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 by 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.The phallotoxins, a family of poisonous bicyclic heptapeptides from the mushroom Amanita phalloides (for review see ref.1), whose main representative is phalloidin (Ta), form tight complexes with F actin from liver cells and from muscle. As Staining of Cells. Cells grown on glass coverslips were rinsed briefly in phosphate-buffered saline (Pi/NaCI) (pH 7.4), fixed for 5 min in 3.7% formaldehyde in Pi/NaCl at room temperature and washed extensively in P1/NaCl. After fixation, cells were either used directly for fluorescent staining or dehydrated in absolute acetone for 4 min at -20°C and air dried or rendered permeable by a 2-min exposure to 0.1% Triton X-100 in Abbreviations: FL, fluorescein; Pi/NaCl, phosphate-buffered saline.t To whom reprint requests should be addressed.

Tritium‐containing affinity‐labelling derivatives of phalloidin, an alkylating iodoacetyl compound (EAL) and a photolabile, carbene generating diazirine (PAL), have been reacted with rabbit muscle actin, the former after protection of thiol groups with N‐ethylmaleimide. Labelled peptides generated by tryptic and/or thermolysin digestion were isolated by paper peptide mapping and characterized by determination of their amino acid sequences. EAL binds to methionine‐119 and methionine‐355; PAL binds to glutamic acid‐117. These residues are located in regions with extremely conserved amino acid sequences. The cleft between the two domains of the actin monomer is suggested as the possible binding site for phalloidin.

By staining of cryo-sections of rat liver with a fluorescent phallotoxin, the distribution of filamentous actin in liver cells could be demonstrated by fluorescence microscopy. While in untreated livers filamentous actin forms an almost continous layer at the cell periphery, the poisoning by phalloidin leads to the formation of actin clusters, preferentially located near the cell membrane.

Ketophalloidin, [Cyclo(~-alanyl-~-threonyl-~-cysteinyl-4-u/~o-hydroxy-L-proly~-L-alanyl-2-mercapto-~-tryptophyl-4-oxo-~-norvalyl)-cyclo(3-t6)sulfid](2), aus Phalloidin (1) durch Periodatoxidation erhalten, wurde mit 2,3-Dimercaptopropionsaure zur Dithiolancarbonsaure 3 oder mit 3-Amino-l,2-propandithiol zum (Aminomethy1)dithiolan 4 umgesetzt. Verbindung 4 diente als Ausgangssubstanz fur weitere Derivate: Mit Bernsteinsaureanhydrid entstand die N-(3-Carboxypropiony1)verbindung 5, mit N-(1odacetoxy)succinimid die N-Iodacetylverbindung 6 und aus ihr durch nucleophile Substitution des Iods durch Azidionen die Azidoacetylverbindung 8. Mit Fluorescein-isothiocyanat wurde aus 4 das fluorescierende Phallotoxin 7 erhalten, das sich spezifisch -wie alle hier beschriebenen Phalloidinderivale -an den Rezeptor Aktin bindet. Die Derivate 6 und 7 konnen fur eine kovalente Bindung an Aktin Verwendung finden (Affinitatsmarkierung), die funktionellen Derivate 3, 4 und 5 wurden mittels eines wasserloslichen Carbodiimids rnit Rinderserumalbumin verbunden; das Amin 4 wurde auch rnit aktivierter Sepharose zu einem Adsorbens fur die Affinitatschromatographie verknupft. Components of the Green Deathcap Toadstool Amanita phalloides, LVIIIl). -Some Dithiolanes Derived from Ketophalloidin as Reagents in Biochemical Research Ketophalloidin[cyclic(~-alanyl-~-threonyl-~-cyste~nyl-4-u/~o-hydroxy-~-pro~yl-~-a~anyl-2-mercapto-~-tryptophyl-4-oxo-~-norvalyl)-cyclic(3-,6)sulfide] (2), obtained from phalloidin (1) by oxidation with periodate, was transformed into the dithiolanecarboxylic acid 3 by reaction with 2,3-dimercaptopropionic acid, and into the (aminomethy1)dithiolane 4 by reaction with 3-amino-1,2-propanedithiol. Compound 4 was the starting material for the follpwing derivatives: the N-(3-carboxypropionyl) compound 5, the N-iodoacetyl compound 6 and, by nucleophilic substitution of iodide by azide, for the azidoacetyl compound 8. A fluorescent phallotoxin (7) was obtained by reaction of 4 with fluorescein isothiocyanate. All phallotoxins described in this paper specifically bind, like phalloidin, to the receptor protein actin. Derivatives 6 and 7, as radioactively labeled molecules, are suitable for a covalent fixation to actin (affinity labeling); the functional derivatives 3 , 4 and 5 have been conjugated to bovine serum albumin, and the amino compound 4 has been covalently attached to activated sepharose in order to generate an adsorbent for affinity chromatography.