phalloidine and 1-anilino-8-naphthalenesulfonate

The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing beta-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.

Topics: Anilino Naphthalenesulfonates; Animals; Apoproteins; Benzothiazoles; Cell Membrane; Cell Nucleus; Circular Dichroism; Coloring Agents; Cytoskeleton; Fluorescent Dyes; Hydrogen-Ion Concentration; Kinetics; Mice; Microscopy, Electron; Mutation; Myoglobin; NIH 3T3 Cells; Phalloidine; Propidium; Protein Structure, Secondary; Spectrometry, Fluorescence; Tetrazolium Salts; Thiazoles; Time Factors; Ultraviolet Rays

We have studied the effects of three compounds on surface oscillations of human red blood cell ghosts: the P-ATPase inhibitor, suramin; the fluorescent dye of a similar structure, 1,8-anilinonaphthalene sulfonate (ANS); and subfragment 1 of skeletal muscle myosin (S1). It has been found that suramin (10 microM), ANS (100 microM) and S1 (2 mg/ml) suppress the surface oscillations reversibly. The shape of the ghosts remains unchanged. We have also found that suramin and ANS inhibit the ghosts' non-transport (presumably, F-actin-associated) ATPase. The results of the present study suggest the important role of actin ATPase in the generation of cell surface oscillations. The effect of S1, the protein which increases the torsional, but not the bending, rigidity of F-actin upon binding to filaments, favours the possibility that just the torsional dynamics of actin protofilaments leads to the observed oscillations of the ghosts' surface.

Topics: Actins; Adenosine Triphosphatases; Anilino Naphthalenesulfonates; Animals; Cytoskeleton; Erythrocyte Membrane; Fluorescent Dyes; Humans; Myosins; Phalloidine; Rabbits; Suramin