pH dependence of sphingomyelinase activity in PMNs. Sphingomyelinase activity was determined using liposomes containing NBD-sphingomyelin. Each sample consisted of PMNs (1 × 10⁶) in 0.25 mL Tris-HCl (pH 7.2 to 7.8; ▪) or Tris-maleate buffer (pH 5.6 to 7.2; ○) containing 25 mmol/L Mg²⁺ or Na acetate buffer (pH 4.0 to 5.6; •), with liposomes containing 10 μmol/L substrate and 30 μmol/L lecithin. Samples were probe sonicated and incubated at 37°C for 30 minutes in a bath sonicator. The fluorescent product, NBD-ceramide, was isolated by partitioning the assay mixture with 2-propanol, heptane and water, and was analyzed on a fluorimeter. Values represent the mean ± SD for three experiments. Inset: Sphingomyelinase assays were conducted at pH 7.4 in the presence or absence of 25 mmol/L Mg²⁺, 5 mmol/L EGTA or 5 mmol/L EDTA. Values represent the mean ± SD for three experiments.