Increase in Osmotic Fragility of Bovine Erythrocytes Induced by Bacterial Phospholipases C

Abstract

Bovine erythrocytes were treated with each of three bacterial phospholipases C; phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens, sphingomyelinase C (SMase) of Bacillus cereus and phosphatidylinositol-specific phospholipase C (Plase) of Bacillus thuringiensis. An increase in osmotic fragility was detected by means of a coil planet centrifugation (CPC) apparatus (Biomedical Systems Co., Tokyo) after the treatment with these enzymes.
The peak of hemolysis normally observed in the untreated erythrocytes at the range between 50 and 100 mOsM shifted to 160 to 200 mOsM with the progress of sphingomyelin hydrolysis by phospholipase C of C. perfringens.
Sphingomyelinase C of B. cereus showed two different effects on bovine erythrocytes: In the absence of divalent cations or in the presence of Ca²⁺ alone, the peak of hemolysis shifted to the region from 130 to 160 mOsM, without appreciable hydrolysis of sphingomyelin, while in the presence of Mg²⁺ or Mg²⁺ plus Ca²⁺, the peak of hemolysis further shifted to the region from 160 to 200 mOsM with the hydrolysis of sphingomyelin.
Abrupt shift in osmotic fragility to a much higher region around 250 mOsM was produced by treatment with increasing amounts of phosphatidylinositol-specific phospholipase C. In this case, a significant amount of acetylcholinesterase was released from the erythrocyte membrane without hot or hot-cold hemolysis.
The mechanism of alteration of osmotic fragility of bovine erythrocytes by treatment with phospholipases C seems to differ from case to case, depending upon the specific action of each enzyme toward the membrane phospholipids.