Analysis of cytotoxicity of lectin and non-lectin proteins from Amanita mushrooms

Hemagglutinating, hemolytic and cytotoxic activities of proteins extracted from five species of Amanita genus, (A. muscaria, A. rubescens, A. phalloid es, A. citrina, A. virosa) were studied. Hemagglutinating lectins were dete cted in extracts of A. muscaria and A. rubescens and purified by affinity c hromatography on immobilized mucin. A. muscaria lectin agglutinated human r ed blood cells at concentration as low as 0.8 mug/ml, such agglutinating ac tivity being inhibited only by salivary mucin or asialomucin. A. rubescens lectin showed a weak agglutinating activity (200 mug/ml). In A. phalloides extracts hemolytic lectin, known as phallolysin, was detected and purified by affinity chromatography on ovomucin. The lowest hemolytic concentration of phallolysin amounted to 5 mug/ml. The hemolytic activity of phallolysin was effectively blocked by ovomucin and human glycophorin A. The toxicity o f protein preparations towards cultured murine leukemia L1210 cells was est imated. Phallolysin exhibited a marked cytotoxic effect (IC50 1.7 mug/ml). Non-lectin protein fraction of A. phalloides was also toxic for L1210 cells (IC50 0.35 mug/ml) and induced cytomorphological changes quite different f rom that caused by phallolysin. Protein responsible for this toxic effect w as identified by preparative slab PAGE and was found to differ considerably from phallolysin. Proteins from other mushrooms, including purified hemagg lutinins from A. muscaria and A. rubescens, showed no cytotoxicity at conce ntration up to 40 mug/ml.