Fig. 2.

(a) Phospholipase activity of SseJ requires the presence of a eukaryotic factor. GST–SseJ, GST–SseJS151V or GST was incubated with DPPC liposomes in the presence or absence of HeLa cell PNS for 2 h at 37 °C, pH 7.4, before released FFA were quantified. Data represent mean±sd derived from three independent experiments performed in triplicate. (b) Phospholipase activity is dependent on SseJ concentration, but limited by the amount of activator. A fixed amount of HeLa cell extract (0.8 mg protein) was incubated with 1–7.5 μg GST–SseJ (•), GST–SseJS151V (▴) or GST for 2 h at 37 °C, pH 7.4, before FFA were quantified. Values of FFA following incubation with GST were subtracted from values obtained by incubation of DPPC with equivalent amounts of GST–SseJ or GST–SseJS151V. These data represent the mean derived from two independent experiments performed in triplicate. (c) Phospholipase activity as a function of PNS protein amounts. GST–SseJ (5 μg) (▪) or GST was incubated with a range of concentrations of HeLa cell PNS from 0 to 1.1 mg protein at pH 7.4. Released FFA were quantified after 2 h incubation at 37 °C. Values obtained with GST alone were subtracted from values obtained with GST–SseJ. These data represent the mean derived from two independent experiments performed in triplicate.