidelines while using this reagent |
Protease inhibitor cocktail | Roche Life Sciences | 11836170001 | dilute it fresh |
Glass beads, acid washed, 425-600 mm | Sigma Aldrich | G8772 | |
Bicinchoninic acid solution | Sigma Aldrich | B9643 | |
Copper (II) sulfate | Sigma Aldrich | C2284 | |
Scintillation counter MicroBeta2 with 1-detector | Perkin Elmer | 2450-0010 | |
Spectrophotometer Biomate 3 | Thermo Scientific | 840208300 | |
BSA stock solution (10 mg/ml) | New England Biolabs | B9001S | |
Scintillation liquid | Research Product International Corp | 111198 | |
S-(5'-Adenosyl)-L-methionine chloride (hydrochloride) | Cayman Chemicals | 13956 | dilute the reagent in 20 mM HCl and freeze aliquots at -80 °C |