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.

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

A method is presented for the rapid separation of cell nuclei from the rabbit cerebral cortex into two populations. The first of these consists largely of nuclei with the morphological characteristics of neuronal nuclei, the second almost entirely of nuclei with the morphological characteristics of glial cell nuclei. From studies based upon sensitivity to the toxin α-amanitin, the ratio of incorporation of different bases, ionic requirements and differential sensitivity to actinomycin D, it is concluded that under both the classical low and high salt conditions described by other workers, two enzymes are active in RNA synthesis. The presence of a third enzyme of low activity cannot be excluded. No qualitative difference in the number of enzymes involved in RNA synthesis in neuronal and glial cell nuclei has been found, but there are quantitative differences in activity between the two nuclear populations.

Studies on rna synthesis in two populations of nuclei from the mammalian cerebral cortex

Publisher Summary This chapter presents the status of RNA polymerases involved in RNA synthesis in mammalian cells, with emphasis on some recent developments in the field of nuclear RNA polymerase. The chapter discusses that the RNA polymerases of eukaryotic nuclei exist in multiple forms. These enzymes are compartmentalized within the nucleus. They have different requirements for cations and salt. The nucleolar enzymes are insensitive to α-amanitin in vitro whereas the nucleoplasmic enzymes are generally inhibited by the toxin, which binds to the enzyme and prevents RNA chain elongation. The nucleolar enzymes are more active with native DNA as the template, whereas the nucleoplasmic enzymes are more efficient with a denatured template; however, addition of a cytoplasmic protein factor can restore the ability of the latter enzyme to transcribe preferentially native DNA. The regulation of the activity of purified nucleolar RNA polymerase by hormones independent of any significant modification in the chromatin template is important, considering the emphasis in the past on template modification as the most prominent means of gene regulation. Only the early effect of hormones on the purified RNA polymerase activity has been examined with purified RNA polymerases. The chapter concludes that finally, the factors that are involved in initiation, termination and release of RNA chains in eukaryotic cells should be explored.

Mammalian RNA polymerases.

Immunological studies of yeast nuclear RNA polymerases at the subunit level

Thesulfur-containing antibiotic thiolutin hasbeenshowntobea potent, reversible inhibitor ofthegrowth ofSaccharomyces cerevisiae. Viability was unaffected overtheconcentration rangeof4to100ug/ml. Atconcentrations as lowas2 sg/ml, thedruginhibited ribonucleic acid(RNA)andprotein synthesis inwholecells andspheroplasts. Atthese lowconcentrations, protein synthesis continued for ashort period oftimeafter RNAsynthesis was completely stopped. Withhigher drugconcentrations (greater than20;g/ml) protein synthesis was inhibited; concentrations ofthiolutin up to100,ug/ml didnotaffect translocation orpeptide bondformation incell-free protein-synthesizing systemsfromyeast. Theeffect ofthiolutin on theactivity ofpartially purified deoxyribonucleic acid-dependent RNA polymerases was examined, andthedrug was found tobea potentinhibitor ofRNA synthesis invitro. Inhibition was greatest whenthe polymerase was preincubated withthiolutin. Several mechanisms arediscussed toexplain themultiple effects ofthiolutin onS.cerevisiae. Since theaction ofthe drugiseasily reversed, thiolutin may provetobeofuse instudies ofvarious stages ofyeastgrowth. Inbacteria, antibiotics haveplayed useful roles inproviding genetic markers foranumber ofmacromolecules andhavealso beenusedin dissecting theroles ofmacromolecules inconcerted reactions. Theuseofantibiotics inyeast hasnotbeenexploited tothesameextent. This isdue,inpart, tothefactthatmanyofthe better-characterized antibiotics donotaffect Saccharomyces cerevisiae, presumably because theyeastcell isimpermeable tothese compounds. Manyantifungal compounds havebeen isolated during theprocess ofscreening for antimicrobial agents, and,although theymay notbemedically useful because oftheir toxicity properties, itisquite possible that someofthese compounds mayhavebiochemical applications. Thisreport describes ourinitial studies onthe modeofaction ofonesuchantifungal agent, thiolutin.

/pdf/mode-of-action-of-thiolutin-an-inhibitor-of-macromolecular-4eqieany5i.pdf

Mode of Action of Thiolutin, an Inhibitor of Macromolecular Synthesis in Saccharomyces cerevisiae

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2871%2980445-3

H. Ponta

Papers

DNA‐dependent RNA polymerases from yeast. Partial characterization of three nuclear enzyme activities