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.
