Abstract
This is an assay designed to examine the radioactive phosphorous incorporation when the molecule is being synthesized, which means that only de novo synthesized phospholipids can be detected. Thus, with this technique it is possible to detect in vitro phospholipid synthesis under different required experimental conditions respect to controls (Guido and Caputto, 1990; Ferrero et al., 2014). There are different types of lipids. Among them we can find phospholipids, which contain glycerol esterified with two fatty acyl chains and a phosphate group that can also be bound to an organic molecule that acts as “hydrophilic head”, as shown in Figure 1 for the case of phosphatidylcholine. This structure confers amphipathic properties to lipid molecules that allow them to form lipid bilayers, making phospholipids the main components of biological membranes. Figure 1. Representation of phospholipid structure. Extracted from: http://bio1151.nicerweb.com/Locked/media/ch05/phospholipid.html
Keywords: Phospholipids Synthesis, Radioactive, In vitro, ATP-P32, Labeling
Materials and Reagents
Equipment
Procedure
Note: To determine under which conditions lipid synthesis is linear with time, prepare different tubes and carry out the reaction at 37 °C for different times. If you are evaluating the capacity of modifying lipid synthesis of a given protein, you must also determine under which conditions lipid synthesis is linear with the concentration of your protein of interest. The person that will carry on the experiments must have strict training with radioactive working, according to rules and governing laws.
Representative data
Figure 3 is a representative example of data that indicates the type of results expected. In this case, the experimental condition measures the amount of phospholipid synthesis in the presence of c-Fos protein, its phosphorylated version or its mutants. In this regard, results are represented as amount of phospholipid labeling (Ferrero et al., 2012). Figure 3. c-Fos phosphorylated by c-Src does not activate phospholipid synthesis. The capacity to activate phospholipid synthesis of recombinant c-Fos, c-Fos phosphorylated by purified c-Src (P-c-Fos), the phosphomimetic Y10/30E mutant of c-Fos and the non-phosphorylatable mutant of c-Fos Y10/30F was examined as described previously (Gil et al., 2004). Incubations were for 60 min at 37 °C. 32P-phospholipid quantification was performed as described previously (Guido and Caputto, 1990). Results expressed as c.p.m. of 32P incorporated into phospholipids/mg of protein are the mean±s.d. of three experiments performed in triplicate; *P<0.002 with respect to control (buffer) as determined by One Way ANOVA analysis. Y10/30F not incubated with c-Src and c-Src incubated without any added substrates were run as controls. Note that the presence of c-Src in the assays did not modify phospholipid synthesis.
Notes
If you decide to freeze after carrying out the reaction in step A6, don’t add the TCA-PTA 10-1 (% w/v) solution as the homogenate might agglomerate and precipitate together with non incorporated [γ32P] ATP molecules, which can lead to erroneous measurements.
Recipes
Acknowledgments
We thank Dr. Beatriz L. Caputto for helpful discussions to adapt and modify the protocol form her previous work (Guido and Caputto, 1990). We also thank CONICET and FONCYT for funding.
References
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