Staphylococcal β-Hemolysin II. Phospholipase C Activity of Purified β-Hemolysin

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Abstract
Sheep erythrocyte ghosts released water-soluble organic phosphorus when treated with purified β-hemolysin. Phospholipid analysis demonstrated that sphingomyelin accounted for 53% of the phospholipids present in sheep erythrocytes. Purified β-hemolysin showed phospholipase C activity when purified ox brain or sheep erythrocyte sphingomyelin was used as substrate. Such studies have also revealed that the disappearance of sphingomyelin from the reaction mixture was accompanied by a comparable increase in the concentration of phosphoryl choline. Thin-layer chromatography of phospholipids, extracted from sheep erythrocytes which had been exposed to β-hemolysin, demonstrated that sphingomyelin was rapidly degraded. Activators of β-hemolysin, such as Mg ++ , enhanced the release of organic phosphorus from erythrocyte ghosts and from sphingomyelin. Inhibitors of β-hemolysin, such as ethylenediaminetetraacetic acid, p -chloromercuribenzoate, and iodoacetamide, also inhibited the release of organic phosphorus from erythrocyte ghosts and from sphingomyelin. These studies strongly suggested that β-hemolysin enzymatically degraded the sphingomyelin of the erythrocyte membrane. Such degradation probably resulted in the eventual lysis of the erythrocyte.