MECHANISM OF THE LEAKAGE INDUCED ON LIPID MODEL MEMBRANES BY THE HEMOLYTIC PROTEIN STICHOLYSIN-II FROM THE SEA-ANEMONE STICHODACTYLA-HELIANTHUS

A potent hemolytic polypeptide, sticholysin II, has been purified to h omogeneity from the sea anemone Stichodactyla helianthus. The protein produces leakage of aqueous contents of model lipid vesicles composed of either phosphatidylcholine or sphingomyelin if cholesterol is prese nt in these membranes. The leakage has been analyzed by measuring the dequenching of the fluorescent dye 8-aminonaphthalene-1,3,6-trisulfoni c acid, coencapsulated with its quencher N,N'-p-xylenebispyridinium br omide, upon dilution of the vesicle contents into the external medium. The protein displays a maximum effect on vesicles containing. 20-25% cholesterol. Leakage is also produced in vesicles composed of mixtures of phosphatidylcholine and sphingomyelin, the maximum effect being ob served for 20-30% sphingomyelin molar content. The extent of the leaka ge is dependent on the molecular moss of the vesicle entrapped solutes in the range 445-960 Da. This suggests the involvement of a pore of a bout 1 nm in diameter based on the limiting size observed for the leak age of the different solutes. Oligomerization of the protein is appare ntly involved in the membrane permeabilization, based on the kinetic a nalysis of the leakage process which is shown to proceed through an al l-or-none mechanism.