amanitins and Hemolysis

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amanita; Amanitins; Amino Acids; Animals; Biological Assay; Carbohydrates; Cytotoxins; Erythrocyte Membrane; Hemolysis; Humans; Kinetics; Protein Binding

Phallolysin from the toadstool, Amanita phalloides, is a basic protein that causes direct haemolysis of red cells. The dose-response curve is steep; the pH optimum is in the weakly acid range. The rate of haemolysis increases with the concentration of the lysin, the optimal temperature is 20 degrees C. The percentage haemolysis-time curves are S-shaped. Haemolysis is of the non-osmotic type. Ca2+ is not required but inhibits haemolysis in a concentration-dependent fashion, as do Mg2+ and Zn2+. The red cell sensitivity of various animal species decreases in the following sequence:mouse greater than rabbit = guniea pig greater than rat greater than man greater than dog approximately or equal to pig greater than sheep = cattle. Red cells of cattle and sheep are largely resistant. Phallolysin is virtually not consumed on haemalysis: the amount of haemoglobin released increases with the number of red cells applied; on repeated addition of fresh red cells the haemolysate retains its full activity. Phallolysin is not inhibited by serum, albumin, cholesterol, lecithin, cephalin or sphingomyelin; inhibition by red cell ghosts of phallolysin haemolysis is considerably less than that of digitonin haemolysis. At sublytic concentrations phallolysin, unlike benzalkonium chloride, liberates practically no membrane lipids from human red cells. Surface activity of phallolysin does not exceed that of bovine serum albumin.-A saponin-like interaction with cholesterol as the basic mechanism of haemolysis can be disregarded. There is also no evidence suggesting a detergent-like effect.

Topics: Amanitins; Animals; Cations, Divalent; Cattle; Dose-Response Relationship, Drug; Erythrocytes; Guinea Pigs; Hemoglobins; Hemolysin Proteins; Hemolysis; Humans; Hydrogen-Ion Concentration; Lipids; Osmotic Fragility; Rabbits; Rats; Sheep; Species Specificity; Surface Tension; Swine; Temperature; Time Factors

The molecular weight of phallolysin, the toxic haemolysin from Amanita phalloides, was established by gel chromatography to be 30000 daltons. The isoelectric point (I.P.) was found in Ampholine pH 7-10 at 8.34. In Ampholine pH 7-9 the gel chromatographically homogeneous phallolysin was separated into phallolysin A (I.P. 8.06) and phallolysin B (I.P. 7.49). Sodium dodecylsulphate-polyacrylamide gel electrophoresis indicated a molecular weight of 33000 daltons for phallolysin A. Phallolysin was thermo- and acid-labile. It was relatively stable in alkaline solutions. 8 M urea as well as 0.1% sodium dodecylsulphate caused irreversible denaturation. On the other hand, phallolysin showed resistance to diverse proteases (pepsin, trypsin, alpha-chymotrypsin, subtilisin, pronase E, bromelin, proteinase K) and also alpha-amylase and pancreatin. Treatment with proteinase K did not change the molecular weight and the isoelectric points of phallolysin. Resistance to proteases was not due to inhibition of proteases by phallolysin.

Topics: Agaricales; Amanita; Amanitins; Animals; Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Female; Hemolysin Proteins; Hemolysis; In Vitro Techniques; Isoelectric Focusing; Mice; Molecular Weight; Peptide Hydrolases; Rabbits; Sodium Dodecyl Sulfate; Urea

Phallolysin, a protein from Amanita phalloides with cytolytic effects in vitro, was highly toxic when given intravenously to rats, mice, rabbits and guinea pigs: i.v. LD50 in rats was 85 Haemolytic Units (HU)/kg, corresponding to 0.05 mg protein/kg b.w. Death ensued from intravascular haemolysis. In rats large doses (600 HU/kg b.w.) caused cardiac death within a few minutes due to liberation of potassium from lysed cells. The serum contained lethal concentrations of potassium. There was also histological evidence of severe renal damage as a result of the haemolysis. In addition, phallolysin directly damaged the isolated guinea pig heart and the isolated rat liver, probably by its action on membranes. Given by mouth, phallolysin was not poisonous to rats.

Topics: Amanitins; Animals; Blood Pressure; Female; Glomerular Filtration Rate; Guinea Pigs; Heart Rate; Hemolysis; In Vitro Techniques; Kidney; Lethal Dose 50; Liver; Male; Mice; Myocardial Contraction; Osmotic Fragility; Perfusion; Potassium; Rabbits; Rats; Regional Blood Flow

Topics: Acetylglucosamine; Amanitins; Binding Sites; Cell Membrane; Erythrocytes; Glucosamine; Hemolysin Proteins; Hemolysis; Lectins; Oligopeptides

Topics: Amanitins; Amino Acids; Animals; Chromatography, DEAE-Cellulose; Chromatography, Gel; Concanavalin A; Glycoproteins; Hemolysis; Isoelectric Focusing; Lethal Dose 50; Mice; Molecular Weight; Rabbits; Rats