Isolation and chemical characterization of Alzheimer's disease paired helical filament cytoskeletons: differentiation from amyloid plaque core protein.
The paired helical filaments (PHFs) of Alzheimer's disease were purified by a strategy in which the neurons and amyloid plaque cores of protein (APCP) were initially isolated. This was achieved by several steps of isocratic sucrose centrifugations of increasing molarity and a discontinuous isotonic Percoll density gradient. After collagenase elimination of contaminating blood vessels, lysis of neurons was produced by SDS treatment. The released PHF cytoskeletons were separated from contaminating APCP and lipofuscin by sucrose density gradient. A final step consisted in the chemical purification of highly enriched PHFs and APCP components via a formic acid to guanidine hydrochloride transition. PHFs and APCPs were fractionated by size exclusion HPLC and further characterized and quantitated by automatic amino acid analysis. We also present some of the morphological and immunochemical characteristics of PHF polypeptides and APCP. Our studies indicate that apart from differences in localization and morphology, PHF and APCP significantly differ in (a) chemical structure (peptide and amino acid composition); (b) epitope specificity (antiubiquitin, antitau, antineurofilament); (c) physicochemical properties (structural conformation in guanidine hydrochloride); and (d) thioflavine T fluorescence emission. These parameters strongly suggest important differences in the composition and, probably, in the etiopathology of PHF and APCP of Alzheimer's disease.
Roher
A E
AE
Department of Anatomy, Wayne State University School of Medicine, Detroit, Michigan 48201.
Palmer
K C
KC
Chau
V
V
Ball
M J
MJ
D000544:Alzheimer Disease / Q000473:pathology*; D000682:Amyloid / Q000032:analysis* / Q000302:isolation & purification; D016229:Amyloid beta-Peptides; D001921:Brain / Q000473:pathology*; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D003599:Cytoskeleton / Q000032:analysis* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000032:analysis; D009474:Neurons / Q000032:analysis* / Q000648:ultrastructure
D000682:Amyloid; D016229:Amyloid beta-Peptides; D009419:Nerve Tissue Proteins