The lytic behavior of pure phospholipases A2 and C towards osmotically swollen erythrocytes and resealed ghosts

Abstract

SUMMARY1. 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 A2 (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: and C did not alter the lytic behavior of these phospholipases.2. Resealed ghosts made in saline in the presence of 2 mM CaC12 retained 16-20% of their hemoglobin and were sensitive to lysis by phospholipase C, but not by phospholipase A2; 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 A2 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 A2.3. Resealed ghosts made in sucrose in the presence of 2 mM CaC12 retained 24-30% of their hemoglobin and showed increasing lysis by phospholipase C with increase in the lysing tonicity prior to resealing. Phospholipase A2 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 A2 or C showed no increased lysis as compared with non-proteolysed cells.

Influence of extracellular media’s ionic strength on the osmotic stability of Sahel goat erythrocytes

“…Sucrose is a non-ionic disaccharide osmolyte which does not penetrate the erythrocyte membrane [28] and osmotic fragility curve in sucrose solutions may demonstrate the osmotic properties of erythrocytes more effectively than saline [29]. Haemolysis was reported to occur more slowly in hypotonic saline than sucrose solution at the same osmotic strength [30].…”

“…Replacing isotonic sucrose with isotonic saline potentiated the lytic effect of phenothiazines [10], whereas the lytic effect of saponin was reduced in isotonic sucrose [31]. Phospholipases A 2 and C were not lytic to intact human erythrocytes in both isotonic and hypotonic (0.6%) saline, but they evoked lysis of intact erythrocytes suspended in hypotonic sucrose (below 120 and 100 mM, respectively) [29,32].…”

Hemolytic Effects of Snake Venoms

“…However, if the red cell membrane is subjected to certain treatments producing little or no hemolysis per se, the phospholipase becomes able to hydrolyze extensively the phospholipid substrates and hemolysis occurs. WOODWARD and ZWAAL (1972) demonstrated that hypotonically swollen human erythrocytes expose their phospholipids to the action of pancreatic phospholipase A, with subsequent lysis of the cells. LAN KISCH et ai.…”

Molecular sieving action of the cell membrane during gradual osmotic hemolysis

“…Because at least partial dehydration would seem to be necessary for a sugar to interact with the membrane, sucrose and fructose would be more likely to enter the membrane than glucose. The entrance of sucrose into the stressed membrane probably explains the susceptibility of red cells swollen in hypotonic sucrose solutions to phospholipase C and A2 compared to the resistance of cells in saline (Woodward and Zwaal, 1972). When sucrose is present with NlI 4 Cl, additional membrane sites could be occupied in the stressed membrane, and its area could be further expanded prior to hemolysis (Table IX).…”

“…Experimental data support these predictions. Phospholipids are labeled to a greater extent with acetic anhydride in ghosts than in intact cells (Carraway et a1., 1972); phospholipase C from Bacillus cereus and pancreatic phospholipase A2 hydrolyze phospholipids in ghost but not in intact RBC' s when suspended in saline (Zwaal et aI., ] 971; Woodward and Zwaal, 1972); and the differences observed with spin labels between the inner and outer membrane leaflet in intact cells are not present in ghosts unless the hemolysis medium contains Mg++ (Tanaka and Ohnishi, 1976).…”