Theodor Wieland

Amatoxins, phallotoxins, phallolysin, and antamanide: the biologically active components of poisonous Amanita mushrooms.

This review gives a comprehensive account of the molecular toxicology of the bicyclic peptides obtained from the poisonous mushrooms of the genus Amanita. The discussion of the biochemical events will be preceded by a consideration of the chemistry of the toxic peptides. The structural features essential for biological activities of both the amatoxins and the phallotoxins will be discussed, also including the most important analytical data. Similar consideration will be given to antamanide, a cyclic peptide, which counteracts phalloidin. In addition, the phallolysins, three cytolytic proteins from Amanita phalloides will be discussed. The report on the biological activity of the amatoxins will deal with the sensitivity of the different RNA-polymerases towards the toxins and with their action on various cell types. Consideration will also be given to systems in which alpha-amanitin was used and can be used as a molecular tool; in the past, many investigators used the inhibitor in molecular biology, genetics, and even in physiological research. As for the phallotoxins, discussion of the affinity of these toxins for actin is provied. Further discussion attempts to understand the course of intoxication by filling in the gap between the first molecular event, formation of microfilaments, and the various lesions in hepatocytes during the intoxication.

Modification of Actins by Phallotoxins

Phallotoxins bind to filamentous actin (F-actin) from liver or rabbit muscle with Kdiss∼10−8M. By this combination the structure of F-actin is stabilized to such an extent that it will be resistant to the depolymerizing action of 0.6M KI and of deoxyribonuclease I, to denaturation by heat (70 °° C), and to local ruptures caused by ultrasonication or by cytochalasin B. The structural features of phallotoxins essential for affinity to F-actin are specific.

Struktur und Wirkung der Amatoxine

The amatoxins, mainly 0cand fl-amanitine, are the essential lethal poisons of the toadstool Amanita phalloides. Structurally they are cyclic octapeptides whose ring is divided and held in compact shape by a sulfoxide bridge. Chemical changes in the molecule, like scission of one of the rings or alterations at the y=hydroxylated isoleucine side chain, may reduce or even eliminate toxicity. The toxic action can be attributed to a biological property of the toxins operating at molecular level: extremely strong inhibition of one of the eucaryotic RNA polymerases (form II resp. B). Bisnor-deoxy-amanineamide, a synthetic compound having the features of the highly toxic natural products in simplified form, inhibits RNA synthesis by 90% at a concentration of 40 ~tg/ml. I t is t03 times less active than the natural amatoxins and therefore nontoxic.

Spectroscopic evidence for the interaction of phalloidin with actin

Phalloidin and secophalloidin, a derivative which is nontoxic and does not stabilize F-actin [3], were samples from our laboratory. Bovine serum albumin was a product of Behringwerke, Marburg. F-actin, free from troponin and tropomyosin was a gift of P. Dancker and M. Hoffmann, who prepared it from rabbit sketetal muscle according to [4] . The F-actin pellets were homogenized in 0.1 M KC1 to a final concentration of 1.48 X IO-’ M. Concentrations of actin and phalloidin were determined spectrophotometrically. For actin eM was determined at 280 nm as 4.16 X lo4 based on Lowry’s method [S] and on the molecular weight of the actin subunit of 45.000 daltons. The EM at 300 nm for phalloidin was 1.18 X lo4 considering a correction of the original value of 1 .lO X 1 O4 [6] for 3 molecules of water in cristalline phalloidin. Spectroscopy was carried out in an Aminco DW-2UV-VIS spectrophotometer using two tandem cuvettes with a total length of 0.875 cm. Initially both tandem cuvettes contained actin solution as described above in one compartment, and 0.70 X lo-’ to 1.48 X 10e4 M solutions of the cyclic peptide in the other. After recording the baseline, the solutions in the two compartments of one cuvette were mixed and the difference spectrum was measured.

Antamanide and analogs. Studies on selectivity and stability of complexes

Abstract The complexing behaviour of antamanide was studied with various mono- and bivalent cations in different solvents. Complexes of highest stability were found for Na, Ca and Tl(I). Polarity of solvent affects complex stability constants strongly, but the selectivity to a lesser degree. The antamanid-Na complex was isolated as a crystalline perchlorate. UV and CD spectral changes on addition of water indicate a transition between defined solvation states of antamanide, that were referred to a conformational change previously detected. The biological activity of some analogs of antamanide was related to Na complexation. From the analogs synthesized up to now it is evident, that Na complexation or at least an attribute of the molecule running parallel to this, is a prerequisite, but not the only one, for biological activity.

Affinity of antamanide for sodium ions

In a mass-spectroscopic analysis of a sample of AA which had been in contact with Na+ ions during isolation, A. Prox found a peak of m/e = I 169 (= 23 + mass peak 1146 of the cyclopeptide). This peak was more pronounced when a sample of AA, which had been obtained by adding a 5% NaCl solution to an alcoholic solution of the cyclopeptide, was examined. Two additional peaks at 1169 and 1168 (1169-H+) could also be observed (Hd). An interaction of AA with Na+ ions could also be seen in the IR spectra. In the absence of N

A carbene-generating photoaffinity probe for beta-adrenergic receptors

the spectrum (in KI) shows two bands of equal height (at 1630 and 1660 cm-t) in the carbonyl region, whereas in the Na+containing sample the 1630 cm-1 band was about 10% more intense (Hd). IR spectra in solutions (fig. I_) are even more informative, revealing more nonhydrogen bonded NH-groups in the Na-complex (NH

Immunosuppressive activity in the series of cycloamanide peptides from mushrooms

A new radioiodinated (2.2 Ci/mumol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [125I]ICYP-diazirine binds with high affinity (Kd = 60 pM) to beta-receptors from turkey erythrocyte membranes. Upon irradiation, [125I]ICYP-diazirine is covalently incorporated in a Mr 40 000 protein. Stereoselective inhibition of photolabeling by the (-)enantiomers of alprenolol and isoproterenol indicated that the Mr 40 000 protein contains a beta-adrenergic binding site. The yield of specific labeling was up to 8.2% of total beta-receptor binding sites. The Mr 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [125I]ICYP-diazirine resulted in specific labeling of two proteins with Mr 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximately Mr 67 000) was labeled specifically.

Experimental and calculated conformational characteristics of the bicyclic heptapeptide phalloidin

The immunosuppressor activity of the cycloamanides A, B, C, and D, and two of their D-amino acid residue-containing analogues, was examined using PFC (plaque forming cell) and DTH (delayed type hypersensitivity) tests. It was found that cycloamanide A (CyA A, II) [c-(Phe-Phe-Ala-Gly-Pro-Val-)] and its D-Phe-containing analogue III [c-(Phe-D-Phe-Ala-Gly-Pro-Val-)] are the most potent immunosuppressors of the whole series. The retroanalogue of III [c-(D-Phe-Val-Pro-Gly-Ala-)] was found to be less active than III. The immunosuppressor activity of O-carboxymethyl-Tyr6-antamanide (I) was also tested. It was found that the substitution of one of the Phe residues of ANT by O-carboxymethyl-Tyr does not substantially affect the immunosuppressor activity.

Amanitin binding to calf thymus RNA polymerase B

Abstract Several conformations generated from approximate potential energy calculations are presented for the bicyclic heptapeptide phalloidin which are consistent with the conformation-dependent information obtained from proton nuclear magnetic resonance measurements performed on phalloidin in dimethylsulfoxide solution. In each conformation, the cysteine amide proton is intramolecularly hydrogen bonded, the tryptophan amide is internally buried and the methyl group of the alanine residue preceding tryptophan is shielded by the tryptophan ring. Thus, phalloidin appears to be a relatively rigid molecule in solution.