Fig. 3.
CGI-58 shows lysophosphatidic acid acyltransferase activity. Recombinant His12-CGI-58 (1 μg) was incubated with 10 μM [14C]oleoyl-CoA in the presence of various lipid acceptors (each at 50 μM) and 50 mM Tris-HCl, pH 7.5, for 10 min at 30°C prior to solvent extraction and TLC of extracted lipids. A: Phosphorimager scan of silica gel TLC plate showing radioactive solvent-extracted lipids obtained without addition of recombinant CGI-58 (lane 1) or with addition of CGI-58 to reactions with lysophosphatidic acid (lane 2, LPA), lysophosphatidyl choline (lane 3, LPC), lysophosphatidyl ethanolamine (lane 4, LPE), lysophosphatidyl serine (lane 5, LPS), lysophosphatidyl inositol (lane 6, LPI), or glycerol-3-phosphate (lane 7, G3P) as acceptors. Radioactive phosphatidic acid (PA) formed in lane 2 coeluted with an unlabeled phosphatidic acid standard transiently observed following iodine staining. B: Histogram of enzyme activity of lanes depicted in A. Data represent means ± SD for duplicate samples from one of two experiments.