Solubilization and fractionation of paired helical filaments.
Paired helical filaments isolated from brains of two different patients with Alzheimer's disease were extensively treated with the ionic detergent, sodium dodecyl sulphate. Filaments were solubilized at different extents, depending on the brain examined, thus suggesting the existence of two types of paired helical filaments: sodium dodecyl sulphate-soluble and insoluble filaments. In the first case, the number of structures resembling paired helical filaments greatly decreased after the detergent treatment, as observed by electron microscopy. Simultaneously, a decrease in the amount of sedimentable protein was also observed upon centrifugation of the sodium dodecyl sulfate-treated paired helical filaments. A sodium dodecyl sulphate-soluble fraction was isolated as a supernatant after low-speed centrifugation of the sodium dodecyl sulphate-treated paired helical filaments. The addition of the non-ionic detergent Nonidet-P40 to this fraction resulted in the formation of paired helical filament-like structures. When the sodium dodecyl sulphate-soluble fraction was further fractionated by high-speed centrifugation, three subfractions were observed: a supernatant, a pellet and a thin layer between these two subfractions. No paired helical filaments were observed in any of these subfractions, even after addition of Nonidet P-40. However, when they were mixed back together, the treatment with Nonidet P-40 resulted in the visualization of paired helical filament-like structures. These results suggest that at least two different components are needed for the reconstitution of paired helical filaments as determined by electron microscopy. The method described here may allow the study of the components involved in the formation of paired helical filaments and the identification of possible factors capable of blocking this process.
González
P J
PJ
Centro de Biología Molecular (CSIC-AUM), Facultad de Ciencias, Universidad Autonóma de Madrid, Cantoblanco, Spain.
Correas
I
I
Avila
J
J
D000544:Alzheimer Disease / Q000473:pathology*; D001921:Brain / Q000473:pathology / Q000648:ultrastructure*; D001923:Brain Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007382:Intermediate Filaments / Q000737:chemistry / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000302:isolation & purification*; D012016:Reference Values
D016900:Neurofilament Proteins