Phalloidin isolation

Several experimental approaches can be employed to determine the pool sizes of polymerised and non-polymerised actin. Firstly, the enzyme DNAse I is inhibited by monomeric (G) actin, but not by polymerised (F) actin. Secondly, polymerised actin can be directly visualised by use of fluorescent derivatives of phalloidin, a cyclic peptide isolated from the toadstool Amanita phalloides that selectively binds to polymerised actin with high affinity. 

Labeled phallotoxines (phalloidins) The bicyclic peptides isolated from Amanita phalloides mushroom bind selectively to F-actin in nanomolar concentrations. They have advantages over antibodies for actin labeling... 

Phenanthroline like 1,10- and 1,7-phenanthrolines has anticancer properties.374 The toxic effects of phalloidine, an isolate of the fungus Amanita phalloides, are inhibited by 4,7-phenanthroline.536 4,7-Phenanthroline also inhibits proline incorporation into proteins.391,392 3-Methyl-4,7-phenanthroline has been suggested as a diagnostic agent for assisting in the detection of blood in body fluids.537... 

A sample purified of an Ostreopsis ovata extract was lately assayed on actin cytoskeleton of isolated rabbit intestinal cells by Botana s group. The measure of a fluorescent dye bound specifically to actin filaments (Oregon green 514-phalloidin) showed that Ostreopsis ovata toxin also interfered the cytoskeleton as occurred in the two previous cases (Table 6.4). In addition, activity on membrane potential of neuroblastoma cells was studied through the fluorescent probe bis-oxonol. The purified extract displayed a depolarizing effect (the value of relative fluorescence obtained at 350 seconds for cells exposed to the sample was 1.49 0.12 versus control) also similar to palytoxin and ostreocin-D. Therefore, although these studies only shed very preliminary information abont the toxic compound produced by Ostreopsis ovata, they appear to point out some link between its cellular targets and those identified for palytoxin and ostreocin-D in these studies. 

Fig. 3. HIV-1 envelope signaling triggers actin rearrangement. Resting CD4T cells were treated with a laboratory-adapted viral strain, HIV-1 4.3 (a), or with a primary viral isolate, HIV-1 g3ug 4e (b), or with gpl 20IIIB (100 pM) (c) for various times, fixed and permeabilized, and then stained with FITC-phalloidin for F-actin and analyzed by flow cytometry. Shown are histograms of F-actin staining.

Bavaria, where in 1937 Ulrich Wieland and Feodor Lynen were able to crystallize for the first time a substance which was called phalloidin that rapidly killed mice after intraperitoneal application. Three years later Heinrich Wieland and Rudolf Hallermayer crystallized from Amanita phalloides extracts a second toxin, amanitin , which with smaller doses killed the experimental animals only after several days Uke a deadly dish of A, phalloides kills humans. Bernhard Witkop (Plate 49) 1940 in H. Wieland s laboratory recognized that phalloidin is a peptide and isolated a new imino acid, allo-hydroxyproline. Then the Second World War interrupted Amanita research which was resumed in Heidelberg by one of the present authors only ten years later and continued in Mainz, Frankfurt and again in Heidelberg through the following decades. 

A cyclo-nonapeptide, isolated from linseed by Kaufmann and Tobschirbel [48] which also prevents the uptake of phalloidin (and other substances) by hepatocytes, cyclolinopeptide A, shows a certain similarity with AA in its amino acid sequence (Fig. 20) but does not form alkali-metal complexes. With the accumulation of aromatic amino acids between proline residues as an active region in mind, Kessler et al. designed some smaller cyclopeptides of which one, c(D-ProPhePheProPhePhe), exhibited somewhat stronger protection and a second less related one, called 008, showed even better protection of liver cells against phalloidin than antamanide [49]. 

In the search for the lethal poison in A phalloides, in the absence of specific indicators, animal experiments were used, first with guinea pigs then with white mice. The fractions obtained in the work-up of extracts from the fungus were, as is the practice in pharmacology, injected into the animals under their skin (subcutaneously, s.c.) or into the veins (intravenously, i.v.) or into the abdomen (intraperitoneally, i.p). A sample was regarded active (toxic) if on its administration the mouse died within a few hours. Guided by this assay it became possible to isolate in 1937, as mentioned above, crystalline phalloidin and later several related peptides, the phallotoxins. The smallest dose which kills a 20 g... 

Ahhreviationy. ATi apoptosis inducing fector, At f5 autophagy protein 5, LC3B microtubule-associated protein 1 light chain 3B, mtHSPJO heat shock protein 70 mitochondrial isoform, p62/SQSTMp62/sequestosome 1 Phalloidin is a toxin isolated from the deadly Amanita phalloides that binds directly F-actin... 

In isolated hepatocytes the first toxic lesion is a change in cell shape. About 10 minutes after administration of phalloidin to the medium hepatocytes develop numerous blebs on their surface (11). We compared the kinetics of bleb formation in vivo with the kinetics of actin polymerization from the thymosin (34 complex in vitro, and found that the two processes develop at a comparable rate. Since polymerization occurs at a rate somewhat faster than the development of surface blebbing in vivo, the morphological lesions in the... 

Weiss, G., Sterz, I.,Frimmer, M., and Kroker, K., 1973, Electron microscopy of isolated rat hepatoc 4es before and after treatment with phalloidin. Beitr. Pathol. 150 345-356. 

WiELAND and CO workers 64, 114, 429) independently reported oxidation of the indole-2-thioether function with H2O2 in acetic acid and applied the oxidative reaction to phalloidin. The isolation of the... 

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