Functional interaction between Cerebratulus lacteus cytolysin A-III and phospholipase A2. Implications for the mechanism of cytolysis.
A study on the interaction between bee venom phospholipase A2 and Cerebratulus lacteus cytolysin A-III, a major hemolysin secreted by this organism has been carried out. The hemolytic activity of A-III in phosphate-buffered saline is increased 5-fold in the presence of phospholipase A2 from bee venom. Dansylphosphatidylethanolamine (DPE) labeled, phosphatidylcholine-containing liposomes and human erythrocyte membranes were employed to study the interaction between these two proteins. In DPE-liposomes, A-III alone had no effect on DPE fluorescence nor did it enhance either the phospholipase A2-dependent fluorescence increase or blue shift in emission maximum, indicating that the cytolysin is not a major phospholipase A2-activator. However, when DPE was incorporated into erythrocyte membranes, A-III alone induced a 40% fluorescence increase and a 5 nm blue shift, implying a transient activation of an endogenous phospholipase A2. Further studies using synthetic lysophosphatidylcholine and free fatty acids demonstrated that the hemolytic activity of A-III is potentiated by free fatty acids, a product of phospholipid degradation catalyzed by phospholipase A2. Together, sublytic concentrations of A-III and nonlytic concentrations of oleic acid cause extensive cell lysis. Subsequent analysis of this phenomenon by gel filtration chromatography, analytical ultracentrifugation, chemical cross-linking, and measurement of [14C]oleic acid binding by the cytolysin demonstrated that binding of oleic acid to A-III causes aggregation of the toxin molecules to a tetrameric form which has a higher alpha-helix content and a greater activity than the monomer.
Liu
J W
JW
Department of Biochemistry and Molecular Biology, University of Cincinnati College of Medicine, Ohio 45267-0522.
Blumenthal
K M
KM
D001514:Bee Venoms; D003603:Cytotoxins / Q000378:metabolism*; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008387:Marine Toxins / Q000378:metabolism*; D010714:Phosphatidylethanolamines; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013050:Spectrometry, Fluorescence
D001514:Bee Venoms; C026601:Cerebratulus lacteus toxin A-III; D003603:Cytotoxins; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D008081:Liposomes; D008387:Marine Toxins; D010714:Phosphatidylethanolamines; C024284:dansyl phosphatidylethanolamine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2