Phosphate analysis and dephosphorylation of modified tau associated with paired helical filaments

Hanna Ksiezak-Reding, Wan Kyng Liu, Shu Hui Yen

Research output: Contribution to journalArticlepeer-review

171 Scopus citations

Abstract

We performed phosphate of tau proteins isolated fromal human brain, tau associated with paired helical filaments (PHF-tau), Alzheimerr 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 gradieent 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 fragmetss of PHF-tau. The shift in mobility was not observed with the C-terminal fragments of 25-26 kDa, whth 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.

Original languageEnglish (US)
Pages (from-to)209-219
Number of pages11
JournalBrain Research
Volume597
Issue number2
DOIs
StatePublished - Dec 4 1992
Externally publishedYes

Keywords

  • Alkaline phosphatase
  • Alzheimer disease
  • Paired helical filament
  • Phosphate analysis
  • Tau protein

ASJC Scopus subject areas

  • Developmental Biology
  • Molecular Biology
  • Clinical Neurology
  • Neuroscience(all)

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