Subcellular localization of neutral sphingomyelinase and ceramide in resting human PMNs. Isolated, DFP-treated PMNs at 1.5 to 5 × 107 cells/mL, were disrupted by nitrogen cavitation. Ten milliliters of postnuclear supernatant was layered on a three-layer Percoll density gradient and centrifuged as described in Experimental Procedures. The gradient was fractionated into 35 fractions by aspiration from the bottom of the tube. Fractions were assayed for neutral sphingomyelinase, myeloperoxidase (MPO), lactoferrin, gelatinase, HLA class I, and latent alkaline phosphatase (latent AP). Numbers are average of four experiments, normalized to a cell number of 3 × 108 cells, and expressed in percent of the total amount measured in the fractions 1 through 35. The subcellular distribution of ceramide is shown in the bottom panel. Ceramide was measured as described in Experimental Procedures after removal of Percoll by ultracentrifugation. The absolute value for neutral sphingomyelinase activity in the peak fraction was 65.5 pmol sphingomyelin hydrolyzed/min/mL. The peak value for ceramide was 0.44 nmol/mL. Results of one representative experiment are shown.
