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Fibrillogenesis and Cytotoxic Activity of the Amyloid-forming Apomyoglobin Mutant W7FW14F
- Source :
- Journal of Biological Chemistry. 279:13183-13189
- Publication Year :
- 2004
- Publisher :
- Elsevier BV, 2004.
-
Abstract
- 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.
- Subjects :
- Circular dichroism
Time Factors
Membrane permeability
Amyloid
Phalloidine
Ultraviolet Rays
Phalloidin
Tetrazolium Salts
amyloid cytotoxicity
macromolecular substances
Fibril
Biochemistry
Anilino Naphthalenesulfonates
Protein Structure, Secondary
Mice
chemistry.chemical_compound
Animals
Benzothiazoles
Propidium iodide
Coloring Agents
apomyoglobin fibrillation
Molecular Biology
Cytoskeleton
Fluorescent Dyes
Cell Nucleus
Myoglobin
Circular Dichroism
Cell Membrane
Fibrillogenesis
Cell Biology
Hydrogen-Ion Concentration
Kinetics
Microscopy, Electron
Thiazoles
Spectrometry, Fluorescence
chemistry
Mutation
NIH 3T3 Cells
Biophysics
Thioflavin
Apoproteins
Propidium
amyloid fibril formation
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 279
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....b7c776f0730926e0f88489464814a137