Binding of phospholipase A to the direct lytic factor revealed by the interaction of Ca 2+ with the haemolytic effect

Abstract

1. 1. Direct lytic factor fractionated from cobra venom by CM-cellulose chromatography contains traces of phospholipase A probably bound by complex formation. Direct lytic factor has been prepared free of phospholipase A activity by rechromatography in the presence of 5 M urea. 2. 2. The presence of a trace amount (about 0.1%) of phospholipase A in direct lytic factor preparations does not affect the direct haemolytic effect of direct lytic factor unless Ca 2+ is added. However, when Ca 2+ is present in concentrations of about 5 mM in incubation mixtures of direct lytic factor and red cells traces of phospholipase A can be detected: in their presence Ca 2+ will enhance whereas in their absence it will depress haemolyis. 3. 3. High concentrations of Ca 2+ (45 mM) inhibit haemolysis by direct lytic factor with or without phospholipase A. 4. 4. Several findings suggest that the effects of Ca 2+ and of direct lytic factor on phospholipase A activity are based on different mechanisms. Ca 2+ is essential for the enzymic reaction proper, whereas direct lytic factor, unable to replace Ca 2+, changes membrane structures exposing phospholipids to the action of phospholipase A.