Palmitoyl coenzyme a

Acyl-CoA and fatty alcohol specificity of TrWSD4 and TrWSD5 was determined by the same spectrophotometric assays as described for WS/DGAT activity assay. Acyl-CoA specificity was measured using hexadecanol as acyl acceptor, whereas alcohol specificity was determined with palmitoyl-CoA as acyl donor. In assays for acyl-CoA specificity of TrWSD4 and TrWSD5, different molecular species of acyl-CoAs were added to the above reaction mixture. Acyl-CoA substrates used in this study include: octanoyl-Coenzyme A, decanoyl-Coenzyme A and palmitoyl-Coenzyme A which were purchased from Sigma-Aldrich, and lauroyl Coenzyme A, myristoyl Coenzyme A, stearoyl Coenzyme A, (6Z,9Z,12Z-octadecatrienoyl) Coenzyme A, (5Z,8Z,11Z,14Z-arachidonoyl) Coenzyme A, and (5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl) Coenzyme A which were obtained from Avanti Polar Lipids (Alabama, USA).

Pyridoxal-5’-phosphate, L-Serine, and palmitoyl coenzyme A were purchased from Sigma-Aldrich (St. Louis, MI, USA). Sphingosine-1-phosphate (S1P) was purchased from Avanti Polar Lipids. DMEM culture media and fetal bovine serum (FBS) were purchased from EuroClone Life Science Division (Milan, Italy). l-[³H(G)]-Serine was purchased from PerkinElmer, Inc. (Wellesley, MA, USA).
Human breast cancer cell line MDA-MB-231, from America Type Culture Collection (ATCC), Rockville, MD, USA, was chosen as the source of serine-palmitoyl transferase (SPT) enzyme. Cells were maintained at 5% CO₂, 95% humidity at 37 °C in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin solution as antibiotics. Cells were seeded in 150 mm Petri dishes (3 × 106 cells in 24 mL) and left to grow for at least 3 days, until 80–90% confluence was reached, before being collected for microsomal membranes fraction extraction, due to the localization of the studied enzyme at this level.

Fatty acid-free bovine serum albumin (BSA), fraction V, was from Roche Diagnostics GmbH. Amplex Red was from Life Technologies. Rotenone, FCCP [carbonyl cyanide p-(trifluoro-methoxy)-phenylhydrazone], GDP (guanosine 5′-diphosphate) (sodium salt), pyruvic acid (sodium salt), L(−) malic acid (disodium salt), palmitoyl coenzyme A (lithium salt), dl-carnitine HCl, safranin O, EDTA (ethylenediamine tetraacetic acid), horseradish peroxidase, alamethicin, PFOA (perfluorooctanoic acid), PFOS (perfluorooctane sulfonic acid) (tetraethylammonium salt), and octanoic acid (sodium salt) were all from Sigma-Aldrich Co. PFOS, PFOA, and octanoate were dissolved in 20 mM K⁺–Tes (pH 7.2) with 5 % ethanol. GDP was dissolved in 20 mM Tes (pH 7.2), and the pH of the solution was readjusted to 7.2. FCCP was dissolved in 95 % ethanol and diluted in 50 % ethanol. Ethanol in a final concentration of 0.1 % did not in itself have any effects on the parameters measured.