Figure 5.

A, The effect of pH. Incubations were carried out with 1 ng of purified PLA₂, 0.2 mm 1-palmitoyl-2-[¹⁴C]palmitoyl-PC, 1 mm lubrol PX, and 10 mm CaCl₂ in 75 mm buffer at 30°C for 15 min in a final volume of 50 μL. Buffers used were: acetic acid, pH 4.5 to 5.5; Mes, pH 5.5 to 6.5; Bis-Tris propane, pH 6.5 to 9.5, and Caps, pH 9.5 to 11. Bis-Tris propane, 1,3-bis(Tris[hydroxymethyl]methylamino)propane; Caps, 3-(cyclohexylamino)-1-propanesulfonic acid. B, The effect of Ca²⁺ concentration on PLA₂ activity. Incubations were carried out with 1 ng of purified PLA₂ in 50 mm Tris-HCl buffer, pH 8.0, in the presence of 0.2 mm 1-palmitoyl-2-[¹⁴C]palmitoyl-PC, 1 mm lubrol PX, and with Ca²⁺ concentrations as indicated in the figure in a final volume of 50 μL at 30°C for 15 min. C, Time-course incubations of purified PLA₂ with and without BSA. Purified PLA₂, 0.5 ng, was incubated in the presence of 0.2 mm 1-palmitoyl-2-[¹⁴C]palmitoyl-PC, 1 mm lubrol PX, 10 mm CaCl₂, 50 mm Tris-HCl, pH 8.0, in the absence of BSA (•), with 50 μg of BSA (○), or with 250 μg of BSA (×), in a final volume of 50 μL for the indicated times at 30°C. D, Effect of substrate concentration at various lubrol PX/phosphatidylcholine molar ratios. Purified PLA₂, 1 ng, was incubated in the presence of 10 mm CaCl₂, 50 mm Tris-HCl, pH 8.0, 0.02 to 1 mm 1-palmitoyl-2-[¹⁴C]palmitoyl-PC without detergent (•) or with a lubrol PX/phosphatidylcholine molar ratio of 2 (○) or 6 (×) in a final volume of 50 μL for 10 min at 30°C. conc., Concentration.