Fig. 3.

Effect of ion strength, cationic amphiphilic drug, or pH on LPLA2 activity. A: The reaction mixture contained 0-500 mM NaCl, 48 mM sodium acetate (pH 4.5), 10 μg/ml BSA, liposomes (127 μM phospholipid), and either 14.5 ng/ml of recombinant mouse LPLA2 (•) or 7.8 μg protein/ml of the soluble fraction obtained from MDCK cells transfected with mouse LPLA2 (+) in 500 μl of total volume. Before starting the reaction, liposomes consisting of DOPC, sulfatide, and NAS (3:0.3:1, molar ratio) were preincubated for 5 min at 37°C. The reaction was initiated by adding the recombinant LPLA2 or the soluble cell fraction and carried out at 37°C. The reaction products were extracted and separated by an HPTLC plate using a solvent system consisting of chloroform-acetic acid (9:1, v/v). One of the products, 1-O-oleoyl-NAS, quantified by scanning the plate, was plotted against NaCl concentration. B: Liposomes consisting of DOPC sulfatide and NAS were preincubated with a different concentration of cationic amphiphilic drug, amiodarone, for 5 min at 37°C and then incubated with the recombinant LPLA2. C: The reaction was carried out in sodium citrate/sodium phosphate buffer over a pH range. For the transacylase assay of LPLA2 (•), the reaction mixture contained liposomes (127 μM phospholipid) consisting of DOPC, sulfatide, and NAS (3:0.3:1, molar ratio), sodium citrate/sodium phosphate buffer, 10 μg/ml BSA, and 14.5 ng/ml of recombinant mouse LPLA2 in 500 μl of total volume. For the esterase assay of LPLA2 (—), the reaction mixture contained 200 μM p-nitro-phenylbuturate (pNPB), sodium citrate/sodium phosphate buffer, 10 μg/ml BSA, and 14.5 ng/ml of recombinant mouse LPLA2 in 500 μl of total volume. The reaction was initiated by the addition of recombinant LPLA2 and run for 10, 20, 30, and 40 min at 37°C. One hundred μl of the reaction mixture was taken at each time point and mixed with 100 μl of cold 0.2 M Na₂CO₃. Absorbance of the mixture at 400 nm was measured immediately. Error bars indicate SD. (n = 3).