A new highly cytotoxic protein, toxophallin, was recently isolated from the fruit body of the death cap Amanita phalloides mushroom [Stasyk et al. (2008) Studia Biologica 2, 21–32]. The physico‐chemical, chemical and biological characteristics of toxophallin differ distinctly from those of another death cap toxic protein, namely phallolysin. The interaction of toxophallin with target cells is not mediated by a specific cell surface receptor. It induces chromatin condensation, as well as DNA and nucleus fragmentation, which are typical for apoptosis. However, caspase III inhibitor [benzyloxycarbonyl‐Asp(OMe)‐fluoromethylketone] did not stop toxophallin‐induced DNA fragmentation. Thus, toxophallin uses a caspase‐independent pathway of apoptosis induction. In the present study, we applied a complementary approach based on a combination of proteomics and molecular biology tools for the protein identification of toxophallin. The primary structure of toxophallin was partially studied via direct sequencing of its tryptic peptides, followed by PCR‐based cloning of the corresponding cDNA. A subsequent bioinformatic search revealed a structural homology of toxophallin with the l‐amino acid oxidase of the Laccaria bicolor mushroom. This demonstrates the usefulness of our approach for the identification of proteins in organisms with unknown genomes. We also found a broad substrate specificity of toxophallin with respect to oxidizing selected amino acids. Ascorbic acid inhibited the cytotoxic effect of toxophallin, most likely as a result of scavenging hydrogen peroxide, which is the product of oxidase catalysis. Thus, in addition to highly toxic cyclopeptides and toxic lectin phallolysin, the death cap fruit body contains another cytotoxic protein in the form of an enzyme, namely l‐amino acid oxidase.
Siuloglycoproteins (SGPs) from plasma membranes of hen red blood cells were isolated by preparative SDS-PAGE and their carbohydrate composition and some immunochemical properties were investigated and compared with that of human glycophorin A. Ft was shown, that two principal SGPs with molecular weights of 28 kDa and 55 kDa are monomeric and dimeric forms of the same molecule and are immunochemically closely related. The amount of carbohydrate component consists about 33 % of molecular mass (in human glycophorin A it is 52.3 %). The analysis of monosaccharide composition of hen SGPs indicates, that they possess O-linked and N-linked oligocaccharide chains. In comparison with human glycophorin A hen SGPs have a higher amount of N-glycosidic chains and lower amount of O-glycosidic chains, the last are tentatively of more branched structure.
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