title: Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments.
abstract: We performed phosphate analysis of tau proteins isolated from normal human brain, tau proteins associated with paired helical filaments (PHF-tau), and Alzheimer tau not associated with PHF. These tau fractions were of high purity. Normal and Alzheimer tau were purified by heat treatment, acid extraction and calmodulin-affinity chromatography with or without HPLC. Fractions containing primarily PHF-tau polypeptides of 60, 64 and 68 kDa and their degraded fragments were purified either on a sucrose density gradient as filaments (PHF) or by heat treatment and acid extraction as amorphous proteins (PHF-tau). PHF and PHF-tau were found to contain 6-8 mol phosphate/mol protein while normal and Alzheimer tau proteins contained 1.9 and 2.6 mol phosphate/mol protein, respectively. Upon 2-h incubation with alkaline phosphatase, PHF lost two of the phosphate groups without apparent changes in the stability and morphology of PHF. The released phosphate originated from the N-terminal half of PHF-tau as determined by immunoblotting with antibodies to epitopes blocked by phosphorylation. Tau-1 and E-2, and by a prominent shift in the electrophoretic mobility of some fragments of PHF-tau. The shift in mobility was not observed with the C-terminal fragments of 25-26 kDa, which retained the epitope to Tau 46. The results suggest that the phosphorylation sites not affected by phosphatase may be located in the 25-26 kDa C-terminal region of PHF-tau and may play a role in structural stability of PHF.
authors: Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461.
authors: H
authors: H
authors: Ksiezak-Reding
authors: W K
authors: WK
authors: Liu
authors: S H
authors: SH
authors: Yen
mesh_terms: D000469:Alkaline Phosphatase; D000544:Alzheimer Disease / Q000378:metabolism* / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D010710:Phosphates / Q000032:analysis*; D010766:Phosphorylation; D012016:Reference Values; D016875:tau Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010710:Phosphates; D016875:tau Proteins; D000469:Alkaline Phosphatase