Biomedical subjects
Publications and source records attributed to H Faulstich.
Amatoxins are the sole cause of human Amanita poisoning, inhibition of transcription eventually leading to cell necrosis. The toxins are easily excreted into the bile fluid, a fact which postulates the interruption of the enterohepatic circulation. All therapeutic means should be applied in order to decrease the serum concentration of the toxins as soon as possible.
Using [3H]-demethylphalloin as a tracer the uptake of phallotoxins by the liver of young (6, 12, 16 days old) and adult rats was determined in relation to the dose of toxin administered. The maximum amount taken up by the livers of the young rats was only about 50% of that in adults. Nevertheless, with a dose as high as 55 mg/kg body weight the toxin concentration in the young liver reached more than 30 micrograms/g, being markedly higher than the minimum concentration (approximately 20 micrograms/g) required to cause irreversible damage of the liver in adult rats and death of the animals. This suggests that the tolerance of young rats to phallotoxins cannot solely be explained by the reduced uptake of the toxin.
A fluorescent derivative of phalloidin has been synthesized possessing high affinity to filamentous actin. This compound was used for visualization of actin-containing structures in eukaryotic nonmuscle cells. Due to its low molecular weight (1250), fixation for formaldehyde was sufficient to render the membrane permeable for the labeled peptide. Bundles of microfilaments are the predominant pattern in the flat rat kangaroo PtK1 cells, whereas a net of concentric fibers characterizes the more spherical bovine kidney MDBK cells. Specificity of staining was confirmed by competition experiments with unlabeled phalloidin.
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.
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Phalloidin reduces the critical concentration [G]c = Kc-1 for the depolymerisation of rabbit muscle actin. Adding 1 equivalent of toxin reduces the [G]c by a factor of 30; adding 2 equivalents reduces the [G]c by a factor of 90. The depolymerisation of actin was measured by the exchangeability of 45Ca and [14C]ADP in equilibrium dialysis. Half dissociation of both the metal ion and the nucleotide were found at the same concentration. From this value we calculate the critical concentration for actin [G]c=1.05 x 10(-6)M. The analogous value in presence of 1 equivalent of toxin is [G]'c=3.7 x 10(-8)M. The dissociation from actin for a labelled phallotoxin, [3H]demethylphalloin, was likewise studied by equilibrium dialysis. The apparent KD for this toxin, as well as for the natural toxin phalloidin, is 3.6 x 10(-8)M. The value is identical to that of [G]'c. This indicates that the dissociation of the toxin and the breakdown of the filaments together with a concomitant release of Ca and ADP are interdependent events.
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Phallotoxins protect F-actin from heat denaturation at a temperature of 70 degrees C for 3 min. This has been shown by difference spectroscopy. G-actin is not protected from heat denaturation by phalloidin. Among the various phallopeptides investigated, 4 toxic ones showed the same protecting ability, whereas non-toxic seco-compounds has no effect. The non-toxic (S)-phalloidinsulfoxide A exhibited only a partial protecting activity, corresponding to an affinity for F-actin of about one tenth of that of the toxic peptides. Evidence for this was obtained from the easy displacement of the sulfoxide A from F-actin by phalloidin as well as by a spectroscopic dilution titration.
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