The lytic behavior of pure phospholipases A 2 and C towards osmotically swollen erythrocytes and resealed ghosts

Abstract

1. 1. Phospholipase C (phosphatidylcholine cholinephosphohydrolase, E.C. 3.1.4.3) from Bacillus cereus evoked hemolysis of intact human erythrocytes in hypotonic sucrose solutions at sucrose concentrations below 120 mM, whereas pancreatic phospholipase A 2 (phosphatide acyl-hydrolase, E.C. 3.1.1.4) became lytic below 100 mM sucrose. Treatment of intact cells with proteolytic enzymes prior to the incubations with phospholipases A 2 and C did not alter the lytic behavior of these phospholipases. 2. 2. Resealed ghosts made in saline in the presence of 2 mM CaCl 2 retained 16–20 % of their hemoglobin and were sensitive to lysis by phospholipase C, but not by phospholipase A 2; resealed ghosts made in saline in the presence of 1 mM EDTA retained only 1–2 % of their hemoglobin and were lysed by both phospholipases A 2 and C. Resealed ghosts prepared in the presence of Ca 2+ after increasing durations of lysis prior to the resealing procedure, showed a decrease in lysis due to phospholipase C, but no lysis due to phospholipase A 2. 3. 3. Resealed ghosts made in sucrose in the presence of 2 mM CaCl 2 retained 24–30 % of their hemoglobin and showed increasing lysis by phospholipase C with increase in the lysing tonicity prior to resealing. Phospholipase A 2 showed a minimum in the lysis of resealed ghosts, which were hemolysed at 20 mM sucrose prior to resealing. Treatment with subtilopeptidase A of resealed ghosts followed by treatment with phospholipase A 2 or C showed no increased lysis as compared with non-proteolysed cells. 4. 4. Alterations in membrane architecture occur during the process of ghost formation from intact erythrocytes by a hypotonic lysis procedure. During this process the polar headgroups of the phospholipids become more readily available for hydrolysis by phospholipase C than does the fatty acid-ester linkage for hydrolysis by pancreatic phospholipase A 2.