Relationship between haemolytic and sphingomyelinase activities in a partially purified β-like toxin fromStaphylococcus schleiferi

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Abstract
β-Toxins of staphylococcal species possess dual activity in that they can both lyse erythrocytes (by ‘hot-cold’ lysis) and catalyse hydrolysis of membrane-associated sphingomyelin. However, the precise relationship between these two activities has not been extensively studied. We have partially purified a β-like toxin from culture supernatants of Staphylococcus schleiferi N860375 which exhibits both ‘hot-cold’ lysis of erythrocytes and neutral sphingomyelinase activities. This toxin has a strong preference for sheep erythrocytes, the membranes of which are rich in sphingomyelin. Kinetic analysis suggests that haemolysis and sphingomyelinase activities are very closely associated obeying identical Michaelis–Menten kinetics. However, pre-treatment with antibodies to Staphylococcus aureusβ-toxin, Ca2+, dithiothreitol and phenylmethylsulfonyl fluoride appear to inhibit sphingomyelinase activity significantly more strongly than haemolysis while Mg2+ activates sphingomyelinase activity more strongly than haemolysis. We attribute these effects to differences in binding properties in the two assays. Micropurification by both sphingosylphosphocholine-agarose affinity chromatography and preparative electrophoresis revealed that the 34-kDa toxin associates non-covalently with individual proteins.