Quantitative solubilization and analysis of insoluble paired helical filaments from alzheimer disease

• Published in: Brain research 1996-04, Vol.717 (1), p.99-108
• Main Authors: Smith, Mark A., Siedlak, Sandra L., Richey, Peggy L., Nagaraj, Ramanakoppa H., Elhammer, Ake, Perry, George
• Format: Article
• Language: English
• Subjects: Adolescent; Adult; Aged; Aged, 80 and over; Alkalies; Alzheimer disease; Alzheimer Disease - metabolism; Biological and medical sciences; Cell Fractionation; Cross-Linking Reagents - analysis; Cross-Linking Reagents - metabolism; Crosslink; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Densitometry; Endopeptidases; Formates; Glycation; Glycation End Products, Advanced - analysis; Guanidine; Guanidines; Hemostatics; Humans; Immunoblotting; Medical sciences; Microscopy, Electron; Middle Aged; Neurofibrillary pathology; Neurofilament Proteins - analysis; Neurofilament Proteins - metabolism; Neurofilament Proteins - ultrastructure; Neurology; Paired helical filament; Phosphorylation; Protein Denaturation; Sodium Dodecyl Sulfate; Solubility; Urea
• ISSN: 0006-8993; 1872-6240
• DOI: 10.1016/0006-8993(95)01473-X

In this study, we evaluate the ability of several solvents to solubilize insoluble paired helical filaments (PHF) of Alzheimer disease. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents of PHF. Using sequential categories of protein solvent to analyze insoluble PHF, only alkali or exhaustive proteolysis are effective in completely solubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosphorylation of PHF with hydrofluoric acid does not affect PHF solubility. These findings suggest that the ‘hyperphos-sphorylation’ of PHF proteins is not responsible for PHF insolubility. However the in vitro glycation of τ generates PHF that are insoluble in SDS and soluble in alkali. These findings suggest that protein crosslinks, including advanced glycation endproduct-derived crosslinks which were recently described in Alzheimer disease, play a major role in effecting PHF insolubility in vivo.