Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments.
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.
Ksiezak-Reding
H
H
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461.
Liu
W K
WK
Yen
S H
SH
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
D010710:Phosphates; D016875:tau Proteins; D000469:Alkaline Phosphatase