Relationship between haemolytic and sphingomyelinase activities in a partially purified beta-like toxin from Staphylococcus schleiferi.
beta-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 beta-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 aureus beta-toxin, Ca(2+), dithiothreitol and phenylmethylsulfonyl fluoride appear to inhibit sphingomyelinase activity significantly more strongly than haemolysis while Mg(2+) 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.
Analytical and Biological Chemistry Research Facility and Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Mardyke, Cork, Ireland.
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Etienne
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Sheehan
D001427:Bacterial Toxins / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism; D006461:Hemolysis / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013210:Staphylococcus / Q000201:enzymology / Q000378:metabolism*
D001427:Bacterial Toxins; D010664:Phenylmethylsulfonyl Fluoride; D004492:Edetic Acid; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium; D002118:Calcium; D004229:Dithiothreitol